Investigation of cap-independent in neuronal differentiation

D I S S E R T A T I O N zur Erlangung des akademischen Grades

Doctor rerum naturalium (Dr. rer. nat.)

eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von M.Sc. Larissa Ruhe

Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst

Dekan der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm

Gutachter:

1. Prof. Dr. Nikolaus Rajewsky 2. Prof. Dr. Alexander Löwer 3. Ph.D. Marina Chekulaeva

Tag der mündlichen Prüfung: 17.12.19 ABSTRACT

Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E-BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap- independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.

ZUSAMMENFASSUNG

Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap- abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt. Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt. Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden.

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SELBSTSTÄNDIGKEITSERKLÄRUNG

Hiermit versichere ich, Larissa Ruhe, dass ich die vorliegende Dissertation selbstständig und auf der Grundlage der angegebenen Hilfsmittel und Hilfen angefertigt habe. Ich erkläre, dass ich sämtliche in der Arbeit verwendeten fremden Quellen und Grafiken als solche kenntlich gemacht habe. In Kooperation erhobene Daten und Ergebnisse wurden ebenfalls im Text als solche kenntlich gemacht. Dazu gehört die Probenaufbereitung, Durchführung und Datenaufarbeitung der massenspektrometrischen Analyse durch Dr. Guido Mastrobuoni. Die Experimente zu circMbl habe ich eigenständig durchgeführt, diese waren aber Teil eines Kollaborationsprojektes, welches von Prof. Dr. Sebastian Kadener initiiert wurde und dessen Ergebnisse in Zusammenarbeit mit Dr. Nagarjuna Reddy Pamudurti, Dr. Osnat Bartok, Dr. Marvin Jens, Dr. Reut Ashwal-Fluss, Christin Sünkel (Stottmeister), Dr. Mor Hanan, Dr. Emanuel Wyler, Dr. Daniel Perez-Hernandez, Evelyn Ramberger, Shlomo Sheniz, Moshe Samson, Dr. Gunnar Dittmar, Prof. Dr. Markus Landthaler, Dr. Marina Chekulaeva und Prof. Dr. Nikolaus Rajewsky entstanden und bei Molecular Cell publiziert wurden [1]. Diese Arbeit oder Teile davon wurden bei keiner anderen wissenschaftlichen Einrichtung eingereicht, angenommen oder abgelehnt. Ich besitze keinen Doktorgrad und habe mich an keiner anderen Stelle um einen Doktorgrad beworben. Die dem Promotionsverfahren zugrunde liegende Promotionsordnung habe ich zur Kenntnis genommen. Weiterhin erkläre ich, dass keine Zusammenarbeit mit gewerblichen Promotionsberaterinnen/-beratern stattgefunden hat und dass die Grundsätze der Humboldt-Universität zu Berlin zur Sicherung guter wissenschaftlicher Praxis eingehalten wurden.

Berlin, 27.05.2019

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TABLE OF CONTENT

ABSTRACT ...... I ZUSAMMENFASSUNG ...... II SELBSTSTÄNDIGKEITSERKLÄRUNG ...... III TABLE OF CONTENT ...... IV LIST OF FIGURES ...... VI LIST OF TABLES ...... VII ABBREVIATIONS ...... VII 1 INTRODUCTION ...... 21 1.1 The canonical mechanism of translation initiation in eukaryotes: 5’end-dependent initiation ...... 21 1.2 Noncanonical mechanisms of translation initiation: cap-independent and internal initiation ...... 23 1.2.1 Translation initiation by internal ribosome entry sites (IRESs) in viruses ...... 23 1.2.2 Translation initiation by internal ribosome entry sites (IRESs) in ...... 25 1.2.3 Cap-independent translation initiation at the 5’end: CITE-like mechanisms ...... 41 1.3 Global and gene-specific translational regulation ...... 46 1.3.1 Molecular properties of the translational repressor initiation factor 4E binding protein 1 (4E-BP1) ...... 47 1.3.2 Biological functions of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) ...... 49 1.3.3 RNA binding and sequence elements in untranslated regions ...... 50 1.3.4 m6A mRNA methylation ...... 53 1.3.5 Upstream open reading frames (uORFs) and start codons (uAUGs) ...... 54 1.3.6 miRNA binding sites ...... 58 1.3.7 mRNA secondary structure ...... 61 1.4 Translational control in physiological and pathological conditions ...... 63 1.4.1 Cell cycle and mitosis ...... 63 1.4.2 Cell stress and apoptosis ...... 69 1.4.3 Cancer and cell transformation ...... 81 1.4.4 Stem cells and differentiation...... 89 1.5 Circular RNA (circRNA) as a new class of cellular RNA ...... 101 1.5.1 Characteristics and functions of circular RNA (circRNA) ...... 101 1.6 Aim of the study ...... 115 2 MATERIAL AND METHODS ...... 116 2.1 Material ...... 116 2.1.1 Chemicals and ...... 116 IV

2.1.2 Buffers solutions and media...... 118 2.1.3 Bacterial strains and eukaryotic cell lines ...... 120 2.1.4 Antibodies ...... 121 2.1.5 Plasmids ...... 121 2.1.6 Primer ...... 122 2.1.7 Kits ...... 128 2.1.8 Devices and consumables ...... 128 2.1.9 Software and online tools ...... 130 2.2. Methods ...... 130 2.2.1 Cell culture ...... 130 2.2.1.1 Cell culture, passaging, freezing and thawing ...... 130 2.2.1.2 Transient transfection ...... 131 2.2.1.3 Stable transfection ...... 131 2.2.1.4 Induction and neuronal differentiation ...... 132 2.2.2 Cloning and plasmid preparation ...... 132 2.2.2.1 Polymerase chain reaction (PCR)...... 132 2.2.2.2 Agarose gel electrophoresis ...... 134 2.2.2.3 Restriction digest and dephosphorylation ...... 134 2.2.2.4 Ligation ...... 134 2.2.2.5 Oligo cloning ...... 135 2.2.2.6 Site-directed mutagenesis ...... 137 2.2.2.7 Bacterial transformation and overnight culture ...... 137 2.2.3 Molecular biology ...... 138 2.2.3.1 RNA extractions ...... 138 2.2.3.2 cDNA synthesis ...... 138 2.2.3.3 Quantitative real-time polymerase chain reaction (qRT-PCR) ...... 139 2.2.3.4 In vitro transcription ...... 140 2.2.3.5 RNA circularization ...... 141 2.2.3.6 In vitro translation ...... 144 2.2.4 Methods of protein biochemistry ...... 144 2.2.4.1 Protein extraction ...... 144 2.2.4.2 Bradford protein assay ...... 145 2.2.4.3 SDS-PAGE ...... 145 2.2.4.4 Western blot ...... 145 2.2.4.5 Mass spectrometry ...... 146 2.2.4.6 Luciferase reporter gene assay ...... 147

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3 RESULTS ...... 147 3.1. Generation and characterization of mouse embryonic stem cells expressing inducible dominant negative 4E-BP1 ...... 147 3.1.1 Working principle of dominant negative 4E-BP1 in cap-dependent translation inhibition ...... 148 3.1.2 ASCL1 induces differentiation of mouse embryonic stem cells into neurons ...... 148 3.1.3 Recombinase mediated cassette exchange to generate stable cell lines expressing dominant negative 4E-BP1 ...... 149 3.1.4 Expression of dominant negative 4E-BP1 reduces cap-dependent translation and promotes cap-independent translation ...... 150 3.2 Identification of upregulated genes upon dominant negative 4E-BP1 expression in mouse embryonic stem cells and induced neurons using proteomic analysis...... 151 3.3 Dominant negative 4E-BP1 resistant genes are linked to DNA repair and neuron projection development ...... 156 3.4 Validation of selected candidates of cap-independent translation in bicistronic reporter assays ...... 156 3.4.1 Bicistronic reporter assays in human embryonic kidney and mouse embryonic stem cells in context of dominant negative 4E-BP1 expression ...... 157 3.4.2 siRNA-based validation of bicistronic reporter constructs in human embryonic kidney and mouse embryonic stem cells ...... 160 3.4.3 Mapping of functional sequence elements in selected genes Fam96b and Riok1 ...... 164 3.4.4 Selected candidate gene Pqbp1 is cap-independently translated in vitro ...... 165 3.5 CircMbl can be translated in a cap- and 5’-end-independent manner in vitro ...... 168 4 DISCUSSION ...... 171 4.1 Potential role of cap-independent translation of Pqbp1 ...... 171 4.2 Role of circRNA translation ...... 175 4.3 Challenges in validation of internal translation initiation ...... 179 4.4 Data of this study in the context of current literature and future directions ...... 185 5 APPENDIX ...... 192 5.1 Supplementary figure...... 192 5.2 Supplementary table ...... 193 6 REFERENCES ...... 195 ACKNOWLEDGEMENTS ...... 241

LIST OF FIGURES

Fig. 1: Model of the canonical translation initiation pathway in eukaryotes...... 22 Fig. 2: Scheme of the 18S rRNA secondary structure including crosslinking sites to mRNA during translation initiation...... 40

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Fig. 3: Overview of regulatory RNA elements in the 5’UTR acting in cis and trans on mRNA translation...... 46 Fig. 4: Inducible cassette exchange stably integrates dominant negative 4E-BP1 and ASCL1 into mESCs...... 149 Fig. 5: Inducible expression of dominant negative 4E-BP1 and ASCL1 promotes cap-independent translation and neuronal differentiation...... 151 Fig. 6: Upregulate genes after dominant negative 4E-BP1 induction in mESCs were selected as candidates for putative cap-independent translation...... 152 Fig. 7: Proteomic analysis identified upregulated genes after dominant negative 4E-BP1 induction in mESCs...... 155 Fig. 8: Candidate genes show activity in bicistronic reporter assays...... 161 Fig. 10: Working principle of siRNA-based validation of bicistronic luciferase reporters...... 162 Fig. 11: Deletion series of Fam96b and Riok1 reveal functionally active elements of transcriptional regulation...... 165 Fig. 12: Pqbp1 is cap-independently translated in vitro...... 167 Fig. 13: CircMbl can be cap-independently translated in vitro...... 171 Fig. 14: Sets of mRNA reporters required to dissect the contribution of different modes of translation initiation that are active for a sequence of interest...... 183

LIST OF TABLES

Tab. 1: Examples of cellular IRESs, which have been reported to be regulated by PTB binding. 37 Tab. 2: Reported genes with cellular IRES-mediated translation and cell cycle-dependent regulation...... 66 Tab. 3: IRES-containing transcripts with important functions in tumor cells...... 84 Tab. 4: Enriched GO terms for candidate genes of cap-independent translation...... 156

ABBREVIATIONS

14-3-3σ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta 40S Eukaryotic small ribosomal subunit 60S Eukaryotic large ribosomal subunit 80S Eukaryotic ribosome 3’ss 3’ splice site 4E-BM Eukaryotic translation initiation factor 4E binding motif 4E-BP Eukaryotic translation initiation factor 4E binding protein 4E-BP1 Eukaryotic translation initiation factor 4E binding protein 1 4E-BP1(4Ala) Dominant negative mutant of eukaryotic translation initiation factor 4E binding protein 1 having four sites replaced by alanine 4E-BP2 Eukaryotic translation initiation factor 4E binding protein 2 4E-BP3 Eukaryotic translation initiation factor 4E binding protein 3 4EGI-1 eIF4E/eIF4G interaction inhibitor 4E-T Eukaryotic translation initiation factor 4E nuclear import factor 1

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5’ss 5’ splice site 5’TOP 5’ terminal oligopyrimidine tract 5’UTR 5’ untranslated region aa Amino acid ADAR1 Ribonucleic acid specific adenosine deaminase AdoMetDC S-adenosylmethionine decarboxylase 1 AGO Argonaute AGO2 Argonaute RISC catalytic component 2 AIRES Artificial internal ribosome entry site AKT AKT serine/threonine kinase 1 ALEX Protein of the Gnas complex gene ALKBH5 Alpha-Ketoglutarate-dependent dioxygenase AlkB homolog 5 AMPK Protein kinase AMP-activated catalytic subunit α1 ANRIL Antisense non-coding ribonucleic acid in the INK4 locus Antp Antennapedia Apaf-1 Apoptotic peptidase activating factor 1 ARE AU-rich elements AS Argininosuccinate synthase ASCL1 Achaete-scute family bHLH transcription factor 1 ASO Anti-sense oligonucleotide asTORi Adenosine triphosphate active-site inhibitors of mechanistic target of rapamycin ATF4 Activating transcription factor 4 ATF5 Activating transcription factor 5 ATXN1 Ataxin 1 AXIIR Annexin II receptor Bag-1 BCL2 associated athanogene 1 BCAT1 Branched chain amino acid transaminase 1 Bcl-2 B-cell chronic lymphocytic leukemia/lymphoma 2 Bcl-xL BCL2-like 1 BiP Immunoglobulin heavy-chain binding protein BOP1 BOP1 ribosomal biogenesis factor bp BTG1 BTG antiproliferation factor 1 C3H10T1/2 C3H mouse embryo pluripotent stem cell line VIII

Cage1 Cancer antigen 1 Caprin-1 Cell cycle associated protein 1 Cas9 Clustered regulatory interspaced short palindromic repeat associated protein 9 CASP3 Caspase 3 CAT Chloramphenicol transferase Cat-1 Arginine/lysine transporter CCND1 Cyclin D1 CCNT1 Cyclin T1 CCR4 Carbon catabolite repressor 4 CDC2L2 Cell division cycle 2-like 2, also known as CDK11A, PITSLRE or p58PITSLRE CDK1 Cyclin dependent kinase 1 CDK11 / CDK11p58 Cyclin dependent kinase 11 CDK2 Cyclin dependent kinase 2 cDNA Complementary deoxyribonucleic acid CDR1 Cerebellar degeneration-related protein 1 CDR1as Cerebellar degeneration-related protein 1 antisense ribonucleic acid CDS Coding sequence C/EBPβ CCAAT/enhancer binding protein beta ceRNA Competing endogenous ribonucleic acid CERT Cytosine-enriched regulator of translation C-FAG C-terminal fragment of apoptotic cleavage of eIF4G cGAS Cyclic GMP-AMP synthase Check1 Checkpoint kinase 1 cIAP1 Baculoviral IAP repeat containing 2 cIAP2 Baculoviral IAP repeat containing 3 circANRIL Circular antisense non-coding ribonucleic acid in the INK4 locus circFoxo3 Circular forkhead box O3 circHECTD1 Circular HECT domain E3 ubiquiting protein ligase 1 circHIPK3 Circular homeodomain interacting protein kinase 3 circMbl Circular muscleblind circPABPN1 Circular poly(A) binding protein nuclear 1 circRNA Circular ribonucleic acid CiRNAs Intronic circular ribonucleic acid IX circSHPRH Circular SNF2 histone linker PhD RING circSLC8A1 Circular solute carrier family 8 member A1 circSRY Circular sex determining region Y circZNF9 Circular CCHC-type zinc finger nucleic acid binding protein CITE Cap-independent translation enhancer c-Jun Jun proto-oncogene, AP-1 transcription factor subunit CLIP-Seq Cross-linking immunoprecipitation high throughput sequencing c-myb MYB proto-oncogene, transcription factor c-Myc MYC proto-oncogene, bHLH transcription factor Co-IP Co-immunoprecipitation CPEB2 cytoplasmic polyadenylation element binding protein 2 Cre Type I topoisomerase from bacteriophage P1 CRISPR Clustered regulatory interspaced short palindromic repeats CrPV Cricket paralysis virus CSDE1 Cold shock domain containing E1 c-Src SRC proto-oncogene non-receptor tyrosine kinase cyp24a1 Cytochrome P450 family 24 subfamily A member 1 DAP5 Eukaryotic translation initiation factor 4 gamma 2, also known as eIF4G2, NAT1 or p97 Dbr1 Debranching ribonucleic acid lariats 1 DCC DCC netrin 1 receptor DDX1 DEAD-box helicase 1 DGCR8 DiGeorge critical region 8 DHX9 DExH-box helicase 9 DICER Dicer 1, ribonuclease III D-IRES Differentiation-linked internal ribosome entry site DKC1 Dyskerin pseudouridine synthase 1 DMD Dystrophin DNA Deoxyribonucleic acid DNase Deoxyribonuclease DROSHA Drosha ribonuclease III DTL Denticleless E3 ubiquitin protein ligase homolog E2f1 E2F transcription factor 1 eEF Eukaryotic translation elongation factor eEF1A Eukaryotic translation elongation factor 1 alpha 1 X eEF2 Eukaryotic translation elongation factor 2 eEF2D Eukaryotic translation elongation factor 2D eEF2K Eukaryotic translation elongation factor 2 kinase EF-1α Elongation factor-1 alpha EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGR2 Early growth response 2 EIciRNAs - circular ribonucleic acid eIF Eukaryotic translation initiation factor eIF1 Eukaryotic translation initiation factor 1 eIF1A Eukaryotic translation initiation factor 1A eIF2 Eukaryotic translation initiation factor 2 eIF2α Eukaryotic translation initiation factor 2α eIF2B Eukaryotic translation initiation factor 2B eIF2B5 Eukaryotic translation initiation factor 2B subunit ε eIF3 Eukaryotic translation initiation factor 3 eIF3a Eukaryotic translation initiation factor 3a eIF3b Eukaryotic translation initiation factor 3b eIF3d Eukaryotic translation initiation factor 3d eIF3l Eukaryotic translation initiation factor 3l eIF3m Eukaryotic translation initiation factor 3m eIF4A Eukaryotic translation initiation factor 4A eIF4B Eukaryotic translation initiation factor 4B eIF4E Eukaryotic translation initiation factor 4E eIF4E1B Eukaryotic translation initiation factor 4E family member 1B eIF4E2 Eukaryotic translation initiation factor 4E family member 2 eIF4E3 Eukaryotic translation initiation factor 4E family member 3 eIF4F Eukaryotic translation initiation factor 4F eIF4G Eukaryotic translation initiation factor 4 gamma eIF4GI Eukaryotic translation initiation factor 4 gamma 1 eIF4GII Eukaryotic translation initiation factor 4 gamma 3 eIF4G2 Eukaryotic translation initiation factor 4 gamma 2, also known as DAP5, NAT1 or p97 eIF4H Eukaryotic translation initiation factor 4H eIF5 Eukaryotic initiation factor 5 XI eIF5B Eukaryotic initiation factor 5B ELAVL1 Embryonic lethal abnormal vision like ribonucleic acid binding protein 1 EMCV Encephalomyocarditis virus EMCV* Nonfunctional encephalomyocarditis virus having the GNRA tetraloop mutated from GCGA into TCCA EMSA Electrophoretic mobility shift assay ER Endoplasmic reticulum eRIP Enhanced non-cross-linking ribonucleic acid immunoprecipitation and microarray analysis technique ERK Extracellular signal-regulated kinases ERK1 Extracellular signal-regulated kinase 1 ERK2 Mitogen-activated protein kinase 1, also known as MAPK1 ESC Embryonic stem cell ETS1 ETS proto-oncogene 1 FACS Fluorescence-activated cell sorting Fak Protein tyrosine kinase 2, also known as PTK2 Fam96b Cytosolic iron-sulfur assembly component 2B FGF1 Fibroblast growth factor 1 FGF-2 Fibroblast growth factor 2 FLuc Firefly luciferase FM3A Murine mammary carcinoma cells FMDV Foot and mouth disease virus Fmn1 Formin 1 FMR1 Fragile X mental retardation 1 FOXO Forkhead box, sub-group O Foxo3 Forkhead box O3 FTO Fat mass and obesity-associated, alpha-ketoglutarate dependent dioxygenase Fus Fused in sarcoma FXR1 FMR1 autosomal homolog 1 G1 Gap 2 G2 Gap 2 G418 Geneticin GABPA GA binding protein transcription factor subunit alpha GARS Glycyl-tRNA synthetase XII

GATA1 GATA binding protein GCN2 Eukaryotic translation initiation factor 2α kinase 4 GCN4 General control nonderepressible 4 gDNA Genomic deoxyribonucleic acid GDP Guanosine diphosphate GMP-PNP Guanosine 5’-[β,γ-imido]triphosphate GO GSK-3β Glycogen synthase kinase 3 beta GTP Guanosine triphosphate Gtx NK6 homeobox 2 h23 Hairpin 23 of 18S ribosomal ribonucleic acid h26 Hairpin 26 of 18S ribosomal ribonucleic acid Hbb β-globin HCV Hepatitis C virus HEK293 Human embryonic kidney 293 cells HeLa Henrietta Lacks, epithelial cervix cell line named after a patient HeLa S3 Henrietta Lacks S3, clonal derivative of the parent HeLa cell line hESC Human embryonic stem cell HIF-1 Hypoxia inducible factor 1 HIF-1α Hypoxia inducible factor 1 subunit alpha HIV-1 Human immunodeficiency virus type 1 HMGN3 High mobility group nucleosomal binding domain 3 hnRNP Heterogenous nuclear ribonucleioprotein hnRNP A1 Heterogenous nuclear ribonucleoprotein A1 hnRNP C Heterogenous nuclear ribonucleoprotein C (C1/C2) Hox Homeobox HOXA Homeobox A cluster HOXB8 Homeobox B8 HPRT Hypoxanthine guanine phosphoribosyl transferase HRI Eukaryotic translation initiation factor 2α kinase 1 HSC Hematopoietic stem cell Hsp70 Heat-shock protein 70 HTD2 3-hydroxyacyl-thioester dehydratase 2 HTT Huntington HuR ELAV like ribonucleic acid binding protein 1 XIII

HuD ELAV like ribonucleic acid binding protein 4 ICE Inducible cassette exchange IcircRNAs Intronic circular ribonucleic acid Id1 Inhibitor of DNA binding 1, HLH protein IGF2 Insulin like growth factor 2 IGFI-IR Insulin like growth factor 1 receptor IL-2 Interleukin 2 iN Induced neuron iPSC Induced pluripotent stem cell IRE Iron response element IRES Internal ribosome entry site IRF3 Interferon regulatory factor 3 IRP Iron regulatory protein IRP1 Iron regulatory protein 1 IRP2 Iron regulatory protein 2 ITAF Internal ribosome entry site trans-acting factor K-562 Chronic myelogenous leukemia cell line KD Knockdown KHSRP KH-type splicing regulatory protein Klf4 Kruppel like factor 4 KO Knockout KV1.4 Potassium voltage-gated channel subfamily A member 4 LARP1 La ribonucleoprotein domain family member 1 LC-MS/MS Liquid chromatography tandem mass spectrometry LFQ Label free quantification LIF Leukemia inhibitory factor LIN28 Lin-28 homolog Lin28a Lin-28 homolog A Lin28b Lin-28 homolog B lincRNA Long intergenic non-coding ribonucleic acid L-Myc MYCL proto-oncogene, bHLH transcription factor lncRNA Long non-coding ribonucleic acid loxP Locus of X-over P1 M Mitosis m6A N6-methyladenosine XIV m1A N1-methyladenosine

7 Me m G 7 G5’ppp5’N, 5’-7-methylguanosine cap Map3k3 Mitogen-activated protein kinase kinase kinase 3 MAPK Mitogen-activated protein kinase Mbl Muscleblind Mbnl1 Muscleblind like splicing factor 1 Mbnl3 Muscleblind like splicing factor 3 MDM2 MDM2 proto-oncogene MEF Mouse embryonic fibroblast MEK Mitogen-activated protein kinase kinase, also known as MAP2K MeRIP-Seq Methylated ribonucleic acid immunoprecipitation sequencing mESC Mouse embryonic stem cell METTL14 Methyltransferase like 14 METTL3 Methyltransferase like 3 Met-tRNAi Initiator methionyl-tRNA M-FAG Middle fragment of apoptotic cleavage of eIF4G MHC Major histocompatibility complex miRISC Micro ribonucleic acid-induced silencing complex miRNA Micro ribonucleic acid MNK1 MAPK interacting serine/threonine kinase 1 Mnt Max binding protein MRE Micro ribonucleic acid response element mRNA Messenger ribonucleic acid MS Mass spectrometry msl-2 Male-specific lethal 2 MTG8a RUNX1 translocation partner 1, also known as RUNX1 or AML1 mTOR Mechanistic target of rapamycin mTORC1 Mechanistic target of rapamycin complex 1 mTORC2 Mechanistic target of rapamycin complex 2 mut Mutant MYC MYC proto-oncogene, bHLH transcription factor Nanog Nanog homeobox NDRG1 N-myc downstream regulated 1 N-FAG N-terminal fragment of apoptotic cleavage of eIF4G NF-κβ Nuclear factor kappa B XV

NGF Nerve growth factor NMD Nonsense-mediated decay n-myc MYCN proto-oncogene, bHLH transcription factor NOT Negative on TATA NPC Neural progenitor cell NRF-1 Nuclear respiratory factor 1 nPTB Neurally enriched polypyrimidine tract binding protein NRF2 Nuclear factor, erythroid 2 like 2 NSC Neural stem cells nt Nucleotide Oct-4 POU domain, class 5, homeobox 1 transcription factor ODC Ornithine decarboxylase OIS Oncogene-induced senescence oligo(dt) Oligo deoxythymidine oORF Overlapping open reading frame ORF Open reading frame ori Origin of replication p Passage p16INK4a Cyclin dependent kinase inhibitor 2A p21 Cyclin-dependent kinase inhibitor 1A p27 / p27Kip1 Cyclin-dependent kinase inhibitor 1B p53 Tumor protein p53 p58PITSLRE Cyclin-dependent kinase 11A, also known as PITSLRE or CDC2L2 p97 Eukaryotic translation initiation factor 4 gamma 2, also known as eIF4G2, DAP5 or NAT1 p120 Catenin δ1 PABP Poly(A) binding protein PABPC1 Poly(A) binding protein cytoplasmic 1 PABPN1 Poly(A) binding protein nuclear 1 Pak2 p21 (RAC1) activated kinase 2 PAR-CLIP Photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation PBS Phosphate buffered saline PCBP2 Poly(rC) binding protein 2 PCNA Proliferating cell nuclear antigen XVI

PCR Polymerase chain reaction PDGF2 Platelet derived growth factor subunit B PeBoW PES1-BOP1-WDR12 complex PERK Eukaryotic translation initiation factor 2α kinase 3 PES1 Pescadillo homologue 1 pH Potential of hydrogen PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase PIC 43S Preinitiation complex PITSLRE Cyclin-dependent kinase 11A, also known as CDC2L2 or p58PITSLRE PKR Eukaryotic translation initiation factor 2α kinase 2 Pol II Polymerase II Pol III Polymerase III PP2A Protein phosphatase 2 phosphatase activator Pqbp1 Polyglutamine binding protein 1 pre-miRNA Precursor micro ribonucleic acid pri-miRNA Primary micro ribonucleic acid PRRC2A Proline rich coiled-coil 2A PRRC2B Proline rich coiled-coil 2B PRTE Pyrimidine-rich translational element pSILAC Pulsed stable isotope labeling by amino acids in cell culture PTB/ PTBP1 Polypyrimidine tract binding protein 1 PTEN Phosphatase and tensin homolog QKI Quaking qRT-PCR Quantitative real-time polymerase chain reaction RA Retinoic acid Rad51 Rad51 recombinase RAN Ras-related nuclear protein Ran-GTP Ras-related nuclear protein guanosine triphosphate RAPTOR Regulatory associated protein of mechanistic target of rapamycin complex 1 RBM4 Ribonucleic acid binding motif protein 4 RBP Ribonucleic acid-binding protein RFP Ribosome footprinting RHA Ribonucleic acid helicase A rHRE RNA hypoxia response element XVII

Riok1 RIO kinase 1 RIP Ribonucleic acid immunoprecipitation RIP-Seq Ribonucleic acid immunoprecipitation sequencing RISC Ribonucleic acid-induced silencing complex RLuc Renilla luciferase RNA Pol II Ribonucleic acid polymerase II RNA Ribonucleic acid RNAi Ribonucleic acid interference RNase Ribonuclease RNA-seq RNA sequencing, also called whole transcriptome shotgun sequencing RNP Ribonucleoprotein ROS Reactive oxygen species RPAD RNase R treatment followed by polyadenylation and poly(A) RNA depletion circRNA isolation method RPF Ribosome protected fragment RPL24 Ribosomal protein L24 RPP14 /MRP subunit 14 RPL10A Ribosomal protein L10A Rpl38 Ribosomal protein L38 RPS6 Ribosomal protein S6 RPS19 Ribosomal protein S19 RPS25 Ribosomal protein S25 RRL Rabbit reticulocyte lysate RRM Ribonucleic acid recognition motif rRNA Ribosomal ribonucleic acid S Synthesis S2 Schneider 2 Drosophila melanogaster cell line S6 Ribosomal protein S6 S6K1 Ribosomal protein S6 kinase B1 S6K2 Ribosomal protein S6 kinase B2 SCA1 Spinocerebellar ataxia type-1 SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SF3B Splicing factor 3B SF3B1 Splicing factor 3B subunit 1 SFPQ/PSF Splicing factor proline and glutamine rich XVIII

SHPRH SNF2 histone linker PhD RING helicase shRNA Small hairpin ribonucleic acid siRNA Small interfering ribonucleic acid SLC8A1 Solute carrier family 8 member A1 SNAT2 Solute carrier family 38 member 2 SNM1 Desoxyribonucleic acid cross-link repair 1A SNP Single nucleotide polymorphism Sos1 SOS Ras/Rac guanine nucleotide exchange factor 1 SOX2 Sex determining region Y-box 2 sprcRNA short polycistronic ribosome-associated coding ribonucleic acid SR Serine and arginine rich SREBP-1a Sterol regulatory element binding transcription factor 1 SRRM2 Serine/arginine repetitive matrix 2 SRY Sex determining region Y SXL Sex lethal TC Ternary complex TEE translation-enhancing element Thr Threonine TIE Translation inhibitory element tiRNA Stress-induced transfer ribonucleic acid TIS Translation initiation site TISU Translation initiator of short 5’ untranslated region TNF-α Tumor necrosis factor TOP 5' terminal oligopyrimidine TRAIL Tumor necrosis factor α-related apoptosis-inducing ligand TRE Tetracycline response element Trim71 Tripartite motif containing 71 tRNA Transfer ribonucleic acid TSS Transcription start site TTP Tristetrapolin Tubb3 Tubulin beta 3 class III U5-15kD Thioredoxin like 4A U5-52K CD2 cytoplasmic tail binding protein 2 uAUG Upstream AUG start codon Ubx Ultrabithorax XIX

UBXD8 Ubiquitin regulatory X domain-containing protein 8 UNR Upstream of N-ras uORF Upstream open reading frame Upf1 UPF1 ribonucleic acid helicase ATPase UT7-mpl Megakaryoblastic cell line expressing the thrombopoietin receptor myeloproliferative leukemia proto-oncogene UTR Untranslated region UTRdb Database of 5’ and 3’ untranslated regions in eukaryotes UV Ultraviolet VEGF Vascular endothelial growth factor WBP11 WW domain binding protein 11 WDR12 WD repeat domain 12 wt Wild type WTAP Wilms tumor 1 associated protein X-DC X-linked dyskeratosis congenita XIAP X-lined inhibitor of apoptosis XLas Extra large as XPO5 Exportin 5 YB-1 Y-box binding protein 1 YTHDF1 YT521-B homology domain N6-methyladenosine ribonucleic acid binding protein 1 YTHDF3 YT521-B homology domain N6-methyladenosine ribonucleic acid binding protein 3 Yy2 YY2 transcription factor Zeb2 Zinc finger E-box binding homeobox 2 Zfp808 Zinc finger protein 808 ZNF9 CCHC-type zinc finger nucleic acid binding protein ZSCAN4 Zinc finger and SCAN domain containing 4

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1 INTRODUCTION

1.1 The canonical mechanism of translation initiation in eukaryotes: 5’end-dependent initiation The mechanism of canonical cap-dependent translation in eukaryotes can be divided into four major steps which are translation initiation, elongation, termination and ribosome recycling. Translation initiation is not only the first but also the most highly regulated step. It requires the activity of at least twelve initiation factors and can be subdivided itself into the four steps of 43S preinitiation complex formation, complex recruitment to the mRNA 5’end, scanning of the 5’UTR and assembly of the 80S ribosome (Fig. 1) [2], [3]. In order for the ribosome to attach to the mRNA, the 40S subunit first needs to form the 43S preinitiation complex (PIC). This requires the delivery of the initiator methionyl-tRNA (Met-tRNAi) to the ribosomal P site by eIF2 and GTP, which together make up the ternary complex (TC). Association of the TC with the 40S subunit involves eIF1, eIF1A, eIF5 and eIF3 and generates the PIC. When the PIC is assembled it can bind the mRNA near to the 5’-7-methylguanosine (m7G) cap. The recruitment is facilitated by eIF3, eIF4B, eIF4H, eIF4F and poly(A)-binding protein (PABP) with eIF4F stimulating the recruitment from the side of the mRNA. eIF4F is a complex that comprises the cap-binding protein eIF4E, the RNA helicase eIF4A and the scaffold protein eIF4G, which apart from being complexed with eIF4E and eIF4A can also bind PABP, eIF3 and the mRNA itself. The mRNA is directly bound by eIF4E, which encloses the cap between two tryptophan residues and which may contact additional cap-proximal nucleotides stabilizing the binding [4]. Its affinity for the cap increases by binding of eIF4G [5], [6]. eIF4G also holds eIF4A in its active conformation and generates a physical link between the mRNA’s 5’end and 3’end by interacting with PABP which results into a so-called closed-loop structure [7]–[10]. The third component of the eIF4F complex, eIF4A, is a RNA helicase with unwinding activity in both 5’ to 3’ and 3’ to 5’ direction which is stimulated not only by eIF4G but also by eIF4B [11]. eIF4A is thought to reduce secondary structures within the 5’UTR to generate a single-stranded landing site on which the PIC can attach [3]. After the PIC is recruited to the mRNA, it starts scanning into the 5’ to 3’ direction. Although the scanning model is consistent with scientific evidence, little is known about the mechanism and its intermediates have never been biochemically assayed [2], [12]. It is for example unclear at which nucleotide the PIC starts to scan and how the mRNA enters the mRNA binding channel of the ribosomal subunit [13]. Some reports suggest that scanning might involve limited relaxations, allowing for scanning in the reverse direction over a distance of a few nucleotides, but it also remains unclear how this might work [13], [14].

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Fig. 1: Model of the canonical translation initiation pathway in eukaryotes. The successive steps of ribosome assembly on a mRNA in the process of canonical translation initiation are depicted. Taken from Jackson et al., 2010 [13].

During scanning through the 5’UTR, the PIC is in an open conformation that is stabilized by eIF1 and eIF1A with the tRNA less tightly anchored in the complex, enabling forward

22 movement and codon selection [15], [16]. The scanning PIC arrests, as soon as an AUG codon in a favorable sequence context appears. Such a sequence is called after Marylin Kozak who identified in the late 1980s the GCC(A/G)CCAUGG motif that promotes highly efficiently translation initiation at the contained start codon [17]–[19]. Pairing between the codon and the anticodon in the ribosomal P site results in closed PIC confirmation leading to the displacement of eIF1 and locking the mRNA into the decoding center [16]. The pairing also leads to hydrolysis of eIF2∙GTP, which requires activation by eIF5 [2]. Subsequently, eIF2∙GDP, Pi and eIF3 are released and the large 60S subunit can join. eIF1A remains associated during joining and is only released afterwards, wherefor it is present throughout every single step of the initiation process [20]. Lastly, hydrolysis of eIF5B∙GTP, triggers the dissociation of eIF5B and eIF1A, which unblocks the ribosomal A site that can now be bound by the eEF1A ∙GTP∙aminoacyl-tRNA complex to begin translation elongation [21].

1.2 Noncanonical mechanisms of translation initiation: cap-independent and internal initiation Apart from the canonical translation initiation process, there exist alternative, less well-known mechanisms that result in protein synthesis by omitting ribosome recruitment to the cap- structure. Hence, these modes of ribosome recruitment are classified as cap-independent. They can be further subdivided into cap-independent but 5’end-dependent or internally mediated translation initiation depending on whether the ribosome enters the mRNA at the 5’end or at an internal sequence stretch.

1.2.1 Translation initiation by internal ribosome entry sites (IRESs) in viruses Eukaryotic translation usually begins by recognition of the m7G cap structure through initiation factors which subsequently stimulate ribosome recruitment. During infection, viruses hijack the eukaryotic translation machinery to enable viral protein synthesis in order to promote propagation and to counteract the immune response of the host cell. For that reason, viruses evolved strategies to cap their own mRNAs or in the case of single-stranded positive-sensed RNA viruses evolved RNA elements, so-called internal ribosome entry sites (IRESs), that allow for non-canonical translation initiation that does not require a mRNA 5’end (i.e. internal initiation) and a m7G cap [22], [23]. The first viral IRESs were discovered in 1988 in the 5’UTR of poliovirus and encephalomyocarditis virus (EMCV), two members of the picornavirus family [24]–[26]. Nowadays, IRESs have been identified in all members of the Picornaviridae family and several other viral orders [23]. Viral IRESs can be classified into four groups according to their structural features and their mode of action leading to translation initiation. Class I IRESs are organized in several 23 domains that are build up by basic structures like stem-loops or bulges which are evolutionary conserved [23], [27]. Ribosomes are recruited upstream of the start codon that is selected by scanning and some upstream AUG codons, which are termed cryptic AUGs, are bypassed [27], [28]. Usually, all initiation factors except of eIF4E and parts of eIF4G are required for the initiation process [27], [29], [30]. Example IRESs from this class are found in poliovirus, other enteroviruses like enterovirus A71 and coxsackievirus B3, as well as in rhinoviruses like human rhinovirus type 2 [23]. Class II IRESs, like class I IRESs, require all initiation factors except for eIF4E and parts of eIF4G for initiation and they are strongly conserved in primary and secondary structures although these structures differ from class I [27]. The structural differences likely account for a different mode of action in which ribosomes are directly tethered to the start codon of type II IRESs without any prior scanning [23], [28]. Prominent examples of type II IRESs are contained in EMCV from the Cardiovirus genus and foot and mouth disease virus (FMDV) from the Aphtovirus genus. Another group of viral IRESs, type III or hepatitis C virus (HCV)-like IRESs, exhibit more complex and compact secondary and tertiary structures like pseudoknots. They require less initiation factors as type I and II IRESs, namely eIF2, eIF3 and eIF5 [30]. Further, HCV-like IRESs directly interact with the small ribosomal subunit placing the start codon into the P site of the ribosome without prior scanning [23]. Interestingly, eIF3 and HCV-like IRESs interact with the 40S subunit at the same binding site, so that eIF3 is rearranged during HCV-like IRES- mediated translation resulting in eIF3 binding the IRES but not the ribosome as during canonical initiation [30], [31]. Class IV IRESs, which are exclusively found in the Dicistroviridae family, are localized in intergenic regions in between two open reading frames (ORFs), contrary to the remaining IRES classes that are localized in viral 5’UTRs [23]. This type of IRES is the most complex, usually containing several pseudoknots which are involved in ribosome recruitment without help by any initiation factors [23]. Certain pseudoknots of type IV IRESs mimic the shape of a tRNA so that they occupy the ribosomal A site during ribosome recruitment [28]. Translation initiation then requires two pseudo-translocation events in advance of the first peptide formation shifting the pseudoknot from the A to the P site before the first true tRNA is shifted from the A to the P site [23]. Both, eEF1A and eEF2 are required for the process and instead of using Met-tRNAi, translation usually starts with alanine-tRNA at authentic non-AUG start codons [23], [30]. Examples of type IV IRESs are found in cricket paralysis virus (CrPV), honey bee Israeli acute paralysis virus and shrimp Taura syndrome virus [28].

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Apart from interacting with eIFs, eEFs or ribosomal subunits to mediate translation initiation, viral IRESs often interact with other factors, called ITAFs, which are mostly RNA- binding proteins [30]. The first ITAFs identified were PTB involved in poliovirus IRES regulation and La involved in FMDV IRES regulation [32], [33]. While some IRESs are heavily dependent on ITAFs, like FMDV IRES requiring PTB, other IRESs show high activity without interacting ITAFs, like EMCV which is only modestly stimulated by PTB [27], [34], [35]. The main function of ITAFs is to stabilize the association of the IRES with the translational machinery; sometimes in cooperation with other canonical initiation factors or further ITAFs [30].

1.2.2 Translation initiation by internal ribosome entry sites (IRESs) in human After the discovery of viral IRESs, also cellular mRNAs were investigated for their potential to mediate non-canonical translation initiation. It started with the finding that BiP mRNA was resistant to cap-dependent translation inhibition during poliovirus infection and follow-up studies reported a few years later in 1991 that BiP 5’UTR mediates IRES-dependent translation initiation [36], [37]. A systematic analysis of polysome-associated mRNAs in poliovirus infected cells suggested that ~3 % of cellular mRNAs use internal translation initiation or other initiation mechanisms that are independent of eIF4F, which is reduced in infected cells due to proteolytic cleavage of eIF4G [38]. Polysome profiling under diverse conditions like mitosis, hypoxia and apoptosis were performed and under each condition ~3-5 % of mRNAs remained associated with heavy, translating polysomes [39]–[44]. But as translationally active mRNAs varied in between conditions, it was assumed that overall 10-15 % of the transcriptome has the potential to be efficiently translated under stress conditions [40]. Nowadays, at least 85 potential cellular IRESs and 39 viral IRESs have been studied in more detail and extensive information can be found at the IRESite database [27], [45]–[47]. In contrast to viral IRESs, cellular IRESs cannot be classified in groups according to their structure or function because they are more diverse in their properties, lack sequence or structural conservation, contain non-contiguous sequence elements and essentially their mechanistic mode of action is rather unexplored [48], [49]. In depth secondary structure analyses of putative IRESs of c-Myc, L-Myc, FGF-2, FGF1, Kv1.4, Bag-1, Igf2, cat-1, Mnt and MTG8a transcripts are available and a number of these putative IRESs do not require a preserved overall structure for full activity, which implies a different biological setup as observed in viral IRESs [45]. Further, basic 5’UTR features as number of upstream AUGs (uAUGs), GC content or length are not sufficient to distinguish IRES containing 5’UTRs from conventional 5’UTRs [45]. Various attempts have been made to computationally predict cellular IRESs but the diversity of reported 25

IRES contexts together with general challenges in modelling dynamic RNA folding during the process of translation from in vitro obtained structural data severely impairs the power of such predictions [45]. An example of one frequently quoted structural motif of cellular IRESs is a Y- shaped stem-loop followed by a second small stem-loop directly upstream of the start codon that was described by Le et al. in 1996 after analyzing unusual folding regions in BiP, Antp and FGF- 2 by use of several RNA folding prediction programs [50]. Apart from the limitation that identification of the Y-shaped RNA motif is based on the analysis of only three putative cellular IRESs, experimental evidence for this motif actively operating in internal initiation is missing [48]. Still it is annotated as a functional motif by UTRdb, where it is used to scan UTR sequences for potential IRES activity and where it is found in ~20 % of all UTR entries [45], [51]. Roughly a decade after the first cellular IRES was postulated, the first concerns about IRES-mediated translation arose due to complications concerning the gold standard technique to detect IRES activity, the bicistronic reporter assay, and concomitant difficulties to interpret the data [52]. The major challenge of the assay is to rule out cryptic promoter or splice site activity of the potential IRES sequence by applying thorough experimental validation. If careful validation is missing a sequence of interest can easily be mis-identified as an IRES, but detailed information about common mistakes in the experimental design of bicistronic reporter assays as well as the interpretation of results is discussed in a separate chapter (see section 4.3). By pointing out general deficiencies in cellular IRES investigations, Marylin Kozak opened the heated debate on the concept of cellular IRESs and in 2002 Han & Zhang provided the first experimental evidence for a poorly identified cellular IRES by developing a new promoter-less bicistronic vector that revealed substantial promoter activity within the 5’UTR of eIF4G, which was previously unrecognized and therefore falsely considered to mediate internal translation initiation [53]. They could even show that a polypyrimidine tract, which was already known to be essential for the putative IRES activity, overlapped with a binding site of C/EBPβ and that two more transcription factors interacted and regulated the promoter within the eIF4G 5’UTR [53]–[55]. Also, the first reported cellular BiP IRES was called into question when succeeding studies found a three to 4-fold stimulation over an empty control vector, in contrast to the original study which found a ~15-fold stimulation over a 400 nt inverted sequence of Antp that was used as negative control [52], [56]. Eventually the BiP IRES was tested in a control vector depleted of potential 5’ss in the first cistron and in this context the putative BiP IRES didn’t score as an IRES as it mediated translation activity with comparable power as an empty control vector [57]. Further, BiP 5’UTR showed some weak promoter activity when tested in a promoter-less construct [58].

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Similarly, a number of other popular IRES candidates could be disproven or are still under discussion due to inconsistent results which await further experimental verification [27], [59]–[61]. One example of a rather well investigated cellular IRES is c-Src but also here additional experimental verification is required to prove its ability to mediate internal translation initiation. Despite the fact that c-Src activity was tested exclusively by use of RNA reporters, which are unsusceptible to artefacts generated by cryptic promoters or splice sites that are active in DNA transfection assays, the investigators did not exploit the full potential of reporter assays to comprehensively dissect the putative IRES activity [62], [63]. For instance, c-Src cap-dependency was determined by use of m7G- and A-capped monocistronic RNA reporters in RRL which revealed that c-Src mediates cap-independent translation more efficiently than cap-dependent translation [62]. However, the assay was not repeated in vivo, wherefore it is unclear if cap- independent translation by c-Src is indeed more pronounced as cap-dependent translation because RRL is known for not adequately reproducing cellular conditions, e.g. eIF2 is rapidly inactivated so that translation is observed under conditions of limited eIF2, which is especially relevant in case of c-Src (see below) [64]–[67]. Further, contribution of the 5’end to overall translation as well as translation of the first cistron in bicistronic reporters was not determined so that it is impossible to distinguish between a bona fide internal initiation or 5’end-dependent but cap-independent translation mechanism in which sequence elements of c-Src 5’UTR could act as cap-independent translation enhancer (CITE). Further in vitro translation experiments could show that c-Src binds the 40S subunit and that it assembles into 80S complexes in the presence of GMP-PNP [62]. Apart from the fact that 40S binding is not equivalent to positioning the 40S subunit on the mRNA in a translation competent conformation, these results suggest that the putative c-Src IRES can mediate translation initiation in an eIF2-independent manner, which is usually required for tRNA delivery and which is released prior to 60S subunit joining by GTP hydrolysis [68]. Another study found that c-Src translation is indeed stable under stress conditions accompanied by eIF2α phosphorylation and limited ternary complex (eIF2-GTP-tRNAi) concentration [63]. In addition, they found that c-Src stem-loop I directly interacts with eIF2A, which assembles into a complex with the 40S subunit and tRNA in filter binding assays and which is required for c-Src translation under stress but not under normal conditions [63]. Hence, it is suggested that eIF2A undertakes tRNA delivery to c-Src when eIF2α is inhibited. However, another study which investigated eIF2-independent translation by ribosome profiling during eIF2α phosphorylation following 0.5 h of arsenite treatment observed reduced translation efficiency of c-Src [69]. As the original study determined c-Src translation during eIF2α phosphorylation following 2 h of tunicamycin treatment it is possible that c-Src translation is stimulated by other tunicamycin-specific stress 27 conditions or that eIF2A levels vary in between the cell lines used in both studies (Huh-7 vs. HEK293T). Further, an independent study reports that c-Src translation is inhibited by impaired rRNA methylation, which seems to differentially affect cellular and viral IRESs, so that differential rRNA methylation under arsenite or tunicamycin initiated cell stress could also have an impact on c-Src translation [70]. Taken together, this example of a putative cellular IRES, investigated by circumventing the common pitfalls of conventional reporter assays still lacks some additional experimental validation to create a clear picture of its mode of action. Provoked by unsatisfying experimental evidences supporting the cellular IRES concept, several objections were raised that question the strength and existence of cellular IRESs in general. For example putative cellular IRESs do not stimulate translation as efficiently as viral IRESs when compared with each other in bicistronic reporter assays [67], [71], [72]. While EMCV IRES stimulates the ratio of cap-independent over cap-dependent reporter expression around 100 – 250-fold in bicistronic mRNAs, putative cellular IRESs rarely promote a stimulation over 10-fold [27]. Also, when the translation efficiency is compared between bicistronic and monocistronic mRNAs, putative cellular IRESs promote translation from the downstream ORF of a bicistronic reporter usually with <2 % of the efficiency of translation from the ORF in a monocistronic reporter [27]. In contrast, EMCV IRES is 50 – 125 % as active in the bicistronic setting as in the monocistronic setting [27], [67], [73]. In addition, viral IRES activity is entirely lost after introduction of point mutations while cellular IRES activity is partially diminished or sequentially lost after deletion of larger sequence stretches [64]. Picornavirus family members, for example, express either class I or II viral IRES elements and even though structural differences exist between the two classes, both share a GNRA (N = any nt, R = purine) tetraloop and another C-rich loop [74]. In EMCV IRES the GNRA tetraloop consists of a GCGA sequence that was mutated into TCCA in this study which greatly reduced EMCV IRES activity about 91 %. This indicates that viral IRESs in their spatial organization act as a unit to promote internal initiation. Contrary, the reduced level of IRES activity displayed by remaining, partially deleted cellular IRESs suggests a modular structure of cellular IRESs in which each domain has the potential to contribute to internal initiation. Cellular IRES sceptics interpret the low translation efficiency as a lack of conserved structural features and the different sequential arrangement of cellular IRES as indication for the non-existence of cellular IRESs. The missing structural homology between cellular and viral IRESs as well as little structural homology among cellular IRESs themselves might reflect the great diversity of cellular regulatory contexts and the differential evolutionary constraints on an element that is contained within a capped transcript that can in principle be translated by the canonical translation initiation mechanism [45], [75], [76]. In line with this, putative cellular IRESs have been reported in many 28 transcripts encoding key regulators of essential cellular processes like apoptosis or cell growth so that an alternative, individually controlled or less efficient translation mechanism of these transcripts may match cellular needs to preclude detrimental effects of protein overexpression under physiological conditions [77]. Moreover, the lack of homology might be explained by a great number of false positive IRES candidates that was routinely included in homology studies and hamper identification of motifs in bona fide cellular IRESs. Further, cellular IRES-mediated translation is regarded as a backup mechanism that comes into play during cellular stress conditions in which global translation is inhibited to allow for specific translation of crucial regulators that establish the cellular readjustment to changed conditions. Nowadays, methodical improvement enabled the investigation of transcriptome-wide translation efficiencies by ribosome profiling or polysome profiling coupled RNA-seq and it appears that global translations is less extensively inhibited during stress conditions as previously anticipated. Often global translation is reduced by 20-70 % under stress conditions, meaning that cap-dependent translation is often active with around a third of its usual efficiency [78]–[80]. This raises the question if alternative mechanisms are really required in such a scenario and if an inefficient IRES-mediated mechanism is powerful enough to yield effective protein levels in this (competitive) context. Again, a less effective alternative mode of translation might be beneficial for transcripts encoding proteins that fulfill critical cell functions not least because deregulated expression of putative cellular IRES-controlled proteins contributes to disease development or cancer [77]. Regarding IRES-mediated translation under specific cellular conditions one should also consider experimental conditions under which IRES activities are studied. Neither cell free translation systems nor cell culture experiments of cells growing in log-scale can comprehensively reproduce the translational environment of most human tissues [71]. Thus, an alternative cap- independent translation mechanism might have the chance to work efficient under less competitive in vivo conditions present in the less translationally active tissues and cell-types of the human body. Another point under discussion is that bi- or multicistronic transcripts are rather rare in eukaryotes which infers that intergenic IRESs, like CrPV IRES, which mediate direct internal initiation are usually not required [81]. However, bi- or multicistronic transcripts in form of upstream ORF (uORF) containing mRNAs are actually very common among human genes [82]– [86]. They are translated by the conventional cap-dependent mechanism which means that ribosomes enter at the 5’end, translate an uORF and reinitiate translation at the canonical start codon of the main ORF. Ribosomes can also scan through one or more uORFs before they initiate translation at the main ORF, which is termed leaky scanning and is as well preceded by 29 recruitment of ribosomes at the 5’end. Canonical initiation and subsequent ribosome scanning can be regulated in cis and trans to finetune protein output [87]. Accordingly, uORFs often do not encode a functional peptide, but instead evolved as regulatory elements controlling main ORF expression especially in response to stress conditions [86], [88]. Further, transcripts can contain two overlapping ORFs in which translation of one ORF starts internally of the other ORF and ORFs can also be found downstream of coding sequences (CDSs) within the 3’UTR [89]–[94]. Several bioinformatic analyses predicted two-ORF- containing transcripts in ranging in numbers from a few hundred to several thousand [92]–[94]. Some of them are conserved in mice and some were found to be translated by ribosome profiling and MS analysis [89], [90], [95]–[97]. While the majority of them produces extended or truncated versions of one protein, only few dual coding transcripts with two separate proteins produced by one transcript have been identified and studied in detail [87]. One example of a dual coding transcript is RPP14 mRNA which encodes the 124 aa RPP14 subunit of RNase P as well as the 168 aa HTD2 that is located 121 nt downstream of the RPP14 ORF [98]. First attempts to clarify the mechanism of RPP14 and HTD2 translation by in vitro translation assays were unsuccessful [98]. Although the translational mechanism remains unknown an independent study found that both RPP14 and HTD2 translation are decreased upon oxygen and glucose deprivation, whereas uORF translation under the same condition was upregulated leading to translational downregulation of the downstream coding ORF [99]. This indicates that translation of this dual coding transcript is differentially regulated than inhibitory uORF translation i.e. not by means of leaky scanning and reinitiation. This is in line with the common observation that reinitiation occurs exclusively following the translation of small uORFs and not following a whole CDS [100]–[102]. The reason for the failure of ribosomal reinitiation is unknown, but it is hypothesized that initiation factors gradually dissociate from the elongating ribosome so that following short uORF translation factors are still present enabling a second round of translation while they are absent following elongation of a longer ORF [100]. Another example is the XLas/Gas gene which gives rise to a dual coding mRNA that is used as a template for the 78 kDa G-protein α-subunit XLas and the 356 aa proline-rich protein ALEX [103]. ALEX ORF overlaps with the XLas ORF, starting downstream of XLas in another reading frame and stopping exactly at the same position [103]. Both proteins are translated from the rat XLas cDNA in in vitro translation and RNA transfection assays, but the exact initiation mechanism is unknown. As rat XLas sequence differs from human XLas sequence, bona fide dual protein expression from the human transcript remains to be investigated. In human, the start codon of XLas is surrounded by a strong Kozak consensus sequence (GttaugG, Ensembl transcript ID ENST00000371100.8) so that ribosomes are not expected to scan through this 30 codon [92], [104], [105]. According to Klemke et al. the human ALEX protein starts with a MPRREEKYP aa sequence and ALEX ORF is preceded by three uAUGs within the XLas ORF [103]. However, according to Ensemble genome browser release 92 (April 2018) an ORF starting with this aa sequence is located 1096 nt downstream of XLas AUG, containing 14 uAUGs in total. Hence, initiation at this ORF by leaky scanning is highly unlikely so that an internal initiation mechanism would be expected. Ensembl and UniProt contain also records of the ALEX transcript and protein which slightly differ from the Klemke et al. annotation. According to UniProt, ALEX starts with MMARPVDPQ aa sequence (UniProt primary accession number P84996, release 2018_05) and according to Ensembl ALEX starts with one less (MARPVDPQ, Ensembl transcript ID ENST00000306120.3) [105], [106]. This means that ALEX ORF would be located 188/191 nt downstream of XLas AUG, containing two or three (depending on Ensemble or UniProt start with one or two ) uAUGs, of which one is in a strong Kozak context (GNNaugG, N = any nucleotide, Ensembl transcript ID ENST00000371100.8) and two are in an adequate context (ANNaugT, ANNaugA, N = any nucleotide, Ensembl transcript ID ENST00000371100.8). Hence, also in this sequence context translation of ALEX ORF by leaky scanning is rather unlikely [107]. Further investigations to elucidate ALEX translation might have the potential to uncover an internal initiation mechanism. But the genetic locus is complex, giving rise to 58 transcripts, including antisense transcription and imprinting, so that it is advisable to check if ALEX expression might also be controlled by transcriptional regulation [105], [108]. Further, ribosome profiling identified so-called short, polycistronic ribosome-associated coding RNAs (sprcRNAs) by analyzing ribosome footprints on lincRNAs [91]. Some of these lincRNAs were re-named sprcRNAs because they contain multiple small CDSs that can be translated as much as protein coding genes [91]. Future analysis will reveal if small CDSs within sprcRNAs yield functional peptides and if translation initiation at internal cistrons might be regulated by an internal initiation mechanism. In sum, bi- or multicistronic transcripts are not as rare in eukaryotes as envisioned previously. In general, plasticity of the ribosomal scanning mechanism enables translational regulation of a large part of studied natural bicistronic translation events; however, cases are described where an alternative mechanism might be required. In these cases transcripts should be carefully analyzed for alternative promoters or splicing that could generate a second monocistronic transcript, an alternative transcript where both cistrons are fused to a single translation unit or a transcript containing an alternative 5’ UTR [100]. If translation of two CDSs from one transcript is not controlled on transcriptional level, a regulation on translational level is conceivable especially for CDSs which are preceded by a long

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CDS starting with a strong consensus sequence that precludes ribosome reinitiation [17], [101], [102], [109], [110]. But the discussion about the existence of cellular IRESs does not only split the field into advocates and opponents, it also led to the development of alternative concepts explaining such scenarios that hardly fit stiff cap-dependent and IRES-dependent classifications. The IRES concept was and still is often misused or misunderstood as synonymous to a cap-independent translation initiation instead of being perceived as a separate mechanism. Clearly, internal initiation is independent of the cap, however, cap-independent translation does not necessarily initiate internally. Consequently, both mechanisms differ in the bona fide recruitment of ribosomes at an internal sequence of the mRNA or at the 5’end. In case of IRES-dependent initiation this might involve direct binding of the ribosome to the start codon but can also include ribosomal scanning prior to start codon recognition. To reduce confusion and to shift the focus more on specific requirements and the course of events during the initiation process William C. Merrick and colleagues proposed to reclassify the initiation process into four separate aspects, which should be defined for individual transcripts or ORFs [71]. These comprise (1) the initiation effector (active elements including i.e. the cap, RNA motifs, interacting proteins, sequence context of the start codon, structural elements), (2) the role of the effector (mode of action of effectors e.g. direct 40S subunit binding, eIF recruitment, structural rearrangements), (3) binding of the initiator tRNA (which eIFs or alternative proteins are involved in shuttling the initiator tRNA to the P site or possibly the A site of the ribosome) and (4) the location of the start codon (involvement of ribosomal scanning, sequence context like downstream of uORF, overlapping a separate ORF, etc.) [71]. Together these distinctions would allow a better understanding of the process avoiding a stringent stereotypic classification in two different mechanisms of which one accurately describes the vast majority of translation events but the other fails to define translation of many essential regulators in signaling pathways involved in cell survival under various conditions. Likewise, Ivan N. Shatsky and his colleagues Dmitriev, Andreev and Terenin proposed an alternative classification according to which mRNAs are continued to be distinguished by their mode of 40S subunit recruitment, but in contrast to the previous model the new classification distinguishes between four instead of two distinct modes of action [111]. The first class of mRNAs is defined as highly eIF4E- and eIF4G-dependent [111]. This class includes many mRNAs encoding the translational apparatus itself, as well as regulators of cell growth and proliferation [111]. These mRNAs are predominating in polysomes of proliferating cells although their absolute number is relatively small [111]. They are strongly regulated by mTORC1 and eIF4G alone is not sufficient to promote efficient ribosome recruitment. 32

The second class comprises eIF4G-controlled mRNAs [111]. They contain a high affinity binding site for eIF4G within their 5’UTR and do not have or have less requirement for eIF4E during recruitment of 40S subunits. It is also possible that eIF4G binding provokes conformational changes which supports stable eIF4E binding to the mRNA. Provided the latter holds true, the affinity of the mRNA for eIF4E following eIF4G binding would be stronger than that of competitors like 4E-BP. Class three contains mRNAs that are controlled by variants of eIF4E, eIF4G and their combination [111]. Humans express four eIF4E (eIF4E, eIF4E1B, eIF4E2, eIF4E3; GeneCards human gene database version 4.7.1) and three eIF4G family members (eIF4GI, eIF4GII, p97/NAT1/DAP-5/eIF4G2) and relatively little is known about their role and functionality during translation initiation, but few examples of homologs involved in specific translation events have been documented [111]–[113]. Lastly, the fourth class consists of mRNAs that do not use eIF4F for ribosome recruitment [111]. These include mRNAs that might use cellular IRESs for internal initiation but also other mRNAs that might use truncated forms of eIF4G or other functional eIF4G analogs. The mode of 40S recruitment might not necessarily be cap-independent as eIF4F replacing factors could interact with the cap through RNA-binding proteins (RBPs) or other auxiliary factors. Such concepts that divide transcripts into classes according to their dependencies on initiation factors agree well with prevailing findings that during stress conditions translation is neither shut down completely nor that all transcripts are translationally repressed with the same intensity. In addition, the alternative classifications leave more room for targeted regulation of the initiation step which is supported by studies that identified certain groups of transcripts that are dependent on specific factors and variations thereof like e.g. ribosomal subunits or rRNA modifications. For example, Diamon-Blackfan anaemia, a erythroblastopenia characterized by decreased erythroid precursors, is associated with mutations in the RPS19 gene leading to RPS19 deficiency or single aa replacements [114]. RPS19 mutations reduce translation of a subgroup of transcripts including GATA1, BAG1 and CSDE1, which are involved in erythroid differentiation and proliferation, and BCAT1, which is involved in leucine synthesis [115]–[117]. Thus, mutations of an ubiquitously expressed ribosomal protein cause a disease by selectively affecting translation efficiencies of specific sets of transcripts within hematopoietic cells [115]–[117]. A similar effect was observed in X-linked dyskeratosis congenita, a disease characterized by bone marrow failure and increased risk of cancer development [118]. Patients carry mutations in the DKC1 gene encoding dyskerin, a pseudouridine synthetase, leading to decreased rRNA pseudouridylation and decreased translation of tumor and apoptosis associated transcripts like 33 p27, p53, Bcl-xL and XIAP as well as CrPV IRES [118]–[121]. Selective translational defects are likely caused by reduced ribosome affinity for tRNAs and CrPV, which both interact with the ribosomal P site [122]. Another common modification of rRNA is methylation that was also shown to differentially affect translation efficiency of a set of transcripts including p27, p53, SNAT2, CAT- 1 and c-Myc while global translation levels or initiation on viral EMCV, HCV and CrPV IRES elements was unaltered [70], [123]. Reduced rRNA methylation enabled assembly of 48S preinitiation complexes, but abrogated formation of 80S complexes on the affected transcripts [70]. Hence, rRNA methylation and pseudouridylation were both suggested to specifically regulate cellular IRES-mediated initiation in the studies cited above, albeit it remains to be demonstrated that selectively regulated transcript indeed harbor IRES activity. In contrast, alternative concepts would classify selectively regulated transcripts according to their specific requirements of initiation factors, which might yield a translational advantage or disadvantage over canonical transcripts under stress conditions or in context of specialized cell types but not necessarily requires internal initiation to facilitate selective translation efficiency. Like rRNA modifications, also ribosomal proteins were suggested to be involved in cellular IRES translation. One prominent example is RPS25, which is located in the head domain of the 40S subunit. It was shown to directly bind a stem loop of the CrPV IRES and apart from being indispensable for CrPV IRES translation, RPS25 is also involved in HCV and human immunodeficiency virus type 1 (HIV-1) translation [124]–[126]. Following the identification of RPS25 dependency of certain viral IRESs, cellular IRESs were investigated for their requirement of RPS25. Knockdown experiments revealed that global translation levels are unaffected by RPS25 depletion, whereas certain transcripts are indeed less efficiently translated [124], [127]. A recent quantitative proteomic analysis found that RPS25 is sub stoichiometrically distributed in polysomes, indicating that RPS25 is absent from ~40% of translating ribosomes [128]. Further, RPS25 containing ribosomes were enriched on ~100 transcripts encoding genes that mainly function in cell cycle regulation [128]. In sum, the analyses show, that RPS25 is involved in selective translation of a small subgroup of functionally related transcripts. Further, RPS25 is not necessary for the translation of bulk mRNA within the cell. Whether the subgroup of RPS25 dependently translated transcripts is directly interacting with the ribosomal protein and if such interaction decreases the cap-dependency of these transcripts during the initiation process remains to be tested. Nevertheless, the results show that ribosomes as part of the cellular translation machinery are not acting as an invariable unit but are rather versatile in their composition and interaction with target mRNAs. In absolute terms human ribosomes could assemble in 3160 unique 34 configurations concerning only the protein content [129]. At least 212 modified rRNA bases, transiently interacting factors and yet unidentified post-translational modifications add another layer of complexity to the system that likely enables an endless number of configurations which should provide excessive capacity for selective mRNA translation [129]. Hence, heterogenous or specialized ribosomes harbor great potential to shape translation initiation mechanisms fitting the IRES as well as alternative initiation concepts. Using an alternative strategy, several research groups screened and designed synthetic IRES elements to study and elucidate the mechanism of internal translation initiation on the one hand and to develop a more efficient tool to be used in gene therapy on the other hand. The first screen for synthetic IRES elements was conducted in 2001 by Venkatesan & Dasgupta who tested 106 randomized 50 nt sequences within bicistronic reporter plasmids via protoplast fusion of HEK293 cells and FACS sorting for three selection rounds [130]. With this approach they identified two potential 50 nt IRES sequences that lack homology with viral or cellular IRESs and whose cap-independent translation activity was validated by in vitro translation assays [130]. But RNA transfection and quantitative measurements of bicistronic reporters compared to monocistronic reporters and viral IRES elements are required to verify that both elements indeed stimulate internal and not 5’-end dependent translation initiation as well as to determine their efficiency compared to cap-dependent translation and functional viral IRES elements. Nowadays, systematic genome-wide analyses have been conducted to identify human and viral IRESs and their functional elements in mediating translation initiation. One prominent example is an in vitro mRNA display assay in which 1013 random ~150 nt sequences from human total DNA were screened by purification of covalently linked newly translated protein-RNA complexes [131]. This assay identified ~12,300 translation-enhancing elements in the of which 12 % map to regions with annotated genes [131]. Following six rounds of mRNA display the library of 1013 random sequences was dominated by 636 unique sequences of which 225 exhibited full identity with the reference genome [131]. These 225 potential cap- independent translation enhancing sequences were further validated, but introduction of an upstream stem-loop abrogated monocistronic reporter translation of ~80 % of identified sequences, which indicates that these sequences require a free 5’end for ribosome recruitment and are rather unlikely to mediate internal translation [131]. Further, the validation of potential cap-independent translation elements revealed that 40 % are false positive elements containing cryptic promoters [131]. Cryptic splice sites weren’t investigated as candidate sequences were validated by use of the vaccinia virus system, which mediates RNA expression in the cytoplasm. However, the vaccinia virus system creates an artificial cellular environment which is unsuitable for IRES investigations and does not preclude nuclear transcription [34], [132]–[134]. Hence, this 35 data of cap-independent translation enhancing elements within the human genome contains a substantial amount of false positive sequences, whose translation activity lacks validation in a natural in vivo context. Another recent global analysis, which is often cited to have shown that ~10 % of human 5’UTRs harbor cap-independent translation elements, performed high-throughput bicistronic reporter assays to screen 55,000 oligos of 174 nt length containing viral, human and previously reported IRES sequences by lentiviral transfection, FACS sorting and DNA sequencing for IRES activity [135]. To exclude cryptic promoters from their dataset, Weingarten-Gabbay et al. performed high-throughput measurements including a bicistronic reporter vector deficient of the EF1α promoter, removing sequences from the analysis for which >20 % of reads were obtained from the cell population expressing the reporter in absence of the promoter i.e. expressing a cryptic promoter [135]. Further they excluded cryptic splice sites by comparing cDNA and gDNA levels of the sequence of interest and discarded all candidates with a ratio smaller than -

2.45 log2 cDNA/gDNA [135]. Even though they successfully exclude IRES candidates which have previously been reported to contain cryptic splice sites and although this approach diminishes the number of false positives within the dataset, it is not eliminating candidates containing weak cryptic promoters and splice sites. This is problematic, assuming that a capped monocistronic reporter is ~50 x more efficiently translated than its bicistronic complement, then the presence of only ~2 % of aberrantly generated transcripts would double reporter readout [64]. Hence, weak cryptic elements have substantial effects and the cutoff values defined in this study likely don’t discard such elements, wherefore interpretation of the data is ambiguous. However, the team searched the dataset for k-mers that are predictive of IRES activity and identified C/U-rich k-mers as determinants of strong IRES activity, especially when positioned ~50 nt upstream of the start codon [136]. Such polypyrimidine tracts could potentially be part of functional cryptic splice sites which weren’t excluded from the dataset due to the cut-off values discussed above. As the study employed a lentiviral vector with an EF-1α promoter containing an intron, it is possible that splicing deleted the sequence downstream of the EF-1α 5’ ss up to a cryptic 3’ ss located downstream of the polypyrimidine tract but upstream of the start codon generating a capped monocistronic transcript deficient of the first cistron and the potential IRES sequence [137]. They also identified a CAG k-mer, reminiscent of a strong acceptor splice site, immediately upstream of the start codon, although this is showing stronger effects in viral sequences than in human 5’UTRs [136], [138]. Contrary, a functionally relevant polypyrimidine tract was previously identified in the 3’ region of type I and type II picornavirus IRESs, where its accessibility and interaction with PTB is required for optimal activity of poliovirus and FMDV IRES for instance [139]–[141]. Thus, 36 instead of being splicing elements, polypyrimidine tracts might indeed be functional elements of cellular IRESs analogous to certain viral IRESs; however, functional assays including RNA transfections to validate the putative IRES activity of candidates identified in the screen are incomplete or missing for now to create clarity. In line with this, artificial IRESs consisting of CCU repeats resembling the PTB-binding motif were previously constructed and analyzed for their potency to mediate internal initiation [142]–[144]. Although these structures are capable to interact with PTB, a potential IRES activity could not convincingly be verified due to experimental shortcomings [143], [144]. Moreover, artificial (CCU)6-IRESs worked more efficient when organized in a stem-loop structure then in a linear sequence context (tested in bicistronic reporters following DNA transfection), which is in contrast to picornavirus IRESs that showed decreased translation efficiency when their polypyrimidine tract was sequestered into stable stem-loop structures [139], [143], [144]. Hence, an analogous mechanism to natural polypyrimidine elements within viral IRESs seems unlikely. Besides, artificial IRESs lack adequate control experiments (promoter-less assay depicted in non- optimal scale, northern blot analysis without marker, low resolution, too much RNA loaded and extensively trimmed) and RNA transfection assays, to convincingly proof their activity [143]. In general, PTB has been extensively studied and described as the first and even universal ITAF stimulating various viral and cellular IRESs (Tab. 1) [145]–[148].

Tab. 1: Examples of cellular IRESs, which have been reported to be regulated by PTB binding. Cellular Reference reporting PTB Comments on IRES activity IRES interaction Apaf-1 [142] (bicistronic reporter assays by Apaf-1 generated aberrant transcripts in DNA transfections only, promoter- [67]; Apaf-1 mediates 5’-end-dependent less control experiment with translation that is less dependent on the inappropriate scale potentially cap but involves ribosome scanning hiding cryptic promoter activity, [149] Northern blots show aberrant transcripts in generated by two constructs) BAG-1 [150], [151] BAG-1 didn’t score as IRES in RNA transfections [59] BiP [152] BiP didn’t score as IRES when tested by vector depleted of 5’ss [57]; BiP showed weak promoter activity in promoter-less vector [58] Cat-1 [153] inadequate RNA analysis by northern blot [154], low and inconsistent Cat-1 activity in DNA transfection of bicistronic reporters [154], in vitro translation activity of Cat-1 in 37

bicistronic reporter was 1% of monocistronic control reporter [59] CDK11p58 [155] (article was retracted five years later [156]) HIF-1α [157] HIF-1α contains cryptic promoter activity [73], in RNA transfections low IRES activity = 0.4% of cap-dependent translation [73] IGF-IR [158] (bicistronic reporter assays by DNA transfections only) Mnt [143] Mnt IRES was discovered by DNA transfections only without any controls for cryptic elements [159], c-myb [143] c-myb IRES activity was predominantly investigated by DNA transfections including one RRL in vitro translation assay in which c-myb has similar activity as an empty vector [143] c-myc [160] c-myc showed promoter activity in promoter-less vector [73] p27Kip1 [161] p27Kip1 showed promoter activity [162] p53 [163] (p53 IRES activity in DNA p53 IRES activity of bicistronic reporter transfection of bicistronic reporter 3 orders of magnitude lower than cap- looks like unspecific background = dependent translation (= 0.3 % of cap- ~4% of cap-dependent cistron, dependent cistron, DNA transfection absolute luciferase values ~1000) via vaccinia virus system) [164], p53 activity in RNA transfection of bicistronic reporters 8.5 times higher than empty vector but translation of cap-dependent cistron inhibited by stable ΔEMCV hairpin structure still 3.5 times higher than p53-mediated 2nd cistron translation, comparison with uninhibited cap-dependent reporter missing [164], dirty northern blot and unsuitable exposure [164], aberrant splicing not convincingly ruled out by RT-PCR [164], [165], bicistronic positive control vector containing EMCV IRES is not working [165] UNR [166] (efficiency of UNR IRES unclear and inconsistent between experiments, compare Fig.1B and Fig.6)

Often putative IRESs regulated by PTB are not comprehensively validated so that it is advisable to first revise the IRES activity before focusing on PTBs role in internal initiation (Tab. 1) [59]. As PTB was originally identified as a splicing factor that interacts with pre-mRNAs within the nucleus, its stimulating effect on putative cellular IRESs might as well result from its function in

38 splicing, potentially generating aberrant reporter transcripts that led to mis-identification of putative cellular IRESs [167], [168]. After the short digression about PTB, I would like to get back to high-throughput screens that aimed to identify IRESs in the human transcriptome or within synthetic sequences. One such screen used only 9 or 18 nt random oligos in bicistronic reporters that were integrated into B104 cells by retroviral transduction [169]. Reporter-expressing cells were FACS sorted, re- cultured and sequenced. The screen was based on the finding of a 9-nt sequence element within Gtx 5’UTR which is complementary to 18S rRNA and which was found to immensely increase translation in bicistronic reporter assays, when introduced in the intercistronic space in multiple copies [170]. The screen identified two candidates that were as well complementary to 18S rRNA and that also showed increased translational efficiencies in bicistronic reporter assays (DNA transfection) when introduced in copies of three or five [169]. Both studies, the screen and the original discovery of the 9-nt element of Gtx 5’UTR, lack appropriate validation by RNA transfections and use northern blots to verify bicistronic reporter expression after DNA transfection, omitting a blot of the most active reporter containing ten repeats of the 9-nt element [169], [170]. Further characterization of the potential 9-nt Gtx IRES revealed that translation rates in monocistronic transcripts (cell-free translation and DNA transfection) decreased with increasing complementarity and that the sequence can bind 18S rRNA as well as the 40S subunit [171], [172]. Binding both 18S rRNA and the 40S subunit is not a proof of functionality per se, as it is unclear where the interaction takes place and if it would allow correct insertion of the mRNA into the mRNA-binding cleft. In fact, the complementary rRNA sequence (1124-1132 nt) is part of a stable hairpin (ΔG = -15.8 kcal/mol), which likely renders it inaccessible for mRNA interaction [173], [174]. However, the 18S rRNA is predominantly organized in hairpin structures and UV crosslinking found that some nucleotides interact with mRNA although they are contained within hairpins [175]. Hairpin 26 (h26), which includes the complementary 9 nt, and hairpin 23 (h23) are located at the E-site of the 40S subunit, where the apical part of h23 is close to mRNA at position -4 and U1107 of h26 is close to mRNA at position -17 [175]. Hence, the potential 9-nt Gtx IRES may directly interact with the small ribosomal subunit in the periphery of the E-site following potential structural rearrangements to render the rRNA accessible. Interestingly, increased complementarity results in decreased efficiency of cap-dependent translation, while multiplication of the complementary element increases both putative IRES- mediated translation of bicistronic reporters and translation of monocistronic reporters [170], [172], [176]. The bicistronic reporter assays were carried out by DNA transfection and lacked controls to exclude cryptic promoters or splice sites. Further, the putative IRES was later 39 renamed a translational enhancer element [177], before it was called a ribosomal recruitment site facilitating ribosome shunting across uAUGs and hairpins [176], until it was described to be consistent with both initiation modes of a model that distinguishes between hypothetic ribosomal tethering (at the cap structure, initiation codon is reached by bypassing intervening mRNA) and ribosomal clustering (at internal sites, initiation codon is reached by dynamic binding and release) [178]. Also here, the underlying experiments included transfected DNA reporters that lacked necessary controls or in addition transformed yeast strains expressing mouse-yeast hybrid 18S rRNA that was incorporated into mono- and polysomes, suggesting translational activity [177]. However, strains expressing only the hybrid 18S rRNA were not viable, suggesting this activity was compromised [177]. Thus, the experiments did not convincingly prove the putative enhancing/internal recruitment/shunting activity of the 9-nt Gtx element.

Fig. 2: Scheme of the 18S rRNA secondary structure including crosslinking sites to mRNA during translation initiation. Contact between 18S rRNA and the mRNA are depicted in 40 close-ups and summarized in a diagram at the bottom which represents the mRNA nucleotide sequence with the AUG start codon corresponding to the positions +1, +2 and +3. Adapted from Pisarev et al., 2008 [175].

Taken together, numerous reports of putative IRESs in eukaryotes exist, but contrary to viral IRESs, the status of cellular IRESs remains controversial [27]. Although it is clear that distinct cellular stress conditions lead to selective translation of subsets of mRNAs, it is unclear to which extend internal translation initiation is contributing to this selective translational activity. One reason is that the majority of IRESs reported today requires ultimate confirmation by more careful validation experiments, as the standard assay applied for IRES identification is prone to create artefacts. Another reason is that in the meantime alternative mechanisms of translation initiation as well as mechanisms of persistent scanning-dependent translation have been discovered that lead to selective protein synthesis without the requirement of internal translation initiation. In the future, comprehensive experimental designs, open-minded research into all possible directions and innovative technology will help to answer as yet open questions on the nature of cellular IRESs and eventually resolve current controversies.

1.2.3 Cap-independent translation initiation at the 5’end: CITE-like mechanisms Alternative translation initiation mechanisms can be independent of the cap structure but still dependent on a free 5‘-end from which the ribosome enters the mRNA for scanning towards the start codon. This mechanism contrasts with IRES-mediated translation in which the ribosome is recruited to an internal site and it contrasts as well with the canonical initiation mechanism in which the 43S PIC is recruited by the eIF4F-bound cap. Although the cap is highly stimulatory and efficient in promoting translation, any mRNA can be translated to a certain extent in mammalian cells even if it lacks a cap [179]. In the 90s Gunnery and Mathews found that this is not only true for in vitro transcribed mRNAs, but also for uncapped reporters synthesized by polymerase III (pol III) [180]. They introduced the tat gene of HIV-1 under a pol III promoter, resulting in a mRNA that was neither capped nor polyadenylated and that was expressed in vitro in a pol III transcription system and translated in vivo in transfected HeLa cells [180]. Mutation analysis of short uORFs and stable stem loops revealed that the uncapped mRNA was scanned from the 5’end suggesting that cap-independent translation still depends on the 5’end [180], [181]. Also uncapped and leaderless mRNA was translated by mammalian ribosomes after the 80S complex and initiator Met-tRNA assembled on the mRNA in the absence of translation initiation factors in toeprinting assays [182]. The mRNA analyzed was cIlacZ from the Λ phage. It had only one nt (G) upstream of the AUG start codon and lacked all regulatory elements inherent to eukaryotic translation initiation. Comparing a capped to an uncapped version of it 41

(starting with ApppN instead of m7G to stabilize the mRNA by preventing degradation by 5’ exonucleases), revealed that cIlacZ had a low cap-dependency compared to conventional mRNAs containing a 5’UTR [183]. In principle the leaderless mRNA can be translated by four different mechanisms in which the initiator tRNA is delivered either by eIF2, eIF2D and its homologs, eIF5B or initiation might even proceed without any of these factors involved [183]. However, it hasn’t been investigated whether all four mechanisms are equally efficient in cap-dependent and cap-independent translation initiation on the leaderless mRNA. But cap-independent translation initiation has also been described for natural mammalian mRNAs. One mechanism resembles the translation initiation of plant positive-strand RNA viruses that employ CITEs to promote protein synthesis from uncapped RNAs. CITEs are usually located at the 3’ UTR of viral RNAs. They can bind a component of the eIF4F complex and form RNA-RNA kissing-loop interactions with a hairpin loop close to the RNA 5’ end [184]. CITEs work as well when they are placed in the 5’UTR [185]. After initiation factors are recruited, ribosomes enter at or near the free 5’ end from where they scan towards the start codon [184]. A similar mechanism has been suggested for mammalian mRNAs like for instance Apaf-1. Feasibility of this mechanism was experimentally demonstrated by inserting the J-K domain of the EMCV IRES, which binds eIF4G, into the 5’UTR of the cap-dependent mRNA of β-globin, rendering β-globin translation cap-independent [186]. Apaf-1 was found to contain a domain, which reduces its dependency on the cap so that an ApppN-capped Apaf-1 mRNA still displays ~15 % of the translational activity of a m7G-capped Apaf-1 mRNA in RNA transfection assays in HEK293T cells [67], [187]. However, further analysis revealed that Apaf-1 mRNA translation is 5’-end dependent and involves ribosome scanning, as insertion of an uAUG or uORF reduces its translational activity, which is only around background level in bicistronic reporters, where it is also not stimulated under stress conditions [67], [187]. Thus the 5’UTR of Apaf-1 is acting like CITEs in plant RNA viruses and careful analyses were required to demonstrate that CITES are functionally different from IRESs in terms of that a free 5’end is required for CITE-mediated cap-independent translation. Roughly two decades ago the Hentze lab performed similar assays by directly tethering IRP-1 fused to the central domain (AA 642-1091) of eIF4G to three repeating iron response elements (IREs) in the intercistronic space of a bicistronic reporter [188]. Like in the proof of principle experiments for CITEs by Terenin et al., recruitment of eIF4G stimulated the translation of both upstream and downstream cistrons, with downstream translation being 2.3 – 5.5 % of cap-dependent translation and negligible compared with viral IRES-mediated reporter translation (~14 % of HCV in DNA transfection assays) [188].

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Apart from eIF4G recruitment during CITE-like translation, other eIFs could play a role in this mechanism. For example, eIF3 could be another potentially recruited factor. eIF3 had been shown to play a role in N6-methyladenosine (m6A)-promoted cap-independent translation and can activate or repress mRNAs of cell proliferation regulators through different modes of binding stem-loops within the respective 5’UTRs [189], [190]. eIF3 encompasses 13 subunits and it can bind the 40S subunit, stimulates 43S formation and 43S attachment to the mRNA as well as subsequent scanning [13]. Two subunits, eIF3d and eIF3l, were reported to have cap-binding activity and eIF3d was even reported to promote an alternative translation initiation mechanism, when eIF4E recruitment to the mRNA is blocked [191]. Hence, eIF3 seems a promising candidate to contribute to CITE-like translation, which could coexist with canonical cap- dependent translation at corresponding mRNAs. Another factor which was frequently reported to play a role in cap-independent or IRES- dependent translation is eIF4G2, also known as NAT1, p97 or DAP5. It was simultaneously discovered by four different research groups in 1997 and is one out of three eIF4G homologues that is N-terminally truncated and therefore misses the binding sites for eIF4E and PABP [192]– [195]. First, it was regarded as a factor that promotes non-canonical translation initiation after caspase cleavage and during cellular stress conditions, but later it was described to activate translation also under physiological conditions [196]–[202]. It’s non-canonical translation regulation was also reported to play a role in the retinoic acid pathway and ESC differentiation [203], [204]. Although eIF4G2 was described to play a role in translation of several putative IRES-containing mRNAs (Apaf-1, Bcl-2, CDK1, c-IAP1/HIAP2, c-Myc, HMGN3, p53, XIAP and its own transcript), it is unclear whether it actually does so, as some of these IRES evaluating experiments would require additional experimental support, while other putative IRESs have been refuted later (Apaf-1, XIAP) [57], [149], [197], [199], [204]–[209]. So, it is unclear if and to what extend eIF4G2 is involved in non-canonical translation initiation. However, eIF4G2 plays role in translation of not only a few selected, but rather a larger group of mRNAs, as depletion of eIF4G2 leads to a reduction in global protein synthesis of about 20-30 % (siRNA silencing in human breast cancer cells) [210]. Interestingly, the depletion of eIF4G1 also leads to a reduction in protein synthesis of about 20-30 %, while simultaneous depletion of both eIF4G1 and eIF4G2 leads to a reduction of about 60 % [210]. Overexpression of eIF4G2 could not compensate eIF4G1 depletion, showing that the factors could not physiologically substitute for each other and that they regulate translation of different mRNA subgroups [210]. Further, it was shown in vitro that eIF4G2 can stimulate translation of distinct m7G- and A-capped reporters and also stimulates their translation when cap-dependent translation is repressed by the presence of 4E- BP1 [206]. Due to these properties and its homology to eIF4G1 it might well be that eIF4G2 43 directs cap-independent or CITE-like mediated translation of distinct mRNAs. This might likely include as well other auxiliary factors or alternative eIF isoforms, but further analyses are required validate this hypothesis. There have also been a few approaches to detect cap-independently translated sequences genome wide. Wellensiek and colleagues for example used an mRNA display strategy by which in vitro-transcribed and in vitro-translated uncapped mRNA reporter fragments of 150 nt length were photo-ligated to a DNA-puromycin linker so that the mRNA got covalently linked to a peptide affinity tag encoded in their own ORF during translation [131]. Fusion products were isolated and functional RNA reporters underwent another selection cycle. After six rounds, more than 12,000 cap-independent translation-enhancing elements (TEEs) were identified within the ~1013 fragments from total human DNA that were included in the assay [131]. Roughly 12 % of these TEEs mapped to genomic regions of known genes. Selected TEEs were validated in vivo by monocistronic reporters with 5-terminal hairpins which were expressed by the vaccinia virus expression system. Roughly 20 % of the sequences passed this test and roughly 60 % of sequences passed the promoterless reporter test [131]. Apart from the fact that a substantial proportion of false positives was identified, the experimental design of this validation assay was not targeted for cap-independent translation. The comparison between m7G- and A-capped reporters would have been straightforward. Insertion of hairpins close to the cap instead evaluates if a free 5’end is required for translation initiation. A follow-up study characterized some putative TEEs in greater detail and a conserved 13-mer motif was identified that is partially complementary to the 18S rRNA [211]. This 13-mer is part of a larger conserved region that spans ~80 nts and contains also a conserved AUG triplet [211]. When the 13-mer is separately tested for its translation efficiency in luciferase reporter assays (HeLa cells transfected with the vaccinia virus expression system) without the larger conserved sequence context, it has only 10 % of the activity of the full length sequence [211]. Further, one of the exemplarily examined TEEs (HGL6.985) didn’t display substantial cap-independent translation efficiency in a mRNA transfection assay (~25 % of capped mRNA reporter) and the 13-mer motif did not enhance translation of the authentic start codon but enhanced uAUG translation generating an elongated protein instead [211]. Thus, it’s unclear whether the TEE would produce a functional protein, when introduced in the 5’UTR of natural mRNAs. One other drawback of this study is that AUGs of the conserved AUG triplet within putative TEEs were generally considered single start codons and not regarded as an entity together with their respective ORF generating patterns of overlapping and non-overlapping uORFs, which themselves could constitute a regulatory unit. Overall, the question of whether the putative cap-independent TEEs work in their genomic context remains unanswered. 44

Another high-throughput approach to decipher cis-regulatory elements driving cap- independent translation was performed by Weingarten-Gabby and colleagues. Sequences with the length of ~170 nt from viruses, human genes, systematically mutated previously reported IRESs and native 5’UTRs were introduced into a synthetic oligo library comprising 55,000 oligonucleotides in total [135]. The library was cloned into a lentiviral bicistronic reporter plasmid and infected H1299 cells, each with a single oligo integration, were sorted into different reporter expression bins by FACS followed by deep sequencing. According to this high-throughput analysis, about 10 % of human 5’UTRs harbor cap-independent translation activity [135]. The assay demonstrates one of the major problems in identifying non-canonical translation elements: the activity levels of the 583 human sequences, which harbor translational activity, range from 1.5 to 8 on a log2 scale relative to an empty vector. Hence, there exist a huge variety of activity and the focus of such analyses should not be primarily on identifying this activity above a selected threshold but rather on showing if this is meaningful in its natural context or during a stress condition. Therefore, a comparison with the monocistronic mRNA would be necessary to evaluate the stimulation from the 5’cap, showing if cap-independent translational activity compared to cap-dependent translational activity could lead to relevant protein output. In a high-throughput assay, thresholds need to be defined for splicing and promoter activity. When it comes to reporter assays, this is an issue as already tiny amounts of aberrant transcripts can lead to significant read-out. Thus, the use of a cut-off value for promoter activity of more than 20 % of reads coming from the cell population that expressed the second-cistron although the expression vector lacked an intrinsic promoter and a cut-off value for splicing activity of ≤-2.5 (log2) of cDNA compared to gDNA reads likely leaves room for false positives [135]. Hence, additional control experiments were performed, but qRT-PCR assays are not the best suitable control, as they are not sensitive enough to rule out the presence of weaker splice sites. The investigators also performed control reporter assays for selected candidates using DNA transfection, which is prone to generate artefacts from cryptic promoter or splice sites, wherefore only mRNA transfection assays are suitable control. Despite the lack of such controls, the identification of novel and previously described short U-rich IRES elements was reported (UACUCCC, UUCCUUU) [135]. The activity of these elements varied between reporter transcripts and also between different sites within a single transcript, indicating that the element on its own might not be a fully functional IRES but might require surrounding structures [135]. This also leads to the question of how active the short motifs are in their natural context that should be quite different from the artificial intercistronic one used in reporter assays. All in all, the authors didn’t clearly distinguish between cap-

45 independent and IRES-dependent translation within their assays and the identified translation elements require further experiments to validate and clarify their mode of action. Taken together, eukaryotic mRNAs can in principle be translated in the absence of the cap structure, although cap-dependent translation is much stronger. Translation enhancing elements in the 5’UTR have been described that can boost cap-independent but 5’end-dependent translation. So-called CITES have been proposed to act in concert with canonical eIFs or specific variants of canonical eIFs like eIF4G, eIF4G2 or subunits of eIF3. The best studied mRNA with efficient cap-independent but 5’end-dependent translation is Apaf-1. Other mRNAs that are cap- independently translated but unable to mediate significant IRES-dependent translation (translation levels around background values), might be translated through a CITE-like translation initiation mechanism under distinct cellular conditions. High-throughput genome- wide assays are not well suited to study the details of non-canonical translation initiation mechanisms as stringent validation experiments are required to rule-out false positives which can easily arise with gold-standard bicistronic reporters. Hence, tedious experimental settings are required to investigate single candidates, which is likely a reason why only few examples of well- described cap-independently translated mRNAs exist and outstanding questions remain. Future analyses might uncover if other isoforms of canonical eIFs are involved in CITE-mediated translation and if CITEs can also promote cap-independent translation when located within the 3’UTR as it has previously been shown in plant viruses.

1.3 Global and gene-specific translational regulation A variety of cis-acting regulatory RNA elements as well as trans-acting regulatory factors is modulating mRNA translation both globally and gene-specifically. Expression levels or phosphorylation states of canonical eIFs and RBPs like eFI2α or 4E-BP1 can globally downregulate levels by acting in trans on the entire pool of mRNAs. However, individual transcripts can escape global translational inhibition or can be particularly severely affected due to the presence of certain cis-acting RNA elements (Fig. 3).

Fig. 3: Overview of regulatory RNA elements in the 5’UTR acting in cis and trans on mRNA translation. RG4: RNA G-quadruplex, RNP: ribonucleoprotein. Adapted from Leppek et al., 2018 [212]. 46

The extend of selective mRNA translational regulation is not yet completely understood, but latest discoveries like translational control through m6A RNA modifications or heterogenous ribosome compositions are shedding light on the diverse modes still to be uncovered. A selection of the most commonly known elements will be described in more detail.

1.3.1 Molecular properties of the translational repressor eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) One family of well-known translational repressors that has been extensively studied in the last two decades since their discovery are 4E-BPs. 4E-BPs were first identified in rats in 1994 and humans and mice are expressing three homologs termed 4E-BP1, 4E-BP2 and 4E-BP3 [213]– [216]. The three proteins share ~60 % of sequence identity and all of them exhibit the canonical eIF4E binding motif (4E-BM) consisting of a YX4LΦ sequence (Y = tyrosine, X = any amino acid, L = leucine, Φ = hydrophobic residue) [215]–[218] . Mammalian 4E-BPs are widely expressed in most tissues, but expression levels can vary substantially for different family members. So, 4E-BP1 is most abundant in adipose tissue, pancreas and skeletal muscle, while 4E-BP2 is predominantly expressed in brain and lymphocytes, whereas 4E-BP3 shows its highest expression levels in skeletal muscle, heart, kidney and pancreas [216], [219]–[221]. According to their differential expression patterns, it was hypothesized that albeit functionally similar, 4E-BPs might be regulated in a tissue-specific manner and that overlapping 4E-BP expression constitutes a safeguard mechanism that ensures cooperative eIF4E activity control. In line with this, homozygous KO mice of 4E-BP1 or 4E-BP2 are viable and don’t show evidence of tumor development, suggesting some redundancy in 4E-BP function [220], [222]. However, 4E-BP1 KO mice show defects in adipose tissue development and energy homeostasis, like reduced white adipose tissue mass, hypoglycemia and increased metabolic rate, while 4E-BP2 KO mice show deficits in hippocampal long-term potentiation leading to learning and memory defects as well as to autistic-like behavioral alterations [220], [222]–[225]. Double KO mice lacking both 4E-BP1 and 4E-BP2 display increased sensitivity to diet-induced obesity and insulin resistance, resulting from increased adipogenesis in conjunction with alterations in fat metabolism and reduced energy expenditure [226]. Taken together, the studies of KO mice reveal that despite being functionally redundant, 4E-BPs are involved in additional tissue-specific translational regulation that cannot be backed up by other members of the protein family. 4E-BPs exert their repressive function by competing with eIF4G for binding to the cap- binding protein eIF4E, thereby blocking eIF4F assembly and cap-dependent translation initiation [215], [227]. 4E-BPs exhibit the same canonical 4E-BM as eIF4G and both proteins interact with 47 the same hydrophobic binding site on eIF4E that is located on the opposite site of the cap- binding pocket [218], [227], [228]. Binding of 4E-BP to eIF4E does not inhibit its interaction with the mRNA 5’ cap, actually there is even evidence that affinity of 4E-BP to cap-bound eIF4E is increased compared to unbound eIF4E and that vice versa binding of 4E-BP to eIF4E increases eIF4E affinity for the cap [229]–[231]. However, 4E-BP binding is regulated by phosphorylation, which has been predominantly studied on 4E-BP1 and 4E-BP2. While in the hypophosphorylated state 4E-BP is tightly bound to eIF4E, sequential phosphorylation leads to dissociation and the relieve of translational repression. In detail, hypophosphorylated 4E-BP first binds eIF4E by a noncanonical binding motif on its lateral surface, followed by competition with eIF4G for binding to the canonical motif on the orthogonal surface [232], [233]. Canonical and non-canonical binding sites are connected by an elbow linker, allowing the bipartite binding [232], [234]. In its unbound state 4E- BP is highly unstructured but when bound to eIF4E, the canonical motif undergoes a transition from intrinsically disordered into a helix structure [5], [235]–[237]. mTOR mediated signaling leads to phosphorylation of the two N-terminal phosphorylation sites Thr-37 and Thr-46, while 4E-BP is still bound to eIF4E [238]. This phosphorylation event is required for the phosphorylation of Thr-70, which again is required for phosphorylation of Ser-65 by either mTOR or PI3K/Akt pathways [238], [239]. Phosphorylation of these four sites together with phosphorylation of Ser-112, which is constitutively phosphorylated in HEK293 cells, eventually leads to release of 4E-BP from eIF4E [239], [240]. The first phosphorylation events at Thr-37 and Thr-46, which are both located directly upstream of the canonical binding motif, induce a conformational change of the middle part of 4E-BP, which folds into a four-stranded β-domain masking the canonical 4E-BM and rendering it inaccessible for eIF4E [237]. Phosphorylation sites Thr-70 and Ser-65 are located in the elbow linker and are not directly involved in eIF4E binding, instead their phosphorylation likely effects the competition with eIF4G by minimizing the competitive advantage of 4E-BP through a conformational transformation [232]. Phosphorylation of 4E-BP is regulated by mTORC1 in response to various stimuli like growth factors, hormones, mitogens, nutrient or oxygen levels, the availability of amino acids, genotoxic stress or inflammation to adjust protein synthesis, which is thought to be the most energy consuming process within cells, to altered environmental and metabolic conditions [241]– [245]. Several signaling pathways like e.g. PI3K/AKT or MAPK/ERK pathways are involved in the transduction of the diverse signals upstream of mTORC1 [246]. The direct downstream substrates 4E-BP and S6K1, which in contrast to 4E-BP promotes protein synthesis, both play central roles in adapting translational activity to control main cellular functions determining cell 48 growth and proliferation [247], [248]. Moreover, a couple of other kinases have been described to phosphorylate 4E-BP1 independent of mTOR, further emphasizing the importance of the translational regulator [249].

1.3.2 Biological functions of eukaryotic translation initiation factor 4E binding protein 1 (4E- BP1) Given the essential role in controlling so many major cellular processes, the deregulation of mTOR signaling also contributes to many pathological conditions such as cancer, obesity, type 2 diabetes and neurodegeneration [241], [245], [250]. For instance, cancer cells commonly display elevated levels of protein synthesis, which is significantly determined by the eIF4E/4E-BP axis [241], [250]–[256]. Overexpression of eIF4E, low levels of 4E-BP, hyperactivation of mTOR and subsequent hyperphosphorylation of 4E-BP deregulate cap-dependent translation and cell proliferation thereby promoting tumor formation, progression and maintenance [257]–[259]. Briefly, eIF4E is thought to be the least abundant translation initiation factor within cells, therefore constituting a bottleneck in the entire process of translation initiation [260], [261]. Early on it was recognized that eIF4E has oncogenic properties and that its overexpression induces cell transformation, which can be counteracted by 4E-BP [262], [263]. Hence expression level and phosphorylation status of either eIF4E or 4E-BP was used in certain types of cancers to predict survival. Nowadays it became more and more clear that the combined analysis of both regulators has more predictive power than individual factor analysis, because the ratio of eIF4E/4E-BP is crucial in determining the availability of eIF4E and defining the total translational outcome [264], [265]. In this regard, 4E-BP was generally considered to be a tumor suppressor, but the picture became more complex with the discovery that upregulated translation in tumor cells did not only occur in a quantitative but also in a qualitative manner. So it was found that global changes in translation were overall rather mild but certain subsets of mRNAs depicted a preferential, disproportional increase in translation in response to oncogenic mTOR signaling [266]–[271]. This pool of eIF4E sensitive mRNAs often exhibits longer and more complex secondary structures within 5’UTRs and in many cases encodes growth factors, cyclins or other regulators further advancing tumor formation [241], [272]–[274]. Thus, 4E-BP may exert pro-oncogenic functions enabling selective translation of key genes that are only poorly translated during physiological conditions and which support the adaption of malignant cells to hypoxia or nutrient deprivation within the tumor microenvironment [241], [251], [256]. One interesting group of transcripts that is specifically sensitive to mTOR signaling is the family of 5’terminal oligopyrimidine (TOP) containing mRNAs. The TOP motif starts with a C 49 just downstream of the cap followed by a polypyrimidine stretch ranging in length from 4 to 15 nts [275]. The 5’TOP motif is evolutionarily conserved among vertebrates and can be found in ~90 human mRNAs encoding all ribosomal proteins and other translation initiation and elongation factors, PABP and two more proteins which are not part of the translational machinery [276]. Using rapamycin or an active-site inhibitor of mTOR, 4E-BPs were identified to be crucial effectors of selective TOP and TOP-like pyrimidine-rich translational element (PRTE) mRNA translation [79], [269]. However, further studies of TOP mRNAs under stress conditions indicated that translational repression does not fully depend on 4E-BP so that other RBPs were suggested to play a role as trans-acting regulators. None of the ones studied initially could meet all necessary requirements, but lately LARP1 could be identified to be involved in TOP mRNA translational regulation (see next section) [276]–[278]. In line with this, another more current study highlighted two additional functionally distinct mTOR-sensitive mRNA subgroups, which lack TOP motifs and instead display short eIF4E-sensitive 5’UTRs (<30 nts) or long 5’UTRs which harbor both eIF4E- and eIF4A- sensitivity [279]. Hence, not only short regulatory motifs but also non-TOP 5’UTR features seem to shape mTOR-sensitivity and translational control by the eIF4E/4E-BP pathway contributing to the diversity in mTOR-regulated mRNA translation [270], [280], [281]. Taken together, 4E-BPs are master regulators of protein translation in response to various physiological and pathophysiological conditions. Understanding their mechanistic regulation and how diverse external and internal stimuli are unified upstream of mTOR-mediated phosphorylation are important aspects of fundamental research. Furthermore, eIF4E is an attractive target in the development of anti-cancer treatments and 4E-BP mimics have been proposed as therapeutic tools to counteract oncogenic eIF4E activity [232].

1.3.3 RNA binding proteins and sequence elements in untranslated regions RBPs associate co-transcriptionally on nascent mRNA precursors as soon as these arise in order to mediate early RNA processing events like capping, splicing or polyadenylation [282], [283]. Database and literature mining defined a consensus of ~1,500 human RBPs, which constitute 7.5 % of all protein-coding genes [284]. Two independent studies provided experimental evidence for the functionality of ~800 of these predicted RBPs in HEK and HeLa cells by protein occupancy profiling or interactome capture using UV crosslinking of RNA-protein complexes and subsequent oligo(dT) affinity purification [285], [286]. In general, RBPs are able to bind thousands of transcripts via ~600 structurally distinct RNA-binding domains (RBDs) [284]. The vast majority of RBPs is ubiquitously expressed and only 6 % show tissue-specific expression patterns [284]. Whereas the function of ~ 1/3 of all 50

RBPs is still unknown or insufficiently inferred from homologues, approximately 700 RBPs bind to mRNA and act in mRNA metabolic processes [284]. Most often these mRNA interacting RBPs bind within 3’UTRs of selective mRNAs to direct mRNA-specific translational repression [287]. Translational regulation by protein-RNA interaction within 5’UTRs is uncommon and IRE-regulated mRNAs constitute a well-studied example, which is described in a later chapter (see section 1.3.7) [13]. Even though regulation by RBPs is almost invariably inhibitory, PABP interaction with the poly(A) tail is an exceptional and well-known example of translational stimulation [13]. Human PABP contains four non-identical RNA-recognition motifs (RRMs) at the N-terminus of which the first and the second one are able to bind eIF4G, while the last two RRMs exhibit high affinity for poly(A) sequence [288]–[290]. The interaction of PABP with eIF4G and the poly(A) tail of an mRNA results in a so-called “closed loop” configuration leading to mRNA circularization [288], [289]. In the circularized configuration, the affinity of eIF4F for the mRNA cap increases by an order of magnitude, thus facilitating translation initiation and possibly ribosome recycling [289]. The interaction of PABP with eIF4G tethers the eIF4F complex to the mRNA so that it does not need to be recruited de novo when interaction of eIF4F with the cap gets lost, establishing a significant translational advantage [13]. An alternative prominent example of RBP-mediated translational repression is the msl-2 gene, an important factor in dosage compensation in Drosophila. Translational repression in female flies is achieved by the female-specific RBP SXL, which on the one hand regulates msl-2 splicing and on the other hand inhibits msl-2 translation [291]. The msl-2 transcript contains two poly(U) rich sequence elements in the 5’UTR as well as four poly(U) elements in the 3’UTR and efficient translational repression requires association of SXL to binding sites in both UTRs [291], [292]. When bound to the 3’UTR, SXL prevents recruitment of the 43S complex, while SXL binding to the 5’UTR blocks scanning of those complexes that potentially escaped the control at the 3’UTR [291]. The suppression of 43S complex assembly on msl-2 transcripts further involves the co-repressor UNR, which is recruited to the 3’UTR by SXL where it interacts with poly(A) tail-bound PABP and possibly additional unknown factors [291]. The case of msl-2 regulation is in line with a generic model of translational repression in which a RBP binds specific sequence elements in a transcripts 3’UTR to cooperate with another repressive protein that interacts with the cap structure and possibly additional intermediate proteins that establish a connection between the RBP at the 3’UTR and the cap-binding protein at the 5’UTR, resulting in an inhibitory closed loop formation which prevents translation initiation [283]. The mRNA conformation in a closed loop constitutes a physical framework to

51 account for the fact that RBPs associating with the 3’UTR often control translation initiation events at the 5’UTR [291]. RBP-mediated translational control is usually stimulated by intrinsic or extrinsic signals which require rapid adjustment of gene expression levels to initiate specific cellular response programs. One example group of transcripts, which is collectively regulated in response to nutrient or oxygen deprivation, are mRNAs encoding ribosomal proteins, most translation factors and a few RBPs all containing a TOP motif at their 5’end [278], [293]. A TOP motif consists of a cytosine directly downstream of the m7Gppp-cap structure which is followed by an uninterrupted stretch of 4-14 pyrimidines [79]. TOP mRNAs are specifically sensitive to mTOR mediated translational regulation and how this set of mRNAs is selectively repressed by mTOR inhibition in comparison to all other cellular mRNAs that are also translationally controlled by mTOR remained a mystery until recently [79], [269]. The discovery of LARP1 acting as a downstream repressor of mTORC1 specifically controlling TOP mRNA translation and hence the expression of fundamental players of global mRNA translation eventually elucidated this essential control mechanism of cellular homeostasis [278], [294]. Upon phosphorylation by mTOR, LARP1 associates with mTORC1 through RAPTOR and TOP mRNAs get translated under physiological conditions [278]. When mTOR is inhibited and LARP1 is present in a dephosphorylated state, it displays enhanced binding affinity for TOP mRNAs and directs association with the cap and the adjacent TOP motif, which results in translational repression of TOP mRNAs due to LARP1 blocking eIF4E from recognizing the cap and subsequent eIF4F assembly [278], [293]. Although the tight translational regulation of mRNAs is predominantly controlled at the step of translation initiation, RBPs also effect mRNA stability to influence translational rates. One well-studied example of such a type of gene expression regulation is the interplay of ELAVL1/HuR and TTP with AU-rich elements (ARE) containing mRNAs. AREs are located in the 3’UTR of a mRNA and are frequently assembled in overlapping AUUUA sequence stretches [295]. They are commonly found in mammalian transcripts encoding inflammatory cytokines, oncoproteins and G-coupled receptors and are estimated to occur with a frequency of 8 % in the human transcriptome [295], [296]. HuR is one of several ARE-binding proteins that can interact with thousands of mRNAs, promoting their stability [297], [298]. TTP is another prominent ARE-interacting protein with opposing biological function, promoting the destabilization of mRNAs by recruitment of the CCR4-NOT complex, which leads to mRNA decay [295]. In the case of p21 mRNA, bound HuR protects the transcript from TTP binding but upon ubiquitination, HuR gets displaced by p97 and UBXD8 ATPase complex which results in p21 destabilization [283], [296]. Over 80 % of TTP binding sites in 3’UTRs overlap with HuR, 52 suggesting a wide and complex interplay in the regulation of mRNA stability of thousands of target mRNAs, like it was described for p21 [299]. In summary, throughout their whole lifecycle mRNAs are found in association with RBPs that orchestrate the various processing, modification and localization events of RNA metabolism. Due to their modular structure made of multiple RNA-binding domains organized in various ways, RBPs achieve high target specificity and affinity to facilitate diverse regulatory functions [282]. They form interconnected networks, which collectively act on processes of posttranscriptional gene expression like mRNA translation discussed here [285]. However, we are still far away from completely understanding the complex interplay of mRNA-RBP interaction networks and it remains elusive how a certain RBP that is able to interact with thousands of mRNAs and that is also able to conduct more than one function, fulfills the correct processing event at the appropriate target transcript at the right time.

1.3.4 m6A mRNA methylation Methylation at the N6 position of adenosine is the most prevalent chemical modification of mRNA of higher eukaryotes. Originally discovered in the 1970s, the biological functions of m6A remained largely unknown until recently, when high-throughput techniques were developed to study m6A modifications transcriptome-wide even in single nucleotide resolution [300]–[302]. RNA methylation is a reversible modification, which is catalyzed be a m6A methyltransferase complex, containing METTL3, METTL14 and WTAP, and erased by RNA demethylases as FTO and ALKBH5 within the cell nucleus [303]–[306]. The distribution of m6A residues along transcripts is uneven with the highest level of methylation in the 3’UTR and in close vicinity of the stop codon [301]. Those m6A sites have been shown to function in RNA metabolism, as in splice site regulation or RNA stability and degradation [307], [308]. In contrast, m6A- modifications in the 5’UTR function in translation initiation [189], [308]–[311]. In reconstituted in vitro systems, which lack eIF4s, translation competent ribosomes are assembled on mRNA reporters exhibiting a single 5’UTR m6A residue and a cap analog unable to promote translation, while unmethylated reporters and reporters carrying m6A modifications in the CDS fail to induce cap-independent translation [189]. The initiation of translation is thereby mediated by eIF3, which is a multi-subunit protein complex that directly binds m6A residues and interacts with 48S ribosomes [189]. This mode of m6A-induced translation initiation is cap-independent but still relies on scanning ribosomes as a stable hairpin at the very 5’end greatly diminishes reporter mRNA translation [189]. Further, the methylation within 5’UTRs is dynamically regulated and increases under certain stress conditions, like heat shock or UV treatment [189]. The stress-induced increase in 53 methylation is specific only for m6A sites of the 5’UTR, while methylation in other transcript regions is relatively unaltered. It enhances the translational efficiency of stress response genes, like for example Hsp70, while transcripts with decreased methylation of the 5’UTR under stress conditions are less efficiently translated [189], [311]. An increase in translation efficiency was also described for a subset of mRNAs containing m6A modifications in the CDS and 3’UTR. In this case, elevated translation initiation rates were promoted by YTHDF1, an m6A binding protein which interacts with ribosomal subunits as well as eIF3, thereby enhancing translation of both cistrons of bicistronic IRES reporters in tethering assays [312]. YTHDF1-mediated upregulation of translation is not retained during arsenite stress, but quickly recovered after stress release. Taken together, m6A-modification of mRNA depicts an epitranscriptomic layer of reversible post-transcriptional gene regulation. Dynamic adenosine methylation allows for translational control of specific mRNA subsets in response to certain stimuli, which are mediated by distinct protein interactors with individual m6A residues that are differentially located within transcripts. Interestingly, in mESCs this mechanism was already shown to play a role in stem cell self-renewal. In this case, m6A methylation of certain transcripts, especially transcripts of developmental regulators, hampers the binding of HuR, which results in transcript destabilization through the miRNA pathway and the maintenance of mESCs in a pluripotent state [313]. Hence, it will be interesting to further investigate if m6A-mediated inducible cap-independent translation is a general mechanism used not only in response to cellular stresses but also in other specialized cell conditions that rely on fast and well-defined translational control of key regulatory genes.

1.3.5 Upstream open reading frames (uORFs) and start codons (uAUGs) Mechanisms of uORF-mediated translational control are not only complex and diverse, they are also not entirely understood yet, although uORFs are prevailing regulatory elements within mRNAs of higher eukaryotes: approximately ~50 % of human and ~45 % of mouse transcripts contain at least one uORF [82]–[86]. uORFs can be differentially defined which is mostly because uORFs can be subdivided in either being located completely upstream of the main ORF (a start codon followed by an in frame stop codon located within the 5’UTR) or overlapping with the CDS of the main ORF (a start codon within the 5’UTR followed by an in frame stop codon located downstream of the start codon of the main ORF). The latter is sometimes referred to as overlapping ORF (oORF) [85] or as uAUG [314]. Here, I will use the term uORF regardless of its relative location to the main ORF. Albeit uORFs are widespread among mRNAs, they seem to be particularly abundant in transcripts of genes that play important roles in cell differentiation, cell cycle and stress response, 54 including also two-thirds of common oncogenes [18], [315], [316]. Usually, their presence within transcripts reduces gene expression levels by decreasing the number of scanning ribosomes that initiate at the main ORF or by decreasing transcript stability through activation of decay pathways like nonsense-mediated decay (NMD) [85], [317]. A large scale study on matched mRNA and protein datasets estimated that uORF-mediated translational repression typically results in a decrease of protein expression by 30-80 % [83]. In the developing zebrafish, the presence of uORFs had similar repressive effects on mRNA translation as miR-430-mediated translational inhibition during maternal-zygotic transition [85], demonstrating that uORFs are potent regulatory elements in post-transcriptional gene expression. Moreover, uORFs are evolutionary conserved, though different studies claimed varying levels of conservation. While analyses of stringently selected sets of orthologous transcripts between human and mouse [318] and rat [314] indicated that uORF occurrence is rather a species-specific event, another study of global translation initiation sequencing in MEFs and HEK293 cells found that 64-85 % of upstream translation initiation sites were conserved between both cell lines [96]. Nevertheless, the repressiveness of uORFs is ubiquitous [82], [83], [319] and the conservation of functionally validated and confidently translated uORFs is high among vertebrates [85], [314]. As uORFs are less frequently present within 5’UTRs than expected, it seems that purifying selection triggered the conservation of functionally active uORFs, whereas non-functional AUG codons were eliminated from upstream sequences [314], [320]–[322]. Besides, upstream translation initiation can take place at near-cognate start codons. In fact, recent methodological advances that allowed for global mapping of translation events at nucleotide resolution revealed that not more than a quarter of upstream translation initiation events occurs at AUG codons meaning that the vast majority of uORF translation is indeed initiated at non-AUG codons with CUG (~30 %) being the most prominent one [91], [96]. The regulatory impact of an uORF depends largely on its position within the 5’UTR, adjacent secondary and tertiary mRNA structures, specific features of its encoded peptide or of the 5’UTR, as well as alternative promoter usage, alternative splicing and further cell type-specific trans acting factors [86]. Together these features determine a variety of mechanisms like leaky scanning, reinitiation, stalling and potentially shunting of ribosomes that shape the translation efficiency of authentic downstream ORFs. Leaky scanning through uORFs for example, is greatly influenced by the context of the surrounding Kozak sequence. Especially the -3 and +4 position (A of AUG = +1) of the Kozak sequence have been shown to be substantial in defining the strength of translation initiation at the start codon [17], [323]. Plenty of studies showed that the start codon context of uORFs is overall weaker than the start codon context of main ORFs and 55 that a stronger Kozak sequence context of an uORF correlates with higher levels of translational repression of the main ORF [18], [83]–[85], [96], [314], [318], [319], [324], [325]. Hence, leaky scanning at uORFs is controlled by the Kozak context which thereby controls the translational efficiency of main ORFs. Reinitiation of ribosomes after uORF translation is mainly dependent on the uORF location within the 5’UTR, uORF length and downstream secondary and tertiary mRNA structures. It was shown that reinitiation is more efficient, when the intercistronic distance is long and the length of the uORF is short [101], [102], [109], [326]. The theory behind is that elongating ribosomes are turning over crucial reinitiation factors which need to reassociate in order to recycle post-termination ribosomes for downstream translation. Classical examples how reinitiation after uORF translation controls downstream gene expression in response to cellular stresses are GCN4 in yeast and ATF4 in humans [327] [328]. The ATF4 mRNA contains two uORFs of which the 5’-proximal one encodes a peptide of three amino acids, while the downstream one is overlapping with the ATF4 CDS [328]. After translating the first uORF, ribosomes reinitiate at the second uORF which greatly represses translation of the overlapping ATF4 CDS [328]. However, stress conditions induce the

Met phosphorylation of eIF2α which abrogates eIF2-GTP-Met-tRNA i complex formation. Under such conditions, reinitiation of ribosomes after uORF1 translation is delayed as loading of eIF2-

Met GTP-Met-tRNA i complex takes longer resulting in scanning through uORF2 and reinitiation at the ATF4 CDS instead [328]. Translation efficiency of a downstream gene can also be controlled by ribosome stalling at an uORF. One well-known example is the mammalian AdoMetDC, an important gene of the polyamine biosynthesis pathway. AdoMetDC translation rates are linked to cellular polyamine concentrations by a regulatory feedback loop that involves ribosome stalling at the termination site of a six codon inhibitory uORF [316]. Ribosome stalling thereby depends on the uORF encoded peptide, which interacts in cis with terminating ribosomes [316], [329]. Another example, where an uORF encoded peptide represses main ORF translation in trans, is AS. The AS mRNA shows tissue-specific 5’-UTR isoform expression due to alternative transcription start site usage [316]. Longer isoforms contain a uORF encoding a 44 aa peptide that represses endogenous AS translation in bovine endothelial cells in a dosage dependent manner upon transfection of a AS- uORF construct [25]. Lately, it was shown that uORF-mediated gene expression cannot only be controlled by uORF encoded peptides but also by interacting RBPs. One example is the translational control of msl-2 mRNA in Drosophila, which is repressed by SXL protein. SXL binding downstream of a short uORF promotes recognition and translation of the uAUG, while main ORF translation is 56 inhibited presumably via SXL interference with ribosomal factors inducing conformational changes of the preinitiation complex needed for subsequent elongation [331]. The second example comes from AXIIR, which is very lowly expressed in most cell types [332]. AXIIR translational regulation is complex and involves four uORFs, two hnRNPs and HuR protein which cooperate to build a multiple fail-safe regulation system ensuring AXIIR expression levels [332]. In this system, the RBP binding most likely alleviate ribosome leaky scanning at uORFs with moderately strong Kozak sequence [332]. Of particular interest are reports on uORFs within IRESs that coordinate main ORF expression through functional and structural interdependency. One example is VEGF gene regulation which involves in addition to a short 3 codon uORF and two putative IRESs also an internal promoter, alternative CUG start codons, alternative splicing, miRNA binding sites, a riboswitch, alternative polyadenylation, a G-quadruplex structure and RBP binding to AU-rich elements [333], [334]. Translation initiation at the VEGF 121 isoform takes places at CUG codons only, whereas larger mRNA isoforms are efficiently translated at both CUG and AUG initiation sites [334]. The mechanism of translation inhibition at the AUG initiation site of VEGF 121 isoform involves the exon sequence and parts of the 5’UTR, which contains a uORF within an putative IRES, while the 3’UTR is not part of the regulatory module [333]. Transient transfections of mutation constructs in HeLa cells suggest, that the exon sequence of the VEGF 121 isoform promotes uORF translation and inhibits reinitiation at the main ORF, while longer VEGF isoforms stimulate the translation initiation at the main ORF and permit reinitiation after uORF translation [333]. The mode of translation initiation at the uORF is eIF4E-independent [333]. Probably, ITAFs interact with longer VEGF isoforms that substitute post-termination ribosomes after IRES-mediated uORF translation with initiation factors needed for reinitiation at the main ORF. On the other hand, it is possible that ITAFs interact with the shorter VEGF 121 isoform to block ribosome recycling after uORF translation. Interestingly, it was found that reinitiation after IRES-mediated uORF translation can only happen, when the eIF4F complex, or at least a fragment of eIF4G, is involved in primary initiation, suggesting that their interaction needs to be maintained for ribosome reinitiation [335]. This suggests that VEGF IRES-mediated uORF translation requires eIF4F family members except for eIF4E. One more proposed example of uORF-IRES coordinated gene expression is the human GARS mRNA. GARS is expressed in two mRNA isoforms of which the longer one contains a 32 codon long uORF and cap-independent translation activity in in vitro translation assays [336]. The uORF overlaps with the 5’ proximal start codon of the mitochondrial GARS ORF so that mitochondrial GARS expression is efficiently blocked. However, translation of the cytosolic GARS ORF from a start codon downstream of the uORF is unrepressed so that the long mRNA 57 isoform exclusively expresses cytosolic GARS [336]. Subcellular fractionation revealed that both the short and the long mRNA isoform associate with the ER but only the long mRNA isoform is locally translated [336]. Although it still needs to be verified that cap-independent translation of the longer GARS isoform is indeed mediated by an IRES (supplementary promoterless control experiments reveal cryptic promoter activity of bicistronic GARS reporters), this is still an interesting combination of relaxed cap-dependency, a repressive uORF and localized translation on a single transcript. It will be exciting to see how the interplay of individual regulatory elements works in selective GARS isoform expression. In conclusion, uORF-mediated translational control of gene expression is highly diverse including various modes of repression that can be dynamically regulated in response to altering cellular conditions. Progress in genome wide analyses enabled detection and quantification of uORF translation together with gene translation efficiencies, elucidating the wide range of uORF activities. Of special interest for future analyses are uORF and IRES mediated upregulation of gene expression during global translational repression and eIF2α phosphorylation. Insights in these mechanisms are needed to understand and overcome similar events happening in cell transformation during tumorigenesis. Further, a number of single nucleotide polymorphisms (SNPs) have been identified to disturb uORF-mediated translational control of gene expression resulting in human diseases [83], [315]. However, the impact of uORF translation on pathogenic gene expression is likely still underestimated and desires further in-depth analyses.

1.3.6 miRNA binding sites miRNAs are a class of small 21-25 nt long non-coding RNAs which were initially identified in 1993 by studying C. elegans mutants with timing defects in larval development [337]. Only since then miRNAs have been recognized as fundamental players in post-transcriptional gene regulation. They are evolutionary conserved and approximately one third of the miRNAs identified in C. elegans also has homologs in humans [338]–[340]. Many miRNAs display tissue- specific or temporally restricted expression patterns during development and their expression level can vary from a few hundred to several thousands of molecules per cell [341]–[343]. To date, about 2,500 human miRNAs have been annotated and more than 60 % of human protein- coding genes are predicted to be regulated by miRNA binding [344], [345]. Hence, miRNAs are involved in a wide range of cellular processes where they mediate translational repression or decay of target mRNAs [346]–[349]. miRNA genes can either be located in intergenic regions forming autonomous transcriptional units or they can be located within of other genes [339], [340], [350], [351]. Further, they can be organized in clusters of often but not always functionally related genes 58 which are transcribed into a single polycistronic transcript [339], [340]. For most miRNA genes, transcription is carried out by RNA Pol II that generates pri-miRNA precursor transcripts which can be several kilobases long and exhibit a 5’cap structure and a 3’poly(A) tail [352], [353]. Pri- miRNAs undergo nuclear cleavage by DROSHA, an RNase III-type that constitutes together with DGCR8 the crucial components of the microprocessor that produces ~70 nt long pre-miRNA hairpin structures [354], [355]. Pre-miRNAs are exported from the nucleus by XPO5 and Ran-GTP [356]. In the cytoplasm they are cleaved by another RNase III-type enzyme called DICER, which generates ~22 bp miRNA:miRNA* duplexes [357], [358]. Processing, strand selection and loading into AGO proteins is thought to happen simultaneously as DICER directly interacts with AGO and other dsRNA-binding proteins to form the RNA-induced silencing complex-loading complex (RISC-loading complex) [359], [360]. According to the asymmetry rule, the strand of the miRNA:miRNA* duplex displaying thermodynamically less stable base pairing at the 5’end is selected as the guide strand or mature miRNA, while the passenger strand or miRNA* strand is quickly degraded [359], [361]. Mature miRNAs loaded into AGO will guide the miRNA-induced silencing complex (miRISC) to target mRNAs by complementary base pairing. Particularly important for target recognition is the miRNA seed region which comprises nucleotides 2-7/8 of the miRNA 5’end and which is often perfectly or almost perfectly (1 mismatch) complementary to the target [362]– [364]. Additional 3’ supplementary pairing is atypical so that the 3’ portion of the miRNA usually does not exhibit a higher degree of Watson-Crick base pairing than expected by chance [345], [363], [365]. This is in contrast to plants, where miRNAs typically show perfect or nearly perfect complementary that leads to endonucleolytic mRNA target cleavage [366]. As animal miRNAs lack perfect complementary base pairing, they generally function in mRNA translational repression or deadenylation, decapping and 5’-3’ degradation [366]. Exceptions have been found in cnidarians, which possess both modes of miRNA-mediated mRNA slicing and translational repression [367], [368]. Further AGO2, the only catalytically active protein among mammalian members of the AGO protein family, was shown to be essential in miRNA processing in early embryonic development and was also reported to rarely slice targets in animals as for example HOXB8 mRNA in mouse [369]–[374]. Despite extensive scientific investigation, the relative contribution of mRNA translational repression and mRNA decay to overall miRNA-mediated gene silencing is still under debate. The consensus emerges that miRNA-mediated mRNA destabilization accounts for most of the repressive outcome of gene silencing at steady state (66-90 %), while translational repression accounts for a minor part (6-26 %) [375]–[382]. However, translational repression can be the predominant effect of miRNA function right after induction and individual targets can show 59 higher degree of translational repression than mRNA destabilization [375], [376]. So far it is unclear, whether the temporal order of events i.e. translational repression followed by mRNA decay reflects a mechanistic coupling of both processes or whether it is the result of differential kinetics and to what extend individual targets are regulated by both processes in interplay [383]. Cases have been reported in which translational repression is uncoupled from deadenylation or where miRNA-repressed targets are translationally re-activated, albeit some of these events appeared in specialized cellular environments, like in oocytes for example that depict an extreme case of post-transcriptional gene regulation due to the absence of active transcription [384]–[389]. Further, it remains controversial how miRNAs achieve specific and significant effects in post-transcriptional gene regulation. Even though a single miRNA is able to directly repress hundreds of target genes, repression levels are generally low (~ 2-fold) [345], [375], [376], [390]. However, the impact of individual miRNAs on a target mRNA can be increased by combined targeting through several miRNAs [375]. Accordingly, most target mRNAs contain on average four binding sites for multiple conserved miRNA families in their 3’UTR and additional synergistic effects were observed for target sites that are located in close proximity to each other (~ 40 nt) [375], [391], [392]. Further, target site efficacy can be elevated in AU-rich sequence contexts, in positions outside the center of long UTRs and in distance of at least 15 nts from the stop codon [391]. Besides, miRNAs can increase their influence on gene expression control by simultaneously repressing targets of the same pathway or protein complex [393], [394]. Several models have been proposed on how miRNAs exert their function and there are indications that miRNAs can act as classical switches, buffers or decoys in gene expression regulation [363], [395]. So it was shown by single-cell analysis and mathematical modelling that miRNA regulation can generate a threshold level of target mRNA below which protein production is efficiently repressed, near which protein expression is sensitive to target mRNA transcription and above which miRNA repression of protein production is minimal [396]. In this way miRNAs can act as a switch and as a fine-tuner depending on target mRNA expression level relative to the threshold. Further, it was shown in a follow-up study that in the context of high transcription, miRNAs reduce protein expression noise of lowly expressed genes whereas they add to protein expression noise of highly expressed genes [397]. Thus, miRNAs are thought to confer precision to gene expression programs and promote robustness of the cell system. Moreover, it was hypothesized that miRNAs are essential components of a network of competing endogenous RNAs (ceRNA) in which coding and non-coding species of cellular RNAs, especially mRNAs, lncRNAs and pseudogenes, interdependently regulate each other through competition between shared miRNA-binding sites, also called miRNA response elements (MREs), resulting in transcriptome-wide RNA-RNA crosstalk that greatly influences the 60 protein output of post transcriptional gene regulation [398], [399]. Even though several examples of functional ceRNA networks have been reported, it seems that the ceRNA theory needs to be adapted to the latest findings as these suggest that competition for miRNA binding sites under physiological conditions is unlikely to globally impact gene regulation and that crosstalk rather achieves effects in specialized environments or cell conditions only [400]–[404]. Another controversial aspect of miRNA function is how translational repression is accomplished. Previously, divergent models were proposed claiming that miRNAs can inhibit translation both at the initiation step as well as at the elongation step but nowadays cumulative experimental evidence suggest that miRNAs principally interfere during initiation of cap- dependent translation [377], [405]–[410]. The exact molecular mechanism of repression is still unclear and several studies used IRES reporters to unravel which phase of the initiation process is inhibited by miRNA targeting. Essentially, it was reported that viral IRESs which promote cap- independent translation without a requirement for eIF4A escape translational repression through miRNA targeting [383]. However, some of the results are contradictory so that additional studies are required to elucidate the mechanistic basis of miRNA mediated translational repression and the miRISC interaction partners involved.

1.3.7 mRNA secondary structure Translation efficiency of mRNAs can be regulated by structural features. An important one, which is inherently present in cytoplasmic mRNAs, is the m7G cap structure [411]. Even though the cap is not absolutely necessary for translation, the translation efficiency is positively correlated with its presence and accessibility [411], [412]. Some mRNAs build secondary structures, like histone H4 mRNA e.g. which forms a cap-binding pocket that hides the cap from eIF4E thereby hampering translation initiation [413]. Further, translation efficiency of mRNAs is substantially influenced by 5’UTR length and the secondary structural elements within. The average 5’UTR in human and rodents is around 200 nt long and does not vary much in length within other vertebrates or invertebrates [414]. Interestingly, human mRNAs encoding transcription factors, growth factors, their receptors, proto-oncogenes and other regulatory proteins that are inefficiently translated under physiological conditions were shown to harbor longer 5’UTR sequences than mRNAs of highly expressed genes [415]. Reduced translational efficiency is likely not due to 5’UTR length per se but rather due to the secondary structures present [416]. So were more than 90 % of these 5’UTRs shown to contain stable secondary structures with average free energies less than -50kcal/mol, of which many were actually located within the first 100 nt from the cap [415]. Earlier experiments using designed hairpins of various thermal stabilities placed in various distances to the cap showed that 61 translation efficiency is consistently inhibited by hairpins of 30-35 kcal/mol and that the repressive effect is the strongest within the first 16 bases of the 5’UTR [417], [418]. According to these findings, a model was proposed in which stem structures in close proximity to the cap block ribosomes from entry at the 5’end [411], [419], [420]. However, once the 40S subunit is bound, it is able to melt base pairs during the scanning process [411], [419], [420]. 5’UTRs of human and mouse exhibit in general more secondary structure than the CDS and two out of three human 5’UTRs display ΔG values of -40 kcal/mol or less allowing for substantial regulatory potential [414], [421], [422]. In line with this, not only stem-loop structures around the cap are able to control mRNA translation, but also stems in proximity to the start codon have been shown to reduce translation efficiency [421], [423]. Moreover, 5’UTRs of vertebrates exhibit a GC content of ~60 % [414]. Intriguingly, high GC contents within stem- loop structures of 5’UTRs were reported to negatively affect translation efficiency independent of the thermal stability or the position within the 5’UTR [418]. Further, at least one example exists where secondary structure had a positive effect on translation initiation. In this case, a stem loop of -19 kcal/mol was inserted downstream of an uAUG which significantly increased translation initiation at the uAUG in vitro [420]. The strongest effect was detected when the stem-loop was placed 14 nucleotides downstream of the uAUG, possibly by pausing the ribosome in optimal position on the initiation codon, as ribosomes typically cover ~15 nts up- and downstream from the start codon during initiation [420], [424]. Another prominent example of a small structural element that regulates gene expression in collaboration with trans acting RBPs is the IRE. The IRE is a conserved 30 nt stem-loop with a free energy value of roughly -5.0 kcal/mol that can be found in 5’UTRs of a number of genes involved in iron homeostasis [419], [420]. It is located in proximity to the cap where binding of the regulatory RBPs IRP1 and IRP2 creates a steric block for the 43S complex under conditions of iron deficiency [12]. However, when the IRE is placed more than 60 nt apart from the cap translational repression is attenuated, suggesting the element is efficient in blocking ribosome recruitment but already bound ribosomes are able to displace the interacting RBPs during scanning [12], [13], [292], [420]. Taken together, structural elements within 5’UTRs build an additional layer in gene expression regulation. A high GC content creates the framework for base pairing, whose stability and position or interacting binding factors can repress or enhance mRNA translational efficiency.

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1.4 Translational control in physiological and pathological conditions After giving insights into mechanisms that regulate the process of translation initiation, the next section is providing examples on the role such translational control is playing during physiological states. Compared to transcriptional control, translational control is a fast means of regulating gene expression in response to external and internal stimuli. Cellular processes like cell growths and division, metabolism or differentiation rely on selective translation and protein synthesis to establish changes in cellular conditions. Signaling pathways often activate multiple regulatory mechanisms to control the global rate of protein production, but despite having several mechanisms in place, the system is not failsafe and defects in translational control can lead to developmental disorders and human diseases of for example the nervous system or cancer.

1.4.1 Cell cycle and mitosis Translational regulation of protein expression is important for cell cycle progression and cap- independent translation is thought to play a major role in the process by mediating protein synthesis during mitosis [42], [425], [426]. This dates back to studies performed in the 60s that reported a distinct decrease of protein synthesis in mitotic cells shown by reduced amino acid incorporation rates which were declined by 50 – 75 % [427]–[430]. Shortly after, it was suggested that reduced protein production during mitosis is the consequence of a lowered rate of translation initiation and ribosome loading onto mRNA [431]. It took decades until this theory was supported by findings of the Sonenberg lab showing that the cap-binding protein eIF4E is hypophosphorylated during mitosis which reduces its binding activity for the cap structure and as a result impairs cap-dependent, but not cap-independent or IRES-dependent, translation initiation [432]. Furthermore, they reported that 4E-BP1 is also hypophosphorylated during mitosis, resulting in strong binding of the suppressor protein to eIF4E which leads to disruption of eIF4F complex formation [433]. These findings explain on the one side the suppression of cap-independent translation and on the other side the failure of Mnk1 kinase, despite being activated in mitosis, to phosphorylate eIF4E due to the inability to assemble into eIF4F [433]. However, recent analyses raise concerns about the level to which protein synthesis is decreased, the degree to which initiation factors are phosphorylated and the grade to which translation elongation is involved into translational regulation during mitosis. For example, HeLa cells arrested with colcemid or nocodazole showed a global reduction of translation during mitosis [42], [434], [435], while mitotic HeLa cells treated with CDK1 inhibitor RO-3306 displayed only a modest reduction in translation rates [436]. Contrary, HeLa cells synchronized in a double thymidine block maintained almost similar translation rates throughout the whole cell cycle [437], [438]. 63

Such inconsistencies were not only detected at the level of translational suppression during mitosis, but also apply to the phosphoregulation of initiation factors that were suggested to be involved in this suppression. So, mitotic 4E-BP1 is hypophosphorylated in nocodazole arrested HeLa cells [433], while aphidicolin treated HeLa cells are strongly phosphorylated at Ser- 65 and Thr-70 during mitosis [439]. In addition, mitotic phosphorylation of 4E-BP1 was found to be mediated by CDK1 via a mTOR-independent mechanism leading to the suggestion that hyperphosphorylated 4E-BP1 sustains cap-dependent translation in mitotic cells [440], [441]. Moreover, it was hypothesized that these specific hyperphosphorylated isoforms of 4E-BP1 may have a gain-of-function critically important for mitogenesis [442]. These contradictory findings concerning the translational regulation of mitotic cells can widely be attributed to variations in drug treatment used for cell cycle arrest (indicated here by emphasizing differences in synchronization methods applied in the studies mentioned above) [436], [443]. None of the synchronization methods developed so far seems perfect and mitotic slippage during prolonged treatment, heterogeneity of cell populations and most notably activation of stress response signaling pathways and induction of growth imbalance by the mode of action of the drug in use introduce artefacts that mask the true physiological conditions within the cell [444]–[450]. Even though a genome-wide dataset of protein synthesis throughout the cell cycle of unmanipulated asynchronous cells is not available to date, the impression emerges that cap-dependent translation remains active during mitosis. A global switch from cap-dependent to cap-independent translation is often cited but never convincingly demonstrated [64], [197], [254], [433], [451]–[456], as even in extreme cases where translation is reduced about 75 % following colchicine or colcemid treatment, cap-dependent translation is not completely inhibited. Whether these conditions require an alternative mode of translation is in question [78]. Noteworthy, studies of asynchronously growing yeast cells separated by centrifugal elutriation revealed a rather constant rate of protein synthesis during the whole cell cycle and a multi-omics study of HeLa cells synchronized by a double thymidine block suggests that regulation of protein stability rather than translational regulation is the prevailing mechanism to control protein expression during mitosis [438], [457]–[459]. In addition to the considerable doubts concerning the mode of translation initiation during mitosis, some studies found indications for the translational regulation at the level of elongation in unperturbed or thymidine arrested mitotic cells [443], [456], [460]. Further, alternative splicing and splicing factors might be implicated in regulating the translation efficiency of mitotic transcripts [461], [462]. Hence, there are indications against a global translational switch during mitosis and the support of this theory decreased.

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For instance, it was suggested that phosphorylation of eIF2α at G2/M transition is involved in translational suppression during mitosis as the phosphorylated form of eIF2α functions as inhibitor of eIF2B, preventing eIF2α recycling and ternary complex formation [463]. However, this phosphorylation event was shown to be dependent on aphidicolin or nocodazole treatment [436]. Furthermore, the G2/M-specific phosphorylation of raptor was reported to promote IRES-dependent translation and facilitate cell cycle progression at the G2/M boundary [464]. Even though mitotic phosphorylation of raptor seems to happen naturally, follow-up experiments were performed in the presence of nocodazole, which makes it complicated to distinguish between the unperturbed effects of phosphorylated raptor during mitosis and artefacts introduced by nocodazole treatment [464]. Indeed, phosphorylation patterns of raptor are playing a role in mTORC1 activity and cell cycle regulation [465]–[468], however a requirement of raptor phosphorylation for cap-independent translation is not convincingly demonstrated due to the usage of nocodazole and a phosphorylation-deficient raptor, displaying alanine conversions in seven phosphorylation sites, which together is expected to disturb eIF phosphorylation, mTOR activity and cell cycling to such an extent that differences in protein expression in this context cannot be pinpointed to the presence of an IRES alone without excluding side effects and including more proper IRES controls [436], [464], [467], [468]. Also 14-3-3σ was reported to play a role in mitotic translation by binding a variety of translation and initiation factors leading to suppression of cap-dependent translation and conversely to stimulation of cap-independent translation, in particular IRES-dependent translation of p58PITSLRE required for cytokinesis [451]. IRES-dependent translation in 14-3-3σ KD cells was tested by DNA transfection of bicistronic reporters carrying either the p27Kip1 5’UTR, which was shown to harbor promoter activity, or the HIV-1 5’UTR, which was shown as well to harbor cryptic promoter or splicing activity, wherefore both are not suitable to test IRES- dependent translation in this kind of assay [162], [469], [470]. Further, p58PITSLRE is a shorter, presumably IRES-dependent isoform of p110PITSLRE and the authors did not address how KD of p58PITSLRE affects the longer isoform or translation from gene duplication [471], [472]. Hence, it is not clear if p58PITSLRE-specific expression is required for cytokinesis or how cap-independent translation is implicated in the binucleate phenotype of 14-3-3σ-depleted cells. Thus, it appears that cap-independent translation during mitosis is generally less significant as anticipated by early experiments in this field of research. Despite all that, cap- independent translation during mitosis might still be similarly relevant as during any other phase of the cell cycle although probably not the prevailing translation mechanism. So, a couple of cellular IRESs have been reported to be functionally active during mitosis or at G2/M transition (Tab. 2). 65

Tab. 2: Reported genes with cellular IRES-mediated translation and cell cycle-dependent regulation. The results of bicistronic reporter assays testing translation reinitiation or the presence of cryptic promoters or cryptic splice sides are summarized. Gene with reported Reinitiation Cryptic Reported References cell cycle-dependent promoter/splicing regulator(s) IRES activity - + DAP5 [197], [473] Splice site in 5’UTR; inadequate neg. control; low efficiency of cap- BCL-2 independent and 5’end-dependent translation; under tested stress conditions capped transcript without Bcl-2 5’UTR an order of magnitude more efficiently translated than with Bcl-2 5’UTR - ND DAP5 [197] CDK1 Low efficiency; cap-dependency not tested - + hnRNP C [67], [73], [454], c-myc Promoter activity, low efficiency of cap-independent translation [474] ND - ZNF9, PCBP2 [475]–[477] Inadequate neg. control; capped transcript without ODC 5’UTR ODC more efficiently translated than with ODC 5’UTR; no IRES activity in in vitro translation assay using rabbit reticulocyte and FM3A lysate; - + HuR, HuD, hnRNP [161], [162], p27Kip1 C1/C2, PTB [469], [478] Promoter activity in 5’end; low efficiency - - UNR, eIF2α [81], [455], [471] Potential IRES located within the p110PITSLRE ORF; 5’end- p58PITSLRE dependency not tested; no IRES activity in in vitro translation assay using rabbit reticulocyte lysate; strong candidate for bona fide cellular IRES activity (-) ND ND [479] SNM1 No evidence for reinitiation but SNM1 activity is below background level; inadequate experimental design to test mitosis-dependency - (-) PTB, hnRNP C1/C2, [166], [480] UNR UNR Northern blot to validate aberrant splicing has limited sensitivity; inadequate siRNA control assay targeting the 3’ cistron; inconsistent translation efficiency in between assays ND = not determined; - = negative results; + = positive results; (-) = negative results but doubtful because of inadequate experimental setup; neg. = negative

Many of the genes with hypothesized cellular IRES activity of Tab. 2 were initially identified because their function is related to cell cycle regulation and because they displayed a higher

66 protein expression level than average in synchronized mitotic cells and/or because their 5’UTR sequence contains any feature that was believed to hamper cap-dependent initiation and ribosome scanning. In general, most of the analyzed 5’UTR sequences showed relatively low IRES activity in bicistronic reporter assays, a gold standard in the field to evaluate the efficiency of a sequence of interest to promote internal translation initiation. For instance, Bcl-2-dependent translation of the 3’ cistron was ~10 % of cap-dependent translation of the 5’ cistron of the same bicistronic transcript [473]. The IRES activity of SMN1 was also around 10 % of the cap- dependent activity of a bicistronic transcript, while it was only 0,1 % for CDK1 [197], [479]. Other 5’UTRs scored much higher in the bicistronic assay, like for example p27Kip1, which mediated internal translation initiation at the 3’ cistron with roughly half the efficiency of cap- dependent translation at the 5’ cistron, while c-myc even facilitated a ~50-fold higher expression of the 3’ cistron than the 5’ cistron [162], [474]. However, both p27Kip1 and c-myc sequences were shown to harbor promoter activity, which generated artefacts that significantly contributed to the activity determined by DNA transfection of bicistronic reporter constructs [73], [162]. As pointed out by leading scientists in the field, bicistronic reporter assays are inherently prone to generate artefacts wherefore a careful experimental design including adequate controls is required to demonstrate bona fide IRES activity [27], [65], [66], [72], [481], [482]. Almost all studied potential cell-cycle dependent IRESs listed here were either not comprehensively analyzed or contain a cryptic promoter or splice site, which makes it difficult to evaluate their genuine ability to mediate internal translation before eventually considering their cell-cycle dependency or suggested regulators. Notably, the presence of a cryptic sequence element with transcriptional regulatory function does not exclude the presence of an IRES per se. It simply requires a set of additional controls to unambiguously assess the IRES activity of that sequence. One of the few studies employing a comprehensive experimental setup was performed by Lloyd and colleagues who investigated the 5’UTR of the apoptosis regulator BCL-2 for its ability to promote internal translation initiation during stress conditions. As DNA transfections of bicistronic BCL-2 reporter constructs produced aberrant monocistronic transcripts, in vitro transcribed BCL-2 reporters were used in in vitro translation assays as well as in in vivo RNA transfections. A combination of capped, uncapped and hairpin containing mono- and bicistronic reporter constructs was applied to show that BCL-2 5’UTR has 5’end-independent translation activity which is ~10 % of cap-dependent BCL-2 translation in in vivo assays and 33 % in in vitro assays respectively [473]. BCL-2 also showed internal translation activity which was ~60 % of cap-dependent BCL-2 translation in in vitro assays [473]. But insertion of a stable hairpin in front of the 5’ cistron of the bicistronic BCL-2 reporter reduced internal translation initiation around 2/3, indicating that the internal translation activity was somewhat dependent on the translation 67 activity of the upstream cistron wherefore it is rather unlikely to act as a standalone bona fide IRES [473]. Instead weak translation reinitiation might have contributed to the putative internal translation activity of bicistronic reporters. Further, by comparing capped monocistronic BCL-2 reporters to capped monocistronic reporters lacking the BCL-2 sequence it became clear that the presence of BCL-2 5’UTR reduced reporter expression by 84 % [473]. This is not surprising, as the 5’UTR of BCL-2 displays extensive secondary structure, ten uAUGs and one uORF, reducing its translation efficiency [473]. Moreover, a comparison of the translation efficiency of an uncapped or stable hairpin containing monocistronic BCL-2 reporter with an uncapped monocistronic control reporter missing the BCL-2 sequences under stress conditions (increased etoposide concentrations) revealed that although BCL-2 mediated reporter expression slightly increased, it did not exceed the expression level of the negative control reporter [473]. Further, even though the level of 5’end-independent translation initiation was stimulated by nearly 3-fold during stress, it remained below the level of cap-dependent translation initiation of monocistronic BCL-2 reporters [473]. This raises the question if 5’end-independent translation initiation at the BLC-2 5’UTR is of physiological relevance for naturally capped BCL-2 transcripts. Another interesting IRES candidate of Tab. 2 is a 380 nt long stretch within PITSLRE, also known as CDC2L2 or CDK11, a gene encoding a serine/threonine protein kinase. PITSLRE expresses two protein isoforms, p110PITSLRE and p58PITSLRE, which are both translated from the same transcript by alternative in-frame start codons [471]. Expression of the shorter p58PITSLRE protein is enriched during G2/M phase of the cell cycle and its translation is depending on an AUG codon located within the ORF of the longer p110PITSLRE isoform. Hence, the potential p58PITSLRE IRES is special by residing within the coding region of a gene and not in a 5’UTR like other reported candidate IRESs discussed in this chapter. In total, there are 17 AUGs, seven of them in-frame, located in between the start codon of p110PITSLRE and p58PITSLRE and none of them gives rise to another protein isoform, which supports the theory that translation at

PITSLRE AUG18, the start codon of p58 , is indeed mediated by internal initiation rather than cap- dependent ribosome scanning. Initial data of Cornelis and colleagues showed a strong G2/M- specific upregulation of p58PITSLRE and DNA reporter assays complemented with thorough northern blot analyses suggested that p58PITSLRE expression was IRES-driven, although in vitro translation assays using rabbit reticulocyte lysate were not able to detect IRES activity of p58PITSLRE [471]. But in a follow-up study, the IRES activity of p58PITSLRE appeared weaker, as the ratio of internal initiation to cap-dependent initiation was 0.12 for transiently transfected bicistronic expression vectors in HEK 293T cells [455]. Further, in vitro translation assays of bicistronic reporters carrying the 380 nt long sequence stretch upstream of p58PITSLRE AUG, the 68 stretch, which was shown earlier to contain IRES activity, was presented. The relative IRES activity of this p58PITSLRE fragment was determined to be 0.03 in RRL or 0.017 in HeLa S3 cell extracts, respectively [455]. Raw values are provided for the assay in HeLa S3 extracts and it seems questionable if the relatively low expression levels of the IRES-dependent reporter are within the exponential range of this assay. Moreover, alternative splicing was found to generate several PITSLRE transcript variants, which might enable the expression of truncated p58PITSLRE protein not only by internal translation initiation at the full length transcript but also by cap- dependent translation of alternatively spliced shorter transcript [81], [472]. However, regulating protein expression at the posttranscriptional level is faster than at the transcriptional level and flexible switching between protein isoform expression from only a single transcript might harbor further advantages, which is why internal initiation might still be functionally relevant in this case. Nevertheless, as pointed out by Shatsky and colleagues, a control experiment is missing to verify true internal initiation at p58PITSLRE and to exclude an alternative 5’end-dependent translation mechanism [64]. Therefore capped and A-capped monocistronic reporters with and without the start codon of the longer p110PITSLRE isoform would need to be transfected into cells synchronized at the G2/M transition [64]. Taken together, scientific reports on the relevance of cap-independent translation during mitosis are largely inconsistent. This might mostly be attributed to side effects introduced by the cell synchronization methods used. As a result, there is doubt about a global translational switch in mitotic cells that favors cap-independent translation. Anyhow, a couple of cellular IRES candidates are suggested to be regulated in a cell-phase dependent manner being active at G2/M transition or at mitosis. Among these candidates, none seems sufficiently validated to proof bona fide IRES activity. However, cap-independent or IRES-mediated translation might still be relevant during mitosis and some of the candidates might truly promote internal initiation. It just requires adequate experimental design for unambiguous verification. Future analyses should not only include the necessary controls, they might also investigate the role of localized cap-(in)dependent translation at the mitotic spindle, which might contribute to translational regulation in cytokinesis [483]–[485].

1.4.2 Cell stress and apoptosis Environmental changes can induce a great number of cell stresses like starvation, heat shock, hypoxia or DNA damage, which require precise and immediate cellular responses to adjust to and defend against altered conditions. Translational reprogramming enables cells to rapidly regulate their gene expression by globally reducing translation and by selectively expressing effector proteins of the stress response, leading to homeostatic readjustment [44], [69], [99], [486]–[491]. 69

Prolonged stress exposure or failure to resolve stress-induced cell damage can eventually activate apoptosis, a mode of programmed cell death. As conventional translation initiation is compromised during stress response and apoptosis, cap-independent or IRES-mediated translation provides alternative mechanisms that are proposed to play important roles in specialized gene expression and cell survival [40], [41], [492]–[496]. During stress response, cells globally decrease canonical cap-dependent translation initiation by interfering mainly with the following two processes: eIF4F-mediated cap recognition and eIF2-dependent ternary complex formation [497]. In addition, a in 2009 discovered group of tRNA-derived stress-induced RNAs (tiRNAs) is thought to contribute to stress-induced translational repression [498]–[500]. The angiogenin-cleaved 5’-tRNA halves are able to displace eIF4G from uncapped as well as less efficiently from capped transcripts by a yet unknown mechanism [501]. Further, they cooperate with YB-1 in stress granule formation promoting translational repression [502]. However, the effects of 5’-tiRNAs on translation are mild, resulting in about 20 % decrease of protein synthesis when transfected into MEFs [499]. Apart from this novel tiRNA-mediated repression process, the integrated stress response is centered on the availability of the ternary complex [503]. The ternary complex consists of eIF2,

GTP and Met-tRNAi and is delivered to the P site of the small ribosomal subunit to form the 43S pre-initiation complex [2]. Upon start codon recognition, eIF2-bound GTP is hydrolyzed and eIF2∙GDP is released from the ribosomal subunit. Recycling of eIF2 is catalyzed by eIF2B before a new round of ternary complex formation and translation initiation can start [504]. Various kind of stresses prevent eIF2 recycling through activation of one out of four distinct protein kinases, HRI, PKR, PERK or GCN2, that phosphorylate eIF2α at Ser51 rendering it from a substrate to a competitive inhibitor of eIF2B [2], [505]. Moreover, translation initiation under stress conditions is hampered by reduced formation and/or binding of the eIF4F complex to the mRNA 5’cap structure. Usually, availability of eIF4E, the cap-binding protein, is restricted due to increased association with its inhibitor 4E-BP1 which is dephosphorylated following mTOR pathway inactivation [506]. Interestingly, the mTOR transcript itself was recently suggested to be translated in a cap- independent manner in normal and hypoxic conditions to ensure functionality during inhibition of protein synthesis [507]. Although the authors include several necessary control experiments to confirm bicistronic reporter assays, these show weak promoter and weak splicing activity of mTOR 5’UTR making it impossible to verify its cap-independent translation efficiency without analyzing translation of transfected in vitro transcribed mRNA reporters [507]. Such in vitro assays used monocistronic A-capped and G-capped mRNA, but expression values were depicted as a ratio normalized to empty vector. Hence, the cap-dependency of mTOR remains unclear and 70 although further experiments suggest the mTOR sequence might be able to directly interact with 40S ribosomal subunit, this alone is not sufficient to induce internal translation initiation [507]. Furthermore, it was speculated that 4E-T, a nucleocytoplasmic transport protein of eIF4E, facilitates eIF4E translocation to the nucleus and into P-bodies during distinct cell stresses [508]. Sequestration of eIF4E is predicted to not only disrupt eIF4F complex assembly but also to prevent eIF4E phosphorylation which is catalyzed by eIF4G-bound MNK1 kinase [494], [509]. How eIF4E phosphorylation affects translation initiation is controversial as it was shown to reduce eIF4E affinity for the mRNA 5’ cap on the one side and to promote tumorigenesis and selective mRNA translation in response to different cell stressors on the other side [510]–[514]. Anyhow, eIF4E and eIF4G association was further shown to be limited under stress conditions by phosphorylation of eIF4G through Pak2, which competes with eIF4E for binding to the same region on eIF4G [515]. Long-lasting or severe stress can activate apoptosis to eliminate cells which otherwise could become deleterious for the organism. During apoptosis the phosphorylation status of initiation factors gets modified or factors can be cleaved by caspases in such a way that protein- protein interactions are modulated. For example, TRAIL can inhibit overall translation by stimulating increased association of eIF4E with 4E-BP1 or by stimulating eIF2α phosphorylation [516]. But eIF2α phosphorylation can also be counteracted by PP2A-mediated dephosphorylation of eIF2B5 to regulate the balance of apoptosis and cell survival [494]. Most significantly, CASP3 cleaves eIF4GI into three fragments generating N-terminal, middle- and C-terminal fragments of apoptotic cleavage of eIF4GI (N-FAG, M-FAG, C-FAG) [517]. Parts of the middle region of eIF4GI, which contain the eIF4E binding site, were described as the minimal sequence required for cap-dependent translation in vitro [518]. However, cap-dependent translation is greatly reduced in the process of apoptosis and the M-FAG is much more effective in stimulating cap- independent translation (provided that the mRNA 5’ end is assessable) and IRES-mediated translation initiation than cap-dependent translation initiation [205], [519]. The other two members of the eIF4G protein family, eIF4GII and eIF4G2/p97/DAP5/NAT1 are as well targets of caspase cleavage and specific degradation during apoptosis [516], [517]. While it is speculated that cleaved eIF4GII fragments are unable to support translation it is suggested that both eIF4G2 and its apoptotic fragment p86 are able to facilitate cap-independent translation [196], [198], [199], [520]. Taken together, suppression of eIF4F-mediated cap binding and eIF2-dependent ternary complex formation both result in a decrease of canonical translation initiation during cell stress and apoptosis. However, neither eIF4F nor eIF2 inhibition alone causes a complete shut-down of translation and transcripts are differentially repressed by one or the other mechanism [69], 71

[521]. Moreover, a growing number of transcripts was described to contain specialized initiation mechanisms enabling them to escape either eIF4F- or eIF2-dependent translational inhibition permitting preferential protein synthesis during cell stress and apoptosis. One prominent example of specialized translation initiation during eIF2α phosphorylation is the stress-inducible transcription factor ATF4. ATF4 contains two uORFs in its 5’UTR and is inefficiently translated in normal cells, as the second uORF is overlapping with the start codon of the ATF4 coding region, thus repressing ATF4 protein synthesis [328], [522]. However, during stress conditions ATF4 is efficiently translated [523], [524]. Ribosomes initiate translation at uORF1 of ATF4 mRNA, which only spans three amino acids, and then continue scanning the 5’UTR. As the ternary complex concentration is limited, ribosomes require more time to recruit eIF2-GTP to get translation competent again. Hence, reinitiation is delayed and ribosomes will scan through uORF2 and reinitiate translation at the authentic ATF4 start codon [328], [522]. A genome-wide ribosome profiling analysis identified nine more transcripts with increased or resistant translation upon eIF2α phosphorylation during arsenite induced cell stress and all except for one contain uORFs in their 5’UTR [69]. This indicates that eIF2-mediated control of alternative uORF translation is a functional mechanism for preferential protein synthesis upon stress that affects likely a very low number of mRNAs. One frequently cited IRES-dependent translation event is the synthesis of the HIF-1α subunit of the HIF-1 transcription factor which is a master regulator in activating the expression of many genes of the homoeostatic response to hypoxia [525], [526]. Initial IRES analyses lacked essential controls but a follow-up study could reveal that HIF-1α 5’UTR contains cryptic promoter activity [73]. Thus, RNA transfections are required to evaluate IRES activity and these turned out that HIF-1α has high cap-dependency (uncapped HIF-1α reporter was translated with ~0.6 % efficiency of capped HIF-1α reporter) and very low IRES activity (in a bicistronic HIF- 1α reporter IRES mediated translation was 0.4 % as efficient as cap-dependent translation) in HeLa cells [73]. Moreover, another study was unable to find a hypoxia-induced increase in HIF- 1α synthesis in NIH3T3, HeLa and Hep3B cells but rather diminished rates of translation that reflect the decrease in mRNA levels [58]. Later, an alternative translation mechanism was suggested by Chen and Huang, who proposed that under normoxic conditions HIF-1α translation is reduced due to CPEB2 binding to its 3’UTR and interacting with eEF2 which results in decreased translation elongation [527]. But under arsenite induced oxidative stress CPEB2 dissociates from HIF-1α mRNA rendering its translation efficiency [527]. Another prominent case of IRES-mediated translation was reported by Macejak and Sarnow in 1991, who were the first to postulate internal translation initiation to be mediated by an eukaryotic mRNA [37]. They found that the cellular BiP mRNA was translated during 72 poliovirus infection and that the BiP 5’UTR was able to efficiently promote translation of the 3’ cistron from bicistronic DNA reporters [37]. When BiP 5’UTR was later tested in other cell lines using different controls it showed much weaker IRES activity (<5-fold stimulation over the control compared to ~15-fold stimulation over the control by Macejak and Sarnow), which might be explained by the fact that the first study used an unusual negative control which compared BiP translational efficiency against an antisense sequence from Drosophila Antp gene [59], [81], [198], [199]. Furthermore, assays using a bicistronic reporter plasmid depleted of potential 5’ss resulted in very low IRES activity of BiP 5’UTR and assays with promoter-less plasmids revealed low cryptic promoter activity, so that future analysis would require mRNA transfection experiments to evaluate authentic BiP IRES activity [57], [58]. Similarly, many cell stresses were reported to induce IRES activity of XIAP, an apoptotic suppressor protein which is involved in caspase inhibition and which is suggested to support cell survival upon severe but transient apoptotic conditions [528]. The initial analyses lacked some necessary control experiments (siRNA-targeting, hairpin insertion, RNA transfections) and following more comprehensive analyses then revealed cryptic splicing and promoter activity in two test systems used (FLuc/RLuc and βgal/CAT reporter) [57], [65], [73], [528]–[530]. Subsequent RNA transfections resulted in much weaker XIAP IRES activity as seen in DNA transfections, albeit XIAP-mediated translation was still significantly more efficient than empty vector controls [57], [65], [73]. Moreover, it was found that XIAP 5’UTR is alternatively spliced, leading to expression of different isoforms with IRES activity only assigned to the unusual long one (short: 323 nt, long: 1.7 kb) [531]. Further, several RBPs were suggested to regulate XIAP IRES activity. Among them the well-known splicing factors hnRNP C and hnRNP A1, the latter of which binds to the alternative 3’ss within the XIAP 5’UTR [532]–[534]. Additionally, a polypyrimidine tract, which then turned out as well to belong to the alternative 3’ss, was identified to be necessary for IRES activity of XIAP [528]. Hence, stress induced XIAP expression might not only be modulated by internal translation initiation but also on transcriptional level by alternative splicing. While the contribution of alternative splicing to XIAP translation requires further investigation, Holcik and colleagues focused their analyses on the long XIAP isoform and proposed an internal initiation mechanism that depends on eIF5B during eIF2α phosphorylation and involves ribosome recruitment by eIF3 binding near the start codon and PABP binding to the poly(A) tail [535], [536]. The expression of the pro-apoptotic protein Apaf-1 was also described to be mediated by IRES-dependent translation. The initial study discovered that the uAUG-containing and GC-rich 5’UTR of Apaf-1 is very inefficiently translated when introduced into a capped RNA reporter but that it mediates translation of the second cistron of bicistronic DNA reporters 10 x more 73 efficient than an empty control reporter [537]. However, the study included northern blot analysis to verify reporter integrity and the blot depicted aberrant luciferase transcripts generated by all reporter plasmids used in transfection experiments, wherefore the introduction of artefacts cannot be excluded [537]. Subsequent studies reported sequential binding of UNR and PTB to be important for Apaf-1-mediated translation, as well as caspase-cleaved eIF4G2/p86 to support Apaf-1 IRES efficiency but none of the studies validated reporter assays by RNA analyses to proof bona fide Apaf-1 IRES activity [142], [196], [538]. Half a decade later Andreev et al. performed systematic tests on a group of reported cellular IRESs, including Apaf-1 and found that Apaf-1 5’UTR contains some cryptic splicing or promoter elements [67]. Despite showing the least cap-dependency of all cellular IRESs tested in monocistronic RNA reporter assays, Apaf-1 mediated translation initiation very inefficiently when placed within bicistronic reporters [67]. During inhibition of cap-dependent translation by excess 4E-BP1 or m7G, Apaf-1 monocistronic reporter was still translated with half or quarter the efficiency as under normal conditions [67]. Hence, Apaf-1 5’UTR promotes 5’ end-dependent translation initiation which is relatively less reliant on the cap structure when compared to other cellular transcripts. Further investigations revealed that the 5’ end-dependent initiation mechanism involves ribosome scanning and that a certain structural domain within Apaf-1 5’UTR is important for cap- independent translation under apoptotic conditions [149], [187]. Taken together, Apaf-1 has only low potential to promote internal translation initiation though it is efficiently mediating cap- independent but 5’ end-dependent translation, wherefore it doesn’t fit the IRES concept albeit it allows protein expression when eIF4E is limiting. Hence, Shatsky and colleagues proposed a new model of cap-independent translation involving CITEs, which have been originally described in plant viral RNAs, and which constitute an alternative to the model of cellular IRES-mediated translation. Briefly, modules of RNA structures located within the 5’UTR, like the domain II of Apaf-1, provide cap-independency by locally increasing the concentration of key translation initiation components through direct interactions which stimulates 5’ end-dependent ribosome entry and scanning [61], [187]. Another putative IRES containing mRNA reported to be translated as part of the cellular stress response is one of the key translation initiation factors itself, eIF4GI. Initial studies reporting eIF4GI IRES activity lacked proper controls to verify internal initiation at bicistronic reporters (DNA reporter assays without promoterless control or siRNA control assays, no RNA reporter assays) and found a polypyrimidine tract close to the 3’ end of eIF4GI 5’UTR to be indispensable for IRES activity [54], [55]. It turned out that the polypyrimidine tract was part of a canonical splice acceptor site and that this sequence also overlapped with a C/EBPβ binding site [53], [57], [539]. Hence, the putative IRES had strong promoter activity and was part of an 74 usually spliced out intron [53], [539]. Further, the eIF4GI sequence under investigation came from a cDNA clone that did not contain the complete eIF4GI ORF, which was unknown at that time. Just shortly after the initial eIF4GI analyses, a 5’ terminal extended variant of eIF4GI was described, which was also suggested to mediate IRES-dependent translation during poliovirus infection and also lacked some control experiments (DNA reporter assay with northern blot of low resolution) [540]. Later, the full-length eIF4GI sequence was discovered to be even further extended beyond the previous 5’end and also this eIF4GI variant was reported to contain IRES activity [541], [542]. This time, the IRES activity was suggested to be embedded in the eIF4GI ORF mediating translation of several distinct in-frame AUGs resulting in multiple eIF4GI isoforms [541], [542]. The authors invented the term of a cap-dependent IRES, which they propose most accurately describes the translation mechanism of eIF4GI [542]. Indeed, the in vitro and in vivo analyses of m7GpppG- and ApppG-capped monocistronic and bicistronic RNA reporters indicate that eIF4GI is strongly cap-dependently translated. To evaluate the IRES activity, bicistronic eIF4GI reporter activity was compared with HCV- or reverse HCV-mediated internal translation. Both controls are unsuitable to judge eIF4GI IRES efficiency. A reversed sequence is expected to yield lower levels of background translation since it usually contains AUG codons that could engage ribosomes which consequently decreases initiation at the reporter ORF [64]. In other assays of the same study, an empty vector control was used, which would have been more informative as the reversed HCV sequence in this assay. Further, one or two other cellular mRNAs without suggested IRES activity would have been useful controls to depict the range of unspecific reporter readout. Further, the HCV IRES is a relatively weak viral IRES when compared to EMCV for example [59], [64], [67]. In addition, eIF4GI-mediated translation of the second cistron of a m7GpppG-capped bicistronic RNA reporter transfected into HEK 293T cells was 100-fold less efficient than translation of the cap-dependent first cistron and introduction of a stable hairpin at the 5’ end of a bicistronic eIF4GI reporter decreased translation of the second cistron, although not as severe as translation of the first cistron [541], [542]. Hence, it is uncertain if the level of translation of the second cistron of a m7GpppG- capped bicistronic eIF4GI reporter is of physiological meaning or if it rather represents some background variation. Taken together, eIF4GI-mediated translation is dependent on the cap structure and to a lesser extent on upstream translation. Considering its natural environment as part of the coding region located downstream of an uORF that overlaps with the authentic eIF4GI start codon it might be that the putative IRES sequence is rather a “reinitiation” sequence element that adapted to maintain ribosomes in a scanning or translation competent mode after terminating uORF translation. The introduction of a stable hairpin within the

75 intercistronic space directly upstream of the eIF4GI sequence in a bicistronic reporter could help to demonstrate if eIF4GI is indeed capable to recruit ribosomes internally. Another well-known protein family, which is preferentially translated following heat stress, are heat shock proteins that act as chaperons ensuring the correct folding of proteins during the stress response [543]. Most prominent among them is hsp70, which was the first cellular mRNA postulated to be translated by ribosome shunting, an initiation mechanism in which ribosomes bind to the cap and scan discontinuously through the 5’UTR bypassing large sequence segments, uAUGs or hairpin elements to reach the initiation codon [544]. Later, the same transcript was suggested to mediate translation initiation by IRES activity, as well as by a m6A-mediated cap-independent translation mechanism [189], [311], [545]. It remains to be investigated if and how differential initiation mechanisms cooperate in hsp70 translation during normal and cellular stress conditions. However, IRES activity of hsp70 was initially not properly validated and later it was shown that (unmethylated) hsp70 5’UTR does not exhibit IRES activity in vitro and in vivo, while it can promote cap-independent translation during PI3K-mTORC1 inhibition [545]–[548]. Besides, the mechanistic basics of ribosome shunting are only vaguely understood. In case of hsp70 mRNA, which is like adenovirus mRNA sequence complementary to 18S rRNA, it was suggested that shunting might involve mRNA-rRNA interaction [544]. But direct interaction was not analyzed, and complementary elements have not been tested for necessity in translation initiation. Moreover, complementarity of hsp70 to 18S rRNA was much weaker than complementarity of late andenovirus mRNA [544]. Hence, it remains unclear how hsp70 mRNA might function in ribosome shunting. Another transcript that was described to be exclusively translated by ribosome shunting during normal as well as during distinct stress conditions is cIAP2 [549]. The 5’UTR of this apoptosis inhibitor is extremely long (~2.8 kb), contains 64 uAUGs and stable secondary structures [549]. Interestingly, some structural features like a short uORF upstream of a stable stem and a oligopyrimidine tract directly upstream of the start codon of the coding region are conserved among mammalian orthologs and closely resemble cauliflower mosaic virus (CaMV), which is also suggested to be translated by ribosome shunting [549]. When tested in reporter assays, cIAP2 5’UTR displayed neither cap-independency nor IRES activity, but translation of capped monocistronic cIAP2 reporter constructs increased by 3-fold or 2-fold following arsenite or etoposide treatment, respectively [549]. It is hypothesized that cIAP2 5’UTR folds into a stable stem-loop structure which brings both ends of the 5’UTR in close proximity in that way enabling ribosomes after translation of the uORF to shunt to the pyrimidine-rich acceptor site in order to reinitiate translation at the start codon of the coding region [549], [550]. Increased ribosome shunting during stress response might be regulated by trans-acting factors or modifications of 76 eIFs but the molecular details how cIAP2 5’UTR mediates translation initiation or how ribosome shunting works in general are not identified, yet. A non-canonical translation mechanism was also reported for cIAP1 mRNA, another member of the inhibitor of apoptosis protein family. Similarly to cIAP2, cIAP1 mRNA is unusually long (>1.1 kb), contains 23 uAUGs and stable secondary structures [551]. It was suggested that cIAP1 translation is mediated by an IRES and it was found that cIAP1 5‘UTR exhibits comparable IRES activity as HCV IRES in bicistronic in vitro reporter assays using RRL [209], [551]. However, the cIAP1 IRES is weak and mediates translation of the 3’ cistron of a bicistronic reporter with an efficiency that is three orders of magnitude lower than cap-dependent translation of the 5’ cistron [551]. Further, introduction of capped cIAP1 5’UTR in front of a reporter gene reduces gene expression by approximately 50 % and insertion of a hairpin downstream of the cap reduces reporter gene expression by another 30 % in vitro, suggesting that cIAP1 mediated translation is in general rather inefficient and that it can be stimulated to a low degree by presence of the cap structure [551]. In vivo analysis of cIAP1 requires RNA transfection, as the 5’UTR contains a splice site, and revealed that cap-dependent and 5’ end-dependent translation of cIAP1 is roughly similarly potent and depending on the model system ~10-40 % as efficient as a capped reporter translation without 5’UTR [551]. Noteworthy, the same study also shows that background levels of 3’ cistron translation in a bicistronic transcript vary noticeable depending on the intercistronic spacer region. For instance, reversed HCV IRES (477 nt) did not result in any quantifiable downstream reporter expression in HepG2, HEK 293T or HeLa S3 cells whereas a 27 nt long “empty vector” spacing region yielded downstream reporter expression of ~ 0.1-0.25 % of cap-dependent translation in the same cell lines [551]. Another control reporter containing a 590 nt long coding region of human PKR as intercistronic spacer resulted in second cistron expression of ~ 0.6 % of cap-dependent translation in HEK 293T cells, while no expression was detected in HeLa S3 cells [551]. This shows, that intercistronic control regions differentially affect levels of unspecific downstream reporter expression and that these levels can vary in between model systems. Keeping in mind that cIAP1 5’UTR is 1115 nt and mediates 3’ cistron expression with 0.01 % of the efficiency of cap-dependent 5’ cistron expression in bicistronic reporters translated in HEK 293T cells, it remains unclear if this is a significant level of internal translation initiation or rather a variation in background level expression of this assay [551]. In summary, none of the IRESs whose activity during apoptosis or cellular stress conditions is frequently cited and discussed here was convincingly verified. In case validation analysis were accurately conducted it turned out that the sequence of interest did not support the concept of bona fide internal ribosome entry but rather fits alternative models of non-canonical 77 translation initiation. Even though the IRES activity of certain mRNAs was not comprehensively demonstrated yet, follow-up studies began to report the identification of ITAFs which supposedly function in IRES regulation. One of the most studied putative ITAFs is the RNA-binding protein PTB, a splicing factor which is involved in alternative splicing and which shows cytoplasmic localization in addition to its predominantly nuclear concentration. PTB was reported to act as an ITAF on IRES-mediated translation of numerous cellular and viral transcripts, among them Apaf-1, BiP and HIFα, which are also discussed in this chapter [145]–[147], [152], [157], [552]. For example, Apaf-1, whose translation is nowadays considered to be mediated by a CITE-dependent mechanism, was reported to contain an IRES element that acquires its correct structural conformation by binding of nPTB (neurally enriched PTB isoform) and unr [142], [538]. With this the chaperone theory evolved which claims that ITAFs function as RNA chaperones by altering the secondary structure of an IRES into either active or inactive conformation thus regulating the rate of translation initiation. Accordingly it was suggested that PTB acts as a universal ITAF, after a (CCU)n motif was identified which can be bound by PTB and which was used to design a putative artificial IRES (AIRES) [143], [144]. Further, it was postulated that PTB has a pivotal role during apoptosis as the motif is present within mRNAs displaying increased relative translation efficiency in apoptotic cells and because some of these mRNAs are bound by PTB containing protein complexes whose composition is remodeled during apoptosis [41], [553]. However, when the AIRES is inserted into bicistronic reporters appropriate controls of DNA transfection assays are missing or presented in inadequate manner (RNA analysis by northern blot not suitable to detect spliced transcripts with the applied exposure and resolution, promoterless analysis presented in a scale with too big intervals to see promoterless expression levels, hairpin insertion analysis normalized to empty vector control instead of normalizing each bicistronic reporter to its hairpin containing counterpart) [143]. Also candidate mRNAs containing the (CCU)n motif were tested in bicistronic DNA transfection assays lacking RNA integrity analysis [41], [143]. In the case of CCNT1, potential PTB-regulated IRES activity was tested by use of bicistronic DNA and RNA transfections. While the relative internal translation efficiency of CCNT1 was 50- and 97-fold higher than the empty vector control in normal and apoptotic cells using DNA transfection, it was similar or weaker than the empty vector control in normal and apoptotic cells using RNA transfection [553]. At the same time EMCV-mediated translation was ~15- and 22-fold higher than translation of the empty vector control in normal and apoptotic cells using RNA transfection [553]. The investigators argue that certain cellular IRESs require a “nuclear event” and that the PTB-containing protein complex is remodeled within the nucleus of apoptotic cells so that CCNT1 IRES is expected to be activated only 78 following DNA transfection [553]. Considering that bona fide IRES activity of CCNT1 is not unambiguously verified and that no indications exist for PTB-complex remodeling to happen inside the nucleus, this hypothesis requires further validation. Another reasonable explanation for the phenomenon could be that CCNT1 contains a cryptic promoter or a splice site, which generates aberrant monocistronic transcripts following DNA transfection. This monocistronic transcripts could constitute cap-dependent reporter expression which is wrongly perceived as internally initiated and which is missing in RNA transfections, as these don’t undergo nuclear processing. Moreover, an assay of an earlier study suggests low levels of promoter activity of CCNT1 5’UTR and as PTB is known to act as a splicing factor modulating alternative exon inclusion it might promote alternative splicing of the bicistronic transcript upon induction of apoptosis [41]. Taken together, additional control experiments are needed to proof the chaperone hypothesis for PTB. This applies as well to other mRNAs that haven been suggested to promote PTB-controlled IRES-mediated translation [34]. In this regard, additional mechanisms may act during stress response and apoptosis, that contribute to the global decrease of protein synthesis and selective mRNA translation which might not be regulated by IRESs. For instance, alternative splicing of apoptotic factors generates isoforms with antagonizing functions, Apaf-1 is one of it [554], [555]. During apoptosis, broad transcriptional alterations modulate the composition of functional elements within untranslated regions of apoptotic factors which is expected to change translational efficiency and stability [531], [555]–[558]. Moreover, alternative promoter and alternative transcription start site (TSS) usage have been reported during stress response. After glucose starvation for examples, transcripts were generated with shorter 5’UTRs and transcripts with a C as initiating nucleotide were significantly more repressed compared to those starting with purines, as eIIF4E had the lowest binding affinity to those mRNAs [559]. Hence, the nucleotide composition around the TSS can contribute to the translation efficiency, especially when eIF4E availability becomes limiting. An interesting example of increased translation efficiency during stress conditions, which is independent of an IRES element, is the RNA binding protein PABP. Due to alternative promoter usage an isoform with shortened 5’UTR is expressed, which lacks an auto-inhibitory A stretch, and is therefore efficiently translated even though it contains a TOP motif that is highly sensitive to eIF4E [559]. Furthermore, the integrated stress response involves an increased occupancy of ribosomes at uORFs and 5’ends of the main ORF [69], [99], [560]–[562]. Apart from stress- induced translational regulation by uORFs this could be a sign of prolonged translation elongation or elongation pausing. It was suggested that during heat shock response the regulation of translation elongation is a major component of cellular defense and readjustment and that 79 chaperones or reduced chaperone-ribosome association in particular play major roles in elongation pausing [561]. It was also shown, that eEF2K gets activated in response to diverse cell stresses, leading to the phosphorylation of eEF2, which in turn causes eEF2 inactivation preventing it from ribosome binding and hence decreasing peptide elongation rate [563][564]. Thus, regulation of translational efficiency is not only controlled at the step of translation initiation but likely also at the step of translation elongation. In addition, regulation of mRNA degradation and stability is also involved in selective translation of the stress response, although most stabilized mRNAs are translationally suppressed [43], [565]. Furthermore, mRNA translocation to the ER can play a role in selective translational regulation at least in hypoxia [566]. Especially, genes of the HIF-1 regulated network were found to be preferentially translated at the ER following hypoxic stress [566]. Furthermore, dynamic m6A mRNA methylation in the 5’UTR was shown to play a role in translational regulation during heat shock stress. Newly synthesized transcripts after heat shock exhibited increased levels of m6A modification at their 5’ends promoting selective cap-independent but 5’-end dependent translation initiation [189], [311]. Furthermore, distinct RNA elements within 5’UTRs, other than IRESs, were reported to mediate de novo translation during stress conditions. Lately, the translation initiator of short 5’UTR (TISU) element was found to be enriched in mitochondrial mRNAs, that remain translationally active upon energy deprivation [567]. Even though TISU element-mediated translation initiation is cap-dependent, it conferred stress-resistance due to the cooperation of eIF1 and eIF4GI in translation initiation and start codon discrimination, which favors TISU- directed initiation over canonical translation initiation during AMPK-mediated translation inhibition [567], [568]. As the 5’UTR of TISU mRNAs is very short (median length of 12 nt) the eIF4F complex and the 48S initiation complex are expected to overlap at the AUG [568]. Further, the cap as well as the 5’UTR will be located within the ribosomal exit channel during 80S formation. Experimental evidences support a model in which eIF4F is evicted during 48S initiation complex formation leading to the dissociation of eIF4GI and eIF1 [567]. By this means, TISU mRNAs exhibit a distinct eIF-dependency which differs from canonical cap-dependent translation initiation and which is why they maintain their translation efficiency upon altered eIF availability during distinct cellular stresses. Another RNA sequence element, that was suggested to mediate translation of the NRF2 transcription factor under oxidative stress is a G-quadruplex structure [569]. A short RNA fragment of NRF2 5’UTR closely resembling the consensus G-quadruplex sequence was shown to be bound by eEF1A in a H2O2 dose- and time-dependent manner and proposed to stimulate NRF2 translation initiation [569]. However, bicistronic reporter assays were conducted by DNA 80 transfection and required control experiments were missing which leaves the question open whether NRF2 5’UTR contains a cryptic promoter or splice site instead of a G-quadruplex- dependent translation mechanism [569]. Taken together, a variety of regulatory mechanisms are in play that shape gene expression in response to cell stress and apoptosis on transcriptional and post-transcriptional level. While some of the most prominent transcripts originally reported to be regulated via IRES-mediated translation require more in-depth validation, some of them have been shown to be regulated by alternative non-canonical translation mechanisms in response to distinct stress conditions. Continued development and progress of molecular methodologies to study post-transcriptional gene regulation might uncover even more yet unknown mechanisms and elucidate their contribution to global and gene-specific translation regulation under cell stress.

1.4.3 Cancer and cell transformation Cancer cells emerge by neoplastic transformation during which control mechanisms of physiological cell processes get irretrievably impaired, resulting in abnormal cell proliferation, malignant tumor formation and potentially metastasis. Often genomic abnormalities are an underlying cause of transformation. Systematic large-scale analyses revealed that a typical tumor exhibits ~60-90 DNA mutations, which lead to changes on the amino acid level and that less than 15 of these account for tumor initiation or maintenance [246], [570]. Hence, carcinogenesis was conventionally related to genomic alterations and changes of the transcriptome but nowadays, it became more and more apparent that translational dysregulation is a hallmark of cancers which eventually brought about the provocative hypothesis that all tumors ultimately originate from translation initiation dysfunction and should therefore be named translationopathies [571]–[573]. Indeed, the earliest features identified distinguishing cancer cells from normal ones, were enlarged nucleoli, which are distinct cellular regions within the nucleus where rRNA is transcribed and ribosomal subunits are assembled [574]. Upregulation of ribosome biogenesis is a prerequisite to increase global protein synthesis, which in turn is required to drive rapid cell proliferation, as is the case with cancer cells [251], [575]. Thus, it is currently an open question how oncogenic signaling, ribosome biogenesis and translation initiation are interconnected during tumor development and whether overexpression or loss of specific eIFs or ribosomal proteins, cell growth and quantitative or qualitative changes in translation levels constitute a cause or a consequence of distinct steps in neoplastic transformation [573]. Evidence for the pivotal role of translational dysregulation in cancer is coming from diverse scientific and clinical findings. For instance, the gain or loss of function of individual 81 eIFs, especially eIF4E, is a common feature of various kind of tumors and eIF expression or phosphorylation levels are involved in cancer development or progression and are sometimes used as prognostic marker to predict cancer outcome [572], [576]–[578]. Furthermore, signal transduction pathways directly modulate translation factor activity and mutations resulting in oncogenic signaling of these pathways controlling the translational machinery are most commonly found in human cancers [251], [573]. Additionally, recent findings of ribosome heterogeneity and consequent specialized translational activity re-emphasized the role of the ribosome itself in translational gene regulation in general as well as during cancer development [128], [579]. Already two decades ago, ribosomopathies, inherited human syndromes caused by mutations of ribosomal proteins or enzymes involved in ribosome biogenesis, were found to be accompanied by increased cancer susceptibility, clearly demonstrating a link between the translational machinery and cellular transformation [256], [580]. For example, in X-linked dyskeratosis congenita (X-DC) a mutation of the DKC1 gene, which catalyzes the pseudouridylation of ~100 distinct uridine residues within rRNA, leads to decreased translation of a subset of mRNAs reported to be IRES-dependent, including the tumor suppressors p53 and p27, thereby contributing to tumor formation [573]. At the same time, global protein synthesis is largely undisturbed [573]. Such qualitative and quantitative changes of the translatome are a common motif in translational deregulation in cancer which is not only associated with ribosome dysfunction but also with the hyperactivation of signaling pathways and oncogenic eIF expression. The subset of mRNAs showing disproportional changes in translation efficiency was classically termed ‘eIF4E- sensitive’ and often encodes key factors modulating cell growth and proliferation and other proto-oncogenes like for example ODC, CCND1, c-myc, or VEGF [262], [267], [581]–[584]. These transcripts are characterized by long 5’UTRs displaying a high degree of secondary structure which is the reason why they are thought to be particularly sensitive to eIF4E levels, as eIF4E not only facilitates the recruitment of eIF4A and eIF4F assembly but also stimulates eIF4A helicase activity independent of cap-binding thus selectively enhancing translation efficiencies of ‘weak’ mRNAs with complex 5’UTR structures [272], [585]–[587]. Today’s technological advances enabled system-wide investigation of the translational program of cancer cells by ribosome profiling or polysome fractionation followed by microarray analysis. Such studies uncovered that short linear sequence motifs within 5’UTRs like 5’-TOP, PRTE and cytosine-enriched regulator of translation (CERT) as well as structural motifs like G- quadruplexes can promote selective translational regulation of mRNA subsets in response to oncogenic signaling within transformed cells [79], [269], [280], [588], [589]. How these sequence

82 elements mechanistically function in specialized mRNA translation is largely unknown and remains an exciting venue for future researches. So far it is known, that the pool of translationally regulated mRNAs carrying the afore mentioned motifs is specifically dependent on the function of a certain eIF. While 5’TOP, PRTE and CERT carrying mRNAs are sensitive to eIF4E activity, mRNAs with longer and more structured 5’UTRs exhibiting guanine quartet motifs which can form G-quadruplexes are particularly sensitive to eIF4A activity. Intriguingly, a large part of 5’TOP and PRTE containing mRNAs is redundant and exhibit both motifs, whereas CERT carrying mRNAs hardly overlap with 5’TOP and PRTE containing mRNAs [269], [280]. Also, the pool of eIF4A sensitive mRNAs identified in two recently published datasets shows some diversity, inasmuch as classically studied mRNAs like c-myc or CCND1 were identified in both of them, whereas otherwise both datasets show only little overlap of eIF4A-dependently regulated transcripts [588], [589]. Hence, it seems that short linear motifs or structural elements within 5’UTRs can render mRNAs susceptible to the activity of a distinct eIF and that within this pool of eIF-sensitive mRNAs a variety of different elements exist that can in a combinatorial manner allow for robust and precise differential regulation of smaller subgroups of transcripts. Further, it is tempting to speculate that these elements promote translation initiation through interaction with trans acting factors that might as well be regulated by cell type-specific and/or stress-induced means to facilitate translational fine-tuning as well as target recognition. It is unclear, to which extent global translational upregulation and the upregulation of a subset of specific mRNAs is contributing to tumor development and progression. However, it is well known that during cancer development transformed cells are exposed to varying types of cellular stresses inside the tumor microenvironment, which differentially affect the translational efficiency of individual transcripts. For instance, defects of the tumor vasculature usually cause hypoxic stress as well as nutrient deprivation, while a high frequency of cell division and therapeutic treatments typically lead to genotoxic stress and an increase in the production of reactive oxygen species (ROS) [256], [590]. In response to such hostile conditions, cells reduce overall translation rates, which is predominantly regulated at the initiation step, and selectively synthesize proteins required for stress adaption and cell survival by use of alternative translation initiation mechanisms [13], [20], [497], [590] . For example, in both normal and tumor cells severe hypoxia leads to a decrease in global translation of 40-60 % just one hour after treatment, while glucose starvation can reduce overall protein synthesis by up to 50 % and genotoxic stress following UV irradiation can induce a decrease of translation rates around 35-70 % [590]–[592]. At the same time, translation of selective mRNAs is enhanced. For instance, during hypoxia a few dozen transcripts with 83 increased translation efficiency were identified including VEGF and NDRG1 and many targets of HIF-1 which facilitate adaption to hypoxic stress [44], [590], [593]. Interestingly, a selective cap-dependent translation initiation mechanism was described that mediates protein synthesis of target mRNAs by RNA hypoxia response element (rHRE) recognition through the HIF-2α/RBM4/eIF4E2 complex during hypoxia [594]. Such eIF4E2- dependent transcripts include for example EGFR, which is involved in the progression of non- small cell lung cancer and pancreatic cancer as well as several other malignancies [595]–[597]. Modelling of EGFR activation and inactivation mechanisms suggested that at supraphysiological levels of EGFR and/or EGF, which are frequently found in human tumors, EGFR phosphorylation and ubiquitination are uncoupled indicating an intrinsic weakness of the regulatory system that can be exploited by malignant cells to promote proliferation [598]. Given that EGFR translation is preserved by an alternative cap-dependent initiation mechanism during hypoxia, cancer cells might still be able to maintain a proliferative advantage under hypoxic conditions thus hijacking the translational reprogramming by eIF4E- to eIF4E2-mediated translation initiation of the adaptive stress response to hypoxia [594]. Indeed, a subset of growth factors, tumor suppressors and oncogenes are assumed to be encoded by dual mechanism transcripts. These can be translationally upregulated in the context of elevated eIF4E levels by cap-dependent translation as well as in the absence of eIF4E by cap- independent translation [251]. Walters et al. compiled a list of 23 potential IRES-containing mRNAs playing whose dual translation initiation modes might play important roles in tumor progression and survival (Tab. 3) [599].

Tab. 3: IRES-containing transcripts with important functions in tumor cells. Adapted from Walters et al., 2016 [599].

Gene Reference Comment AML1/Runx1/MTG8a [600] MtG8s without IRES activity in RRL in vitro assay: bicistronic MTG8a reporter shows similar internal translation efficiency as bicistronic reporter of empty pRF control vector [143] Apaf-1 [537] Apaf-1 was shown to mediate CITE-like translation initiation which is cap-independent but 5’end- dependent [67], [187] Cat-1 [154] see Tab. 1 c-IAP1/cIAP1 [551] mediates internal translation initiation with 0.01 % of the efficiency of cap-dependent translation of the

84

same bicistronic reporter in HEK 293T cells transfected with RNA [551] cyp24a1 [601] EGR2 [602] EGFR [603] selective cap-dependent translation by rHRE recognition by HIF-2α/RBM4/eIF4E2 complex during hypoxia [594] Hox [604] bicistronic DNA reporter assays generate truncated reporter transcripts, wherefore RNA reporters are required for unambiguous validation of potential IRES activity [604] Hif1α [526] see Tab. 1 c-Jun [605] c-Jun was found to be translated by an eIF3- dependent but eIF4F-independent translation mechanism [190] c-myc [474], [606] see Tab. 1 and Tab. 2 l-myc [607] l-myc internal translation was studied by bicistronic DNA reporter assays including as a control a northern blot analysis with unclear probe lacking further validation experiments [607] n-myc [608] n-myc internal translation was studied by bicistronic DNA reporter assays including a RNase protection assay designed to detect intact full-length bicistronic reporter rather than truncated transcript lacking further validation experiments [608] p16INK4a/CDKN2A [609], [610] p27 [611], [612] see Tab. 1 and Tab. 2 p53 [164] see Tab. 1 was described to be partially dependent on RPS25 [127] p120 [613] SNAT2 [614] SNAT2 is cap- and 5’end-independently translated in in vitro translation assays, translation activity dependent rRNA methylation [70]

85 c-src [62] potential IRES activity of c-src requires further validation (for details see section 1.2.2) SREBP-1a [615] SREBP-1a inhibits translation of downstream reporter in RRL in vitro translation assay, DNA reporter assays with northern blot as control [615] VEGF [616]–[618] VEGF sequence contains a cryptic splice site or a cryptic promoter [618] XIAP [528], [530] XIAP sequence contains a cryptic splice site [65], was described to mediate cap-independent eIF5B- dependent translation when eIF2α is phosphorylated during stress and IRES translation via eIF3-PABP during physiological conditions [536], [619] Zeb2 [620]

The listed dual mechanism mRNAs are often translationally misregulated within cancer cells and IRES elements within their 5’UTRs are thought to allow for increased translation under stress conditions of the tumor microenvironment in addition to increased translation due to altered cap-dependent translation under pathophysiological conditions of cancer cells. Some of these potentially IRES-containing transcripts have not been comprehensively validated to date and some have been reported to be regulated by alternative non-canonical translation initiation mechanisms which are not dependent on internal ribosome entry. One interesting example is p53 whose potentially IRES-mediated translation is crucial during oncogene-induced senescence (OIS) which constitutes a barrier in the process of neoplastic cell transformation. During OIS a switch from cap-dependent to cap-independent translation was proposed to induce p53 translation, which in collaboration with other cell-cycle inhibitors induces cell-cycle arrest [119], [495], [573]. While initial reports on the IRES activity of p53 require further validation to unambiguously proof internal initiation, further indications for translational regulation of p53 through specialized ribosomes were found: translation of p53 is impaired in in a mouse model of X-DC in which the DKC1 gene that encodes for an rRNA modifying enzyme is mutated and in HeLa cells with stable KO of RPS25 [119], [127], [164], [165]. Although potentially not IRES-driven, p53 is selectively translated during OIS and ribosome composition or rRNA pseudouridylation might play a regulatory role in the translation initiation process. In general, the tumor suppressor p53 is the most frequently altered gene in human cancers and constitutes a good example for the complexity and delicacy of the multistep process 86 of neoplastic cell transformation and the role of translational regulation therein [621]. So, p53- mediated control of senescence and transformation depends among others on the translational regulation by 4E-BPs [622]. 4E-BP deficient but p53 expressing cells undergo premature senescence rendering them more resistant to oncogene driven transformation than wt cells [622]. Contrary, p53 deficient but 4E-BP expressing cells are more prone to neoplastic transformation than wt cells [622]. However, knockout mice, lacking 4E-BP and p53 show even higher incidences of tumor development than mice lacking p53 alone [622]. Thus, synergistic effects of elevated eIF4E activity caused by 4E-BP inactivation combined with the loss of p53 function increase tumorigenesis, demonstrating how the interplay of loss-of-function and gain-of-function alterations raise cancer development rates above the rates caused by single alterations only. Further, translational control by eIF4E activity can play an important role in attaining such synergistic effects. In accordance with this, Pelletier et al. hypothesizes that eIF4F activity is crucial for cellular fitness of cancer cells and that targeting eIF4F activity shifts the translatome of cancer cells out of the Goldilocks zone thus destroying proteastasis and consequently the proliferative advantage of cancer cells [274]. Here, the Goldilocks zone describes the cell state in which the translational output optimally supports tumor homeostasis. As already mentioned earlier, it was found that increased expression levels of eIF4E (~2- to 3-fold) increase cellular transformation [274]. On the other hand, reduction of eIF4E levels to 50 % significantly impede cellular transformation, while normal cell development is unimpaired [280]. The fact that eIF4F activity is limiting during oncogene-induced transformation but not under physiological conditions opens up the possibility to specifically target translational processes of cancer cells without affecting healthy cells of the surrounding. This working principle is also referred to as ‘target window’ or ‘achilles heel’ of tumor cells and can be expanded to targeting not only eIF4F but also the translational machinery as well as ribosome production [574], [575], [586], [623]. This strategy has not only the potential to enable the treatment of various cancer types through the same drug, it also has the potential to target driver cells within a single tumor at the same time overcoming the intra-tumor heterogeneity [624]. One of such strategies to treat cancer uses anti-sense oligonucleotides (ASO) targeting the eIF4E transcript. It was found that the eIF4E-ASO reduced eIF4E expression, as well as the expression of eIF4E-dependent proteins like VEGF and induced apoptosis in cultured human breast cancer cells as well as in mice xenograft models, while global protein synthesis was only slightly affected and xenograft-bearing mice did not show any signs of illness [271]. In fact, eIF4E-ASO reduced eIF4E expression by up to 80 % in normal liver tissue without affecting liver weight or liver transaminase levels, while a reduction of eIF4E expression by 56-64 % 87 significantly limited tumor growth due to increased tumor cell apoptosis [271]. Phase I clinical studies on eIF4E-ASO efficacy were promising, demonstrating that eIF4E-ASO can safely be administered to patients, resulting in eIF4E mRNA and protein reduction within tumors, but Phase II clinical trials were discontinued [274], [625]. Another example is the small-molecule inhibitor 4EGI-1, a synthetic peptide that on the one hand upon binding to eIF4E causes the dissociation of eIF4G by an allosteric mechanism and on the other hand stabilizes the association of eIF4E with 4E-BP1 [626]–[628]. Hence, 4EGI-1 inhibits preferentially cap-dependent translation initiation [626]. Moreover, 4EGI-1 was found to have more profound effects on transformed cells than on untransformed cells, reducing especially the translation of mRNAs that regulate core oncogenic pathways including some 5’TOP and PRTE-containing mRNAs [626], [629]. Tumor suppressive effects were successfully demonstrated for several cancers [629]–[633]. Earlier cancer treatment approaches focused on the inhibition of mTOR signaling, which is frequently hyperactivated in cancers and integrates diverse environmental signals to regulate key cellular processes including cell proliferation by control of ribosome biogenesis and global protein synthesis. However, rapamycin, an allosteric mTOR inhibitor, does not efficiently inhibit mTORC1-mediated phosphorylation of 4E-BP and potentiates mTORC2-mediated activation of Akt due to the loss of a negative feedback mechanism [634]–[637]. Thus, naturally occurring rapamycin and its rationally designed analogs (rapalogs), where found to have only limited success in most anticancer monotherapies despite a small subset of cancers like for example renal cell carcinoma [250], [638], [639]. Hence, a second generation of mTOR inhibitors was developed, the ATP active-site inhibitors (asTORi), which were found more powerful anticancer agents than rapamycin in preclinical studies [245], [258], [259], [269]. However, it was already shown that the efficacy of asTORi relies on the eIF4E-4EBP axis and that an increase in the ratio of eIF4E/4E-BP can induce cancer cells to acquire asTORi resistance [253], [264]. These data suggest that in the future a combination of anticancer drugs with different mechanism of actions and different targets respectively, might constitute an efficient strategy in overcoming present drawbacks and developments of resistances in cancer treatment [640]. Taken together, translational cap-dependent and cap-independent regulation is crucial during neoplastic transformation and cancer homeostasis. Changes in gene expression on the translational level can exceed by far changes on the transcriptional level [641], [642]. Hence, translational regulation emerges as a promising target in cancer therapy and a number of therapeutic strategies exploit the potential to interfere with misregulated mRNA translation by targeting signaling pathways, eIFs, putative IRES sequences and ITAFs for application in cancer therapy [246], [571], [643], [644]. 88

1.4.4 Stem cells and differentiation Embryonic stem cells require a tight coordination of gene expression to maintain pluripotency and to undergo self-renewal or differentiation into specialized cell types in precise spatiotemporal manner during development. Extensive work has been done to uncover the underlying mechanisms of epigenetic and transcriptional regulation in stem cell maintenance and differentiation. Much less is known about mechanisms of translational control. However, recent reports indicate that the complex remodeling of gene expression in differentiating cells involves substantial translational regulation. In general, ESCs maintain protein synthesis and cell metabolism at low levels, especially in the quiescent state, to advance longevity and provide optimal protection of their DNA against ROS for instance [645]. Hence, ESC activation and differentiation is characterized by upregulation of translation, which is associated with morphological changes like an increase in cytoplasmic volume, rough endoplasmic reticulum and content of Golgi apparatus [91], [645]– [649]. Apart from a global increase in translation, also transcription is substantially increased during ESC differentiation [646], [650]. Hence, most of differential gene expression between stem cells and stem cell progeny is the result from regulation at both mRNA expression and translational level. While in differentiating ESCs genes are mostly concordantly upregulated in transcription and translation, genes of more terminal differentiation systems can be discordantly regulated. For instance, during differentiation of hepatic progenitors to hepatocytes transcription is largely downregulated while translation of specific genes is upregulated [651]. Contrary, during differentiation of myoblasts to myotubes a substantial amount of genes is differentially expressed on RNA level while the level of translation is unchanged [652]. Hence, translational regulation can both amplify and dampen the effects of differential gene expression between differentiating cells. So far, only a few gene regulatory subgroups which are selectively regulated by translation have been further investigated. One example are genes which are involved in translation, including ribosomal proteins, that are translationally repressed in early neuronal progenitors or during EB formation even though their mRNAs are highly expressed [91], [647]. Later, translation of many ribosomal proteins is reactivated in early neuronal cells [647]. As mTORC1 is a major regulator of protein synthesis and translation of 5’ TOP mRNAs, which are enriched for genes involved in translation, mTORC1 activity and phosphorylation of its primary targets 4E- BP and S6K1 were investigated. In hESCs, Blair et al. found levels of 4E-BP1, S6K1 and RPS6 to be elevated irrespective of their phosphorylation status when compared to neural progenitor cells (NPCs) or neuronal cells and suggested that mTORC1 signaling causes high translational levels of translation-related genes in hESCs [647]. However, a larger part of studies made 89 different observations according to which ESCs and neural stem cells (NSCs) display low levels of phospho-4E-BP1 that increase during differentiation and mTORC1 activation [646], [653]– [655]. Whether selective translational regulation of translation-related genes and ribosomal proteins is specifically mediated by mTOR signaling and phospho-4E-BP1 requires further investigation. However, it is evident that mTOR activity and its regulatory targets 4E-BP and S6K1 play important roles in stem cell maintenance and differentiation. In hESCs activation of S6K1 induces cell differentiation and in NSCs hyperactivation of mTORC1 and subsequent 4E-BP repression drives premature cell differentiation at the expense of self-renewal whereas mTORC1 activation of S6K1/S6K2 is regulating cell size [654], [655]. 4E-BPs further play a dual role during reprogramming of MEFs into induced pluripotent stem cells (iPSCs). When 4E-BP1 and 4E-BP2 expression is lost, reprogramming is impaired due to increased translation of p21 which acts as a major suppressor of somatic cell reprogramming [653]. But when in addition to 4E-BP1 and 4E-BP2 also the p53/p21 pathway is inactivated, reprogramming of triple KO MEFs is greatly enhanced due to increased translation of endogenous Myc and Sox2 pluripotency factors [653]. In HSCs, deficiency of 4E-BP1 and 4E-BP2 alone or in combination significantly increases protein synthesis and leads to impaired self-renewal, whereas no significant effects can be detected in other hematopoietic progenitors [656]. Another study of HSCs expressing a mutated RPL24 ribosome subunit or a Pten deletion resulting in either 30% decrease or increase in protein synthesis could show that perturbed protein synthesis in either direction impairs HSC function and that restoring protein synthesis levels also restores HSC function [649]. Further, it was shown that inhibition of mTOR in primary mESCs induces a pluripotent cell state as seen in diapaused blastocysts which is characterized by reduced phospho-4E-BP1, suppressed translation and remarkably repressed transcription [657]. In mESC culture systems, LIF is indispensable for mESC maintenance as LIF withdrawal results in mTOR activation, indicated by 4E-BP1 and S6 phosphorylation which leads to decreased expression of pluripotency genes like Oct-4, Nanog and Klf4 promoting cell differentiation [658]. While LIF can maintain mESCs in a pluripotent state in the absence of feeder cells, LIF cannot substitute for feeder cells in hESC culture [659]. Nevertheless, mTOR signaling is the driving force of pluripotency and long-term undifferentiated growth in hESCs by preventing mesoderm and endoderm activities [660]. Hence, translational control, in particular the right balance in global translational suppression but sufficient translational activation of key regulators is crucially important for stem cell maintenance and differentiation. Whether the upregulation of protein synthesis during stem cell activation is a consequence or a cause of differentiation remains elusive [645]. Regardless of which is first, mTOR signaling and global perturbation of protein synthesis 90 have been implicated in disease development like Tuberous Sclerosis Complex, myotonic dystrophy type 1 and schizophrenia in ES-derived NSCs and hiPSC-derived NPCs [661]–[663]. While translation is globally increased during ESC differentiation, investigations of transcript-specific translational regulation revealed that uORF translation is at the same time substantially decreased. uORFs are frequently found in protein coding transcripts and 73 % and 41 % of the annotated transcripts expressed in mESCs contain one or more uORFs starting with a CUG or AUG start codon respectively [664]. It is estimated that at least 10 % of these uORFs, mainly the ones starting with canonical AUGs, are actively translated [664]. A typical transcript displays a 25 % decrease in 5’UTR translation compared to the CDS during differentiation, though [91]. Key pluripotency factors like Myc and Nanog and other genes with regulatory functions in stem cell identity like Lin28b, Trim71 and Dicer1 contain uORFs, which might function as control elements in the translational fine tuning of protein output to maintain stem cell function [91], [664]. In line with this, another study in mESCs found that a subgroup of transcripts with translated uORFs is derepressed in response to NMD inhibition by Upf1 KD and that this subgroup is enriched for transcriptional regulators [317]. This suggests that uORF- dependent translational control is associated with NMD which might specifically regulate transcription factor expression in mESCs. Furthermore, transcript-specific analyses revealed that during ESC differentiation changes in gene-level translation are mainly generated by the expression of alternative transcript isoforms rather than by differential translation of constant transcript isoforms [647]. It has been described earlier that individual transcript isoforms can vary in their translation efficiencies and that untranslated regions play a major role in this regulation [665], [666]. In ESCs and NPCs for example, 10 % and 8 % of detected splice variants were differentially associated with polysomes and 84 % and 95 % of them vary in their UTRs [667]. While long 5’UTRs are associated with low translational levels in all cell types during neuronal differentiation (hESCs, NPCs, early and late neuronal cells), long 3’UTRs are preferentially associated with low levels of translation in neurons [647]. Also, relative 3’UTR length is increased in neurons compared to stem cells and precursors [647]. Taken together, translational regulation during stem cell differentiation is hierarchically organized on global and transcript-specific levels. Furthermore, UTRs have a prominent role in translational control and their function is dynamically regulated on transcript level as well as in cell type-specific manner. A great example of 5’UTR isoform-dependent translational regulation of stem cell self- renewal and differentiation was described for the transcription factor Yy2. Yy2 was found to be the most translationally increased gene in 4E-BP1 and 4E-BP2 double KO mESCs, although global translation was widely unchanged [668]. Its overexpression reduced the expression of 91 pluripotency factors Nanog, Myc and Oct4, whereas its depletion induced apoptosis [668]. Hence, tight control of Yy2 expression is required for mESC maintenance. Interestingly, Yy2 mRNA is expressed in two 5’UTR variants, which both contain an intronic sequence that can either be retained or spliced out. Intron retention is mediated by PTBP1 and regulated in a developmentally and tissue-specific way, with reduced retention upon differentiation of mESCs [668]. Translation of the intron-less variant of Yy2 is markedly more efficient, than translation of the intron containing variants and remains insensitive to 4E-BP activity [668]. In such a way, PTBP1-dependent alternative splicing generates efficiently translated Yy2 isoforms that drive stem cell differentiation, while intron containing Yy2 isoforms are lowly translated in the 4E-BP background of undifferentiated stem cells thereby modulating the gene expression of pluripotency factors. uORF-dependent translation is often regulated by eIF2α phosphorylation during stress conditions when global translation is decreased. However, eIF2α phosphorylation is unchanged in differentiating mESCs and due to the global increase in translation rather unlikely to be mechanistically involved in uORF-dependent translational control of stem cells [646]. But contrary to ESCs, phospho-eIF2α was reported to activate IRES-mediated translation in the differentiation of chronic myelogenous leukemia K562 cells into megakaryocytes [669]. Indeed, phospho-eIF2α was substantially increased in the differentiated cells, while protein synthesis was reduced by 40 % [669]. Otherwise, the claim that IRES-mediated translation is activated during megakaryocyte differentiation requires further validation as IRES-mediated translation was investigated by use of DNA transfection of bicistronic reporters lacking some necessary controls. While an individual endogenous gene, PDGF2, with candidate IRES activity was analyzed, global cap-dependent translation during differentiation was not systematically evaluated [669]. Moreover, PDGF2 was also intensively studied by Elroy-Stein and colleagues who claimed that PDGF2 is the first identified cellular IRES to be activated by a physiological signal, inventing the term differentiation-linked IRES (D-IRES) [670], [671]. Nevertheless, the proposed differentiation-induced IRES activity of PDGF2 remains to be unambiguously confirmed, as bicistronic reporter assays lacked some validation experiments and as the vaccinia T7 hybrid system used in earlier studies is not well suited for IRES translation studies due to the virus encoding ~200 genes potentially effecting the cellular translation machinery and generating extremely high reporter expression levels both likely to create artificial situations [132]–[134]. In addition, PDGF2 contains three uORFs in its 5’UTR which could exert translational regulation and represent potential functionally active sequence elements within PDGF2 as an alternative to the proposed IRES [669].

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Apart from megakaryocyte differentiation, cap-independent translation and non-canonical translation mechanisms were suggested to promote selective protein synthesis during diminished global translation in ESCs, facilitating fate decision and ESC differentiation. Several research groups focused their analyses on the role of eIF4G2, a homologue of eIF4GI which lacks the binding site of the cap-binding protein eIF4E and which was reported to regulate cap- independent translation during mitosis, apoptosis and cellular stress conditions as well as IRES- dependent translation of its own transcript [197]–[199], [206]. It was found that homozygous deletion of Eif4g2 caused embryonic lethality in null mutated mice and morpholino treated zebrafish [201], [203]. Further investigations of Eif4g2 KO mESCs revealed normal growth and translation rates but impaired mESC differentiation due to elevated transcription of retinoic acid (RA) -responsive genes identified by cDNA arrays [203]. Later, ribosome profiling enabled more sophisticated analysis of Eif4g2-regulted genes and identified also translationally regulated genes like Map3k3 and Sos1, which are upstream regulators of Erk1/2 in the MAPK signaling pathway [672]. Together this suggests that eIF4G2 selectively regulates translation of proteins promoting cell differentiation which then likely induce transcription of RA-responsive genes further advancing the cell differentiation process and embryonic development [203], [672]. How eIF4G2 preferentially regulates translation of Map3k3, Sos1 and a dozen further targets is unknown yet. However, it was found that eIF4G2 forms exclusive protein complexes with eIF2, FMR1, FXR1 and PRRC2A/B in addition to its interaction with eIF4A and eIF3 that are both shared binding partners with eIF4G [672]. As some of the uniquely interacting RBPs of eIF4G2 were also found in association with polyribosomes it was hypothesized that eIF4G2 forms specialized translational complexes that mediate non-canonical translation of its targets [672]. Furthermore, cap-independent translation and its regulation by eIF4G2 was also investigated in hESCs. eIF4G2 KD hESCs show a similar phenotype to Eif4g2 KO mESCs like normal cell growth and proliferation under regular culture conditions, but impaired cell differentiation due to elevated expression of pluripotency factors Nanog and Oct4 [204]. RNA- Seq of polysome-associated mRNAs revealed that 122 out of 13,200 detected genes showed selective translational upregulation by eIF4G2, with the two GO annotation clusters oxidative phosphorylation/mitochondria and ribosome/translation especially enriched [204]. Interestingly, eIF4G2 target genes in hESCs do not overlap with eIF4G2 target genes in mESCs. Discrepancies could be explained by differences in between organisms and methodologies applied (polysome profiling vs. ribosome profiling; gene KD vs. gene KO) and the fact that Sugiyama et al. published only the 14 top target genes of the in total 124 identified upregulated genes which might well contain some overlap [204], [672]. The results suggest that eIF4G2 is part of an autoregulatory loop, controlling synthesis of the translation machinery which is required for 93 increased translation during differentiation. Further, eIF4G2-regulated genes are involved in mitochondrial oxidative respiratory activity, which is as well required for increased metabolism and energy supply during cell differentiation. Yoffe et al. speculate that such eIF4G2-dependent translation is cap-independently mediated. However, out of seven tested target genes only HMGN3 shows cap-independent monocistronic reporter translation in RRL, which is ~1.5-fold of an empty vector used as negative control [204]. In vivo reporter assays in eIF4G2 KD hESCs lack controls (positive control reporter, hairpin reporter carrying cap-analog, reporter without hairpin carrying cap/cap-analog to evaluate cap-dependency and 5’ end-dependency), while reporter expression in RRL is weak and the RRL in vitro translation system known for its reduced cap-dependency. Further, raw values or unnormalized expression levels are missing, which is why the efficiency of eIF4G2-mediated cap-independent translation is unclear requiring further verification even though direct eIF4G2 interaction with HMGN3 was confirmed by electrophoretic mobility shift assay (EMSA) [64], [204]. In addition, reduced HMGN3 reporter expression in eIF4G2 KD hESCs could be a secondary effect of reduced translation of ribosomal proteins and other translation related genes. Taken together, eIF4G2 regulates protein synthesis of a subset of genes required for pluripotency and cell differentiation in ESCs. It further regulates transcription of gene regulatory networks promoting differentiation, which makes it difficult to distinguish pure translational regulation from secondary/combined effects on gene transcription. Furthermore, the functional mechanism of eIF4G2-mediated translation remains elusive. Likewise, a study in human hematopoietic UT7-mpl cells suggests that formation of specialized eIF4G complexes drives selective translation in megakaryocyte differentiation. In detail, it was shown that during megakaryocytic differentiation protein synthesis increases while the eIF4F complex is selectively remodeled displaying in addition to eIF4GI association increased recruitment of eIF4GII and phospho-eIF4E without a concomitant change in total amounts of eIF4E [673]. Furthermore, 4E-BP1 is released from eIF4F even though 4E-BP1 expression and phosphorylation levels are constant [673]. Also, total eIF4GI and eIF4GII expression levels are unchanged [673]. In contrast to eIF4G2, eIF4GII shares higher sequence identity with eIF4GI (~50 % vs. 29 %) and contains the eIF4E binding site [674]. Hence, the differentiation specific recruitment of eIF4GII to assemble into eIF4F complexes might as well be regulated through eIF4E. eIF4E itself is largely phosphorylated within the eIF4F complex and its phosphorylation is regulated by MNK1 that is recruited together with eIF4GII, wherefore eIF4E phosphorylation is likely happening after eIF4F complex assembly [673], [675]. How eIF4E phosphorylation effects cap-dependent translation is still not well understood because its activity was linked to both global translational increase and decrease depending on the cellular conditions analyzed [273]. However, phospho-eIF4E shows reduced affinity for the cap and polysome microarray 94 analysis revealed that phospho-eIF4E selectively increases the translation efficiency of a subset of 35 mRNAs which encode pro-tumorigenic proteins, whose translation is preferentially required for tumorigenesis [512], [513]. Taken together, it seems plausible that different variants of eIF4G can form translation competent complexes promoting the selective translation of mRNAs required for cell differentiation. Translation initiation of these mRNAs might not necessarily be cap-independent, as eIF4E could be involved in complex assembly and modified phospho-eIF4E could add another layer of specification. The N-terminally truncated variant eIF4G2 which misses an eIF4E binding site can bind its target mRNAs directly or might bridge the direct mRNA interaction through binding of specific RBPs, which in turn could interact with the cap-structure [676]. By now, it is not verified how selectivity is mechanistically established, but based on accumulating evidence it is hypothesized that cellular mRNAs differ in their dependency on eIF4E and eIF4G and the specific variants thereof, which results in preferential recruitment to ribosomes with regard to concentration and modification of these initiation factors under certain cellular conditions [111]. Thus, selective translation of genes that facilitate differentiation is most likely regulated by differential 5’UTR affinities to key initiation factors of the eIF4F complex and its homologues which can be modified by protein-protein interactions (enhanced affinity of eIF4E to the cap by eIF4G) or post-transcriptional modifications (reduced affinity of eIF4E to the cap by eIF4E phosphorylation) [677], [678]. Recently another cap-dependent translation initiation process has been discovered which is independent of the canonical eIF4F complex but is mediated by eIF3 instead. Although this mechanism was observed in HEK 293T cells under normal culture conditions it might as well play a role in stem cell maintenance and differentiation as translation of c-JUN and BTG1, two regulators of cell proliferation, growth and differentiation, were found to be regulated through eIF3 [190]. eIF3 is a multi-subunit translation initiation factor that contains 13 individual proteins (eIF3a-m) hence being the largest eIF that is with 804 kDa roughly half of the mass of the 40S ribosomal subunit [679]. It is importantly involved in 43S preinitiation complex formation, which comprises the small ribosomal subunit, eIF1, eIF1A, eIF3, likely eIF5 and the ternary complex composed of eIF2-GTP-Met-tRNA [679]. During canonical translation initiation it directly interacts with eIF4G to recruit the 43S preinitiation complex to the mRNA 5’ cap [680]. However, Lee et al. could show that RNA-binding subunit eIF3d contains a cap-binding domain that likely is remodeled during eIF3 complex formation allowing selective mRNA 5’ end recognition and specialized eIF4F independent translation initiation [191]. Therefore, eIF3 is recruited to its target genes by binding to mRNA stem-loops predominantly located in the 5’UTR [190]. So far 479 target genes containing mostly a single eIF3 binding site have been identified 95 and eIF3 binding was shown to either promote translational activation or repression [190]. Exemplified by c-JUN mRNA, eIF3-mediated translation initiation is independent of eIF4E, eIF4G and eIF4A, as well as of cell treatment with mTOR inhibitor INK128 [190]. Furthermore, deletion experiments revealed that c-JUN mRNA contains a 5’terminal 153 nt sequence stretch that is inhibitory to eIF4F complex formation. Thus, eIF3-mediated translation of c-JUN is ensured by specific blockage of canonical eIF4F-mediated translation initiation. It will be interesting to see whether eIF3-specialized translation can be regulated in response to specific stimuli like it is the case for eIF4F-mediated translation and which specific conditions are triggering eIF3-mediated translation. Also, the eIF4F inhibitory element is of special interest as it would be essential for the theory of differential gene expression by variable mRNA cap- dependencies to discover how this element functions mechanistically and whether its inhibitory activity could be reversed or modified and is used in a larger subgroup of transcripts. Another translational regulatory mechanism which involves a different eIF than eIF4F was described for Zscan4 expressed in mESCs and late two-cell stage preimplantation embryos. Zscan4 functions in telomere elongation enhancing the genomic stability and its expression is exclusively activated during zygotic genome activation [681], [682]. In culture only 1-5 % of mESCs are in a transient Zscan4-positive state which is associated with global repression of protein synthesis [683]. Hung et al. found that in addition to Zscan4 also Eif1a-like genes and pseudogenes but not Eif1a itself are upregulated in these cells and that their overexpression markedly reduced global translation levels [683]. As Eif1a-like genes share substantial sequence identity with Eif1a it is likely that eIF1A-like proteins function as natural dominant-negative regulators of eIF1A in translation initiation. However, how EIF1A-like factors interfere with eIF1A that functions in 43S preinitiation complex formation by recruiting the ternary complex awaits further investigation [684]. Moreover, it would be interesting to investigate whether genes escaping the global translational repression are translated from non-AUG start codons using elongator tRNAs, like seen in major histocompatibility complex (MHC) class I antigen synthesis, when ternary complex joining and start codon recognition by eIF1 are impaired [685], [686]. Apart from eIF-specific translational regulation also RBP-specific translational regulation by LIN28 was reported to play a role in ESC proliferation and growth. LIN28 is well known for its role in reprogramming human somatic cells into iPSCs in combination with OCT4, SOX2 and NANOG, where it functions as a non-essential factor accelerating reprogramming efficiency by enhancing cell division rates [687], [688]. Further, as a key developmental regulator LIN28 is well known for its role in suppressing let-7 miRNA maturation in ESCs [689], [690]. But besides that, several genome-wide and gene-specific studies found that LIN28 also acts as a mediator of

96 translational regulation independent of let-7 [691]–[698]. However, due to several discrepancies between these studies the molecular basis of LIN28-mediated regulation remains elusive. For example, six genome-wide studies identified varying numbers of LIN28 target genes ranging from ~1,300 identified in hESCs by RIP-Seq, to ~2,000 identified in hESCs by eRIP, to ~3,000 identified in HEK293 cells by PAR-CLIP, to ~6,000 identified in hESCs by CLIP-Seq, to ~10,000 identified by PAR-CLIP in HEK293 cells and to ~13,000 identified by CLIP-Seq in mESCs [694]–[699]. Thus, it is unclear whether LIN28 regulates rather a specialized subset of target genes or interacts with a large proportion of the transcriptome. Further, the identified consensus binding sites of LIN28 differ in their sequence composition (AAG(N)NG [695], GGAGA(U) [696], AYYHY [697], RGGSWG in 3’UTRs and AAGRWG in CDS [699]) as well as in their structural characteristics (binding site located in the terminal loop of a hairpin with a stem of 5-7 bp [695], unpaired regions of secondary structure [696], small A bulge flanked by two G:C base pairs embedded in a complex secondary structure [700], G-rich sequences forming G- quadruplexes [701]). It is likely that LIN28 target recognition is more dependent on secondary structure than on short linear sequence motifs and that sophisticated structure analyses are required to identify the underlying similarities that promote LIN28 binding. Further it is possible that the interaction of LIN28 with different binding partners remodels its RNA binding domains inducing not only distinct LIN28 binding modes but also diverse physiological functions. In line with this, most studies report that LIN28 binding activates translation of target mRNAs [691]–[694], [696], [699], another study doesn’t find significant translational regulation but increased protein abundance due to mildly increased mRNA stabilization [697], whereas two more studies report that targets are predominantly translationally repressed [695], [698]. Translationally stimulated targets are enriched for genes encoding RNPs, genes involved in translation, including ribosomal proteins and genes participating in cellular metabolism including glycolysis in ESCs [694]. In addition, another study found RBPs and genes involved in RNA metabolic processes, splicing and localization to be significantly enriched among LIN28 targets. Further it was found that an upregulation of splicing factors following LIN28 overexpression caused widespread changes in alternative splicing events [696]. Also, Oct4 and Igf2 mRNA were reported to be translationally enhanced by LIN28 and LIN28-interacting partners RHA and eIF3b were suggested to play a role in this process [692]–[694]. Similarly, another study conducted in HEK293 cells found translationally enhanced target genes enriched in the GO terms ribosome, cell cycle, spliceosome and cancer pathways [699]. Even though, a second study in HEK293 cells did not detect significant LIN28-mediated translational regulation, identified target genes were also enriched for RNA splicing factors as well as RBPs, genes involved in nuclear processes, chromatin components, transcriptional regulators and cell cycle-related genes 97

[697]. The HEK293 studies investigated the function of LIN28B and LIN28A, while all ESC studies focused their analyses on LIN28A only. Hence it is likely that detected variations in LIN28-mediated gene regulation in HEK293 cells reflect a cell type- and/or homolog-specific mechanism. One study which found that LIN28-binding induces translational repression identified targets to be integral membrane proteins, secretory proteins and proteins that are localized within the ER and Golgi apparatus [695]. Further, it was found that LIN28 itself localized to the cytosolic surface of the rough ER, suggesting that it regulates ER-associated translation [695]. The second study reporting LIN28-mediated translational repression was conducted in hESCs undergoing differentiation towards trophoblast and neural lineages. Top target genes with increased LIN28 association but decreased translational efficiency upon differentiation belong to IL-2, TNF-α/NF-κβ and NGF signaling pathways [698]. It is possible that some secretory proteins or membrane-bound proteins of these signaling pathways are repressed by LIN28- mediated translational suppression at the rough ER. Taken together, the variety of let-7-independent regulatory roles of LIN28 described to date illustrates how little is known about the underlying molecular mechanism of LIN28- mediated translation. Beyond any question, LIN28 activity is central for stem cell function especially in the establishment of pluripotency and early cell fate decisions. Clarification of LIN28-binding modes and interaction partners will help to reveal its stimulating or repressive effect on the translation efficiency of targets genes, which in turn has the potential to uncover the relation between processes of pluripotency establishment, cell proliferation and differentiation, which are all critically controlled by the so-called “gatekeeper” LIN28 [702]. In addition to LIN28, also the stemness factor OCT4 has been reported to be involved in specialized translational regulation. More precisely, the isoform OCT4B, which is generated by alternative splicing and which cannot sustain ESC self-renewal, was suggested to be translated by an IRES element that is stimulated under stress conditions [703], [704]. But the putative OCT4B IRES element has substantial promoter activity and reporter assays have been conducted by DNA transfection so that the OCT4B IRES requires verification by RNA transfections, including comparisons with OCT4B cap-dependent translation [703], [704]. Another exciting IRES-mediated translation mechanism was reported for a subgroup of Hox genes during mouse embryonic development. Barna and colleagues found that mouse mutants displaying skeletal patterning defects, especially homeotic transformations, were caused by deletions in the Rpl38 gene, which resulted in selectively reduced translation of a subset of Hox genes while global translation was unaffected [705]. In follow up analyses they postulated that these Hox mRNAs contain an IRES element within their 5’UTRs, which is regulated by 98

RPL38, and a translation inhibitory element (TIE), which simultaneously blocks canonical cap- dependent translation [604]. Even though the patterning defects of developing mice mutants are obvious and RPL38 might selectively regulate Hox mRNA translation, the existence of IRES elements within HoxA genes needs further verification. For instance, mechanistic IRES experiments have been conducted in C3H10T1/2 cells by use of bicistronic DNA reporter transfections, which are prone to produce artifacts [65], [481]. Such artefacts might explain the unusually high stimulation of Hoxa9 bicistronic reporter whose IRES activity was >80-fold higher as the negative control (empty vector), suggesting the generation of truncated m7G-capped reporter transcripts [604]. Although unnoticed by the authors, artifacts were confirmed by shRNA validation experiments that revealed significant production of truncated reporter transcripts (~30 % of downstream cistron expression remained after shRNA-mediated silencing of upstream cistron of a Hoxa9 bicistronic reporter) [604]. Hence, IRES-mediated translation of HoxA genes requires verification by RNA reporter transfections to exclude the translational contribution of aberrantly generated transcripts to reporter readout. Further, the function of the TIE is puzzling. The study reported a TIE in four out of five investigated HoxA genes and all of them efficiently block translation of a Hbb reporter, despite the fact that they don’t share sequence similarity or particular high GC content [604]. Interestingly, deletion of the TIE does not elevate Hoxa9 reporter expression. While removal of the TIE in monocistronic reporter constructs yields similar expression levels of full-length and deletion reporters, removal of the TIE in bicistronic reporter constructs reduces expression levels of the deletion reporter by >50 % compared to the full-length reporter [604]. The authors speculate that the element might fold in a specific structure which interferes with scanning ribosomes or that the element might alternatively target mRNAs into RNA granules to keep them translationally repressed [413]. Anyways, the repressive translational effect of the TIE diverges in the different sequence contexts studied so far but if it is reproducible its mechanistic clarification would have the great potential to enlighten fundamentals of the translation initiation process. More recently, Barna and colleagues reported another case of ribosomal protein dependent IRES regulation. They discovered that translating ribosomes display heterogeneity of core ribosomal proteins and that heterogenous ribosomes preferentially translate certain mRNA subsets [128]. Moreover, it was reported that RPL10A regulates the translation of a subset of mRNAs at least partially through an IRES-specific mechanism. Even though RNA reporter assays were implemented in the analysis, these were only used for CrPV IRES investigations including HBB as a negative control while the opportunity to apply RNA in vitro translation assays also for the evaluation of cellular IRES candidates was missed [128]. Further, DNA 99 reporter analysis of potential cellular IRESs lacked siRNA control experiments or other assays to validate reporter integrity so that the IRES activity of the RPL10A-regulated gene transcripts requires further validation [128]. Nevertheless, the phenomenon of heterogenous ribosomes and ribosomal protein-specific translation of mRNA subsets opens up a relatively new and previously unrecognized layer of translational regulation, whose molecular mechanism is still exciting to be deciphered although it might not involve IRESs [129], [706]. Interestingly, the Hox genes Antp and Ubx of Drosophila have also been reported to be translationally regulated by IRESs in a spatio-temporal manner during fly embryonic development [707]–[709]. Even though both are popular examples of IRES-mediated translation, their potential IRES activity requires more extensive verification. For instance, Sarnow and colleagues demonstrated translational activity of the P2 isoform of Antp by DNA and RNA transfections of bicistronic reporters in S2 cells, but the IRES activity was weaker in RNA transfections than DNA transfections and although reporter integrity was tested by northern blot and RNase protection assay, both are unsuitable to unambiguously rule out the generation of monocistronic transcripts [707]. Also, Utx IRES activity was analyzed by bicistronic reporter assays in S2 cells [709]. Validation by northern blots depicted in addition to the full-length reporter transcript also smaller transcripts containing the second cistron but lacking the first [709]. As the smaller transcript was generated as well by empty control reporters missing the Utx 5’UTR the authors reasoned the start site would be downstream of the intercistronic region and as negative control reporters yielded little reporter expression, the smaller transcripts weren’t considered to contribute to the reporter readout. Still, the assay doesn’t exclude the possibility that smaller monocistronic transcripts are generated which contribute to reporter expression. Further, it is concerning that in all reporter assays of Antp and Utx presented, the expression of the first cistron, which is cap-dependently translated and should therefore be invariable for all reporters, is consistently lower for the empty control construct than for the Antp and Utx constructs [709]. This might indicate a potential function of Ant and Utx sequences as translational enhancers. Taken together, protein synthesis and metabolic processes are generally low in ESCs compared to differentiating cells. Hence, global translational control is crucially involved in stem cell activation, but also selective translational regulation seems to play a role during the coordination of stem cell processes. Well-known canonical translational regulators like 4E-BP, eIF4G and eIF3 have been shown to contribute to directed ESC gene regulation and their specific isoforms or interaction partners might expand the regulatory capacity within ESCs. Further, translational regulation by uORFs and differential isoform expression have been observed during ESC differentiation and recently, the heterogenous composition of ribosomal 100 proteins in translating ribosomes was suggested to add another layer of selective translational regulation in stem cells and during embryonic development. Although a few gene-specific IRES- dependent translation events have been reported in stem cells, stringent experimental controls are awaited to verify true bona fide IRES activity. Thorough future investigations will reveal the mechanistic details of how eIFs, RBPs, ribosomal proteins and possibly specialized isoforms thereof establish the selective translation of targets genes and if differential cap-dependency of targets plays a role in the process during stem cell differentiation.

1.5 Circular RNA (circRNA) as a new class of cellular RNA Can circRNAs encode proteins? Apart from studying non-canonical translation initiation on mRNA molecules, this study also investigated the potential of a single circRNA to be used as a template for ribosomes to produce protein via internal translation initiation. As circRNAs have only recently been recognized as prevailing group of cellular RNA, little is known about their potential regulatory functions and whether giving rise to proteins or translational regulation in trans could be a part of it.

1.5.1 Characteristics and functions of circular RNA (circRNA) Functional single-stranded circRNA molecules have first been discovered in the 1970s by identifying the molecular structure of viroids and the nucleotide sequence of the RNA genome of bacteriophage MS2 [710], [711]. Later, in the 1980s the first animal virus, hepatitis delta virus, with a circular single-stranded RNA genome was identified and introns of cytochrome b and c of mitochondrial yeast genes as well as introns of rRNA precursors in archaebacteria and Tetrahymena were described to be covalently closed RNA circles within unicellular organisms [712]–[716]. In the cytoplasm of HeLa cells, circularized RNA molecules of unknown origin were observed in 1979 but it was not until the 1990s, however, that circular RNA transcripts were found to originate from genes like DCC, ETS1, SRY, Fmn1 and DMD [717]–[723]. In the early days these circRNAs with scrambled exon composition were hypothesized to be a sort of short- lived intermediate transcript that is later resolved into a linear one, the result of an aberrant mis- splicing reaction or noise of the splicing system [720], [724], [725]. Only modern technological advances such as RNA-Seq, selective rRNA depletion and bioinformatic analysis tools enabled the identification of circRNAs as an abundant and prevalent class of RNA expressed throughout all tissues of eukaryotes investigated to date [726]–[732]. So far, several subsets of circRNAs have been classified. The term circRNA refers to circularized RNA transcripts that contain exonic sequences, are generated from pre-mRNA by back splicing and are localized in the cytoplasm. Intronic circRNAs (CiRNAs) originate from 101 lariats, they contain two consensus motifs near the 5’ss and the branchpoint, resist for unknown reasons debranching enzymes and localize within the nucleus [733]. CiRNAs accumulate at concise nuclear spots and at their parent gene locus, they associate with elongating Pol II and stimulate parent gene transcription in a mechanistically unresolved manner [733]. Another subgroup with exclusively nuclear localization are exon-intron circRNAs (EIciRNAs) that consists both of exonic and intronic sequences. Like CiRNAs, EIciRNAs accumulate at genomic loci of their parental genes and other distinct genomic areas. Further, they interact with Pol II and U1 snRNP at the promoter region of their parent gene, where they are suggested to function as transcriptional activators [734]. Lately, another group of intronic circRNAs (IcircRNAs) was identified by a circRNA isolation method called RPAD which uses a modified circRNA identification algorithm [735]. As IcircRNA junction sequences were poorly conserved by RT- PCR validation – contrary to CiRNAs which contain the lariat 2’,5’-phosphodiester bonds within circular boundary reads – it is currently unknown how they are generated. The following chapter will focus on the characteristics and functions of cytoplasmic circRNAs, which is the most abundant and widely studied subgroup. CircBase, a database for circRNAs, lists a total of ~92,000 human circRNAs that have been computationally identified to date and which are likely to be expanded by deeper future analyses [736]. circRNAs are frequently but not pervasively expressed as roughly 5-20 % of actively transcribed protein-coding genes also express circRNA isoforms [727], [729], [737]–[741]. The amount of expressed circRNAs can vary between cell lines (~15,200 in human cervical cancer line HeLa S3, ~7,800 in human foreskin fibroblast lines Hs68 and BJ, ~4,300 in the human neuroblastoma line SH-SY5Y, ~1,300 in primary human myoblasts) and tissues (~39,000 in human frontal cortex, ~4,600 in human whole blood, ~1,600 in human liver, ~500 in human adipose tissue) [727], [729], [742]–[744]. For most genes, the relative expression of circRNA isoforms is in between 1-10 % of the expression of the corresponding linear isoforms [727], [729], [731]. But for hundreds of human genes, circRNAs are the predominantly expressed isoform [726], [742]. However, relative abundances of circRNA to linear RNA isoforms can differ considerably in between studies, like total numbers of circRNA prevalence can vary between comparable published datasets [731], [745]. Apart from tissue-specific isoform expression, such discrepancies can be explained by experimental (circRNA enrichment by RNase R, poly(A) depletion, sequencing depth, read length) and bioinformatic distinctions (filtering criteria, correction for artefacts, definition of high-confidence sets) [746]. Furthermore, the abundance of circRNA isoforms is likely underestimated compared to linear isoforms, as only reads covering the circular junction are taken into account for determining circRNA expression,

102 while reads spanning other transcript regions that are shared between linear and circular RNA are not considered to support circRNA expression. Regardless of variability in circRNA detection methods, it appeared that circRNAs are highly abundant in platelets, where circRNA enrichment was associated with transcriptome degradation, and in neuronal cells, especially in the brain, where circRNAs are specifically enriched in synaptosomes and in synaptic genes [732], [737], [740], [742], [747]–[750]. Brain- expressed genes display the highest circular to linear RNA ratio with hundreds of genes showing dominant circRNA expression: >50 % of all transcripts are circular from ~600 genes of the human frontal cortex [737], [742], [748]. Furthermore, circRNA expression in the brain is regulated by development and neuronal plasticity [737], [742]. Most circRNAs are upregulated during different time points of neuronal differentiation and consistently upregulated circRNAs originate from genes with synapse-related functions [737], [742], [748]. Induction of circRNA expression during development is common: it can be seen in other organs like heart, lung, intestine and stomach or during myoblast differentiation, epidermal stem cell differentiation or epithelial-mesenchymal transition [738], [743], [748], [751]. In the course of differentiation and development, many circRNAs change their expression level independent of their cognate mRNAs, although expression changes are overall correlated between RNA isoforms from the same gene [729], [737], [738], [742], [743], [748]. CircRNAs may comprise a single exon or multiple - typically one to five – which are biased to originate from the 5’ terminal part of the host gene [726], [728], [742], [747], [749]. Furthermore, circularization preferably includes the second exon of protein-coding genes, while the first and the last exon are depleted from circRNAs due to the lack of either a splice acceptor or splice donor site at one of their ends [726], [747], [749], [752], [753]. Apart from coding sequence, circRNAs may also contain exons from the 5’ UTR and to a lesser extent from the 3’ UTR [728], [731], [732], [742], [747], [749], [754]. Furthermore, a small amount of circRNAs is oriented antisense to annotated genes [728], [731], [732], [742]. The size of circRNAs can vary substantially from less than 100 nt to more than 4 kb, but the median length of human circRNAs is ~500-700 nts (which reflects the typical circRNA structure of one to five exons and a median exon length of 133 nts) [731], [736], [755], [756]. Interestingly, single-exon circRNAs tend to arise from exons that are longer than the median exon size [727], [748], [757]. While most genes with circRNA isoform expression produce only one or two circRNA isoforms, “hot-spot” genes may give rise to ten or more circRNA isoforms [729], [740], [747], [749], [757]. The isoform expression is especially diverse in the human brain where a gene gives rise to a median number of three circRNAs, with more than 2,300 genes expressing ten or even more circular isoforms [742].

103

Another distinct feature of circRNAs is their high stability, presumably due to the lack of free 5’- and 3’-ends protecting circularized transcripts from degradation through exonucleases [728], [758]. While the median mRNA half-life is 9-10 h, a set of circRNAs was found to exhibit half-lives exceeding 48 h whereas associated linear transcripts of the same host genes exhibited half-lives below 20 h after actinomycin D treatment [727], [759], [760]. Moreover, as circRNAs lack both a 5’cap and a 3’poly(A) tail, it was speculated they might be resistant to miRNA mediated deadenylation, decapping and 5’-to-3’ decay typically caused by miRNA target recognition [406], [761], [762]. Contrary to what was expected, the most prominent circRNA CDR1as was found to be degraded by miR671-directed slicing and another yet undefined miR-7- dependent mechanism [763], [764]. Such miRNA-mediated slicing requires extensive miRNA pairing which is extremely rare in animals so that circRNAs might in general be unaffected by typical miRNA mediated decay [765]. Although unconventional mechanisms of miRNA- dependent circRNA destruction might exist, circRNAs exhibit overall long half-lives which result from slow turnover rates and accumulation [766]. This phenomenon is most prevalent in post- mitotic and slowly dividing cells, like neurons, and is in line with studies reporting that circRNAs accumulate during development and aging [737], [742], [747]–[749], [767]. Furthermore, circRNAs are evolutionary conserved between closely related species like mouse and rat, human or pig and some of them are conserved in more distant species like Drosophila [727]–[729], [731], [737], [740], [742], [743], [747]. In general, genes expressing circRNAs in one species are more likely to express circRNAs in orthologous genes of a different species and conservation positively correlates with circRNA expression [742], [743]. Additionally, sequence conservation is higher for circularized splice sites and exons than for adjacent splice sites and exons of linear transcripts from the same gene [737], [742], [768]. Some circRNAs not only derive from the same host gene but also from the same circular junction: 4,522 (~29 %) and 4,527 (~29 %) murine circRNAs out of 15,849 conserved circRNAs in human brain share the same splice sites or utilize one identical splice site, respectively [742]. Interestingly, human brain tissue seems to be richer in circRNA isoform expression, although a direct comparison due to differing sequencing depth is missing [742]. Further, humans harbor significantly more species-specific circRNA encoding genes than mouse during fetal development and expressed circRNA gene numbers and isoforms are constantly higher from oocyte to morula, reaching a maximum at the 4-cell stage [752]. Increased circRNA expression during 4-cell stage is in line with the major wave of zygotic gene activation during maternal to zygotic transition during early human development [769]. As mouse zygotic transcription waves burst earlier at the 1-cell and 2-cell stages, which show also an increase in circRNA expression, but less pronounced and not at maximal level, human circRNAs might constitute newly evolved 104 players of gene regulation during zygotic gene activation [752], [769]. In agreement with this, a study found that the earliest transcribed zygotic genes evolved recently and exhibit the fewest shared orthologs between species compared to all other developmental stages investigated [770]. Further, the 4-cell stage of human blastomeres was lately suggested to be the first time point in development of heterogenous gene expression, providing the basis for early cell-fate decisions [771]. As circRNAs have previously been reported to function in a molecular circuitry together with miRNAs and core pluripotency transcription factors to regulate pluripotency and cell differentiation, it is tempting to speculate that circRNAs might actively shape gene expression programs and establish cell fate determination, adding a new layer of complexity and species- specific gene regulation in early human zygotes [772]. CircRNAs are generated by a back-splicing or head-to-tail splicing reaction in which a 5’ donor site of a downstream exon is ligated to a 3’ acceptor site of an upstream exon, giving rise to a covalently closed circular RNA. The exact involvement of the spliceosome in this process is not resolved yet and three partly compatible models have been proposed to explain circRNA biogenesis: intron pairing-driven circularization, RBP pairing-driven circularization and Lariat- driven circularization [761]. Nevertheless, it is evident that the spliceosome catalyzes the reaction as either canonical U2 and U12 splice signals flank the splice junction and as mutation of the 5’ss, the 3’ss or the polypyrimidine tract leads either to the production of alternative circRNAs using new cryptic splice sites as a substitute or to a decrease in circRNA formation [728], [748], [753], [773]. Like canonical and alternative splicing, back-splicing is regulated by cis- and trans-acting factors. Both modulate circRNA biogenesis by either bringing the splice sites utilized in circularization closer together in the three-dimensional space or by impeding such proximity. While exons lack any specific sequence elements facilitating circRNA formation (except for canonical splice sites), circRNA surrounding introns tend to be longer than average and promote circularization by short reverse complementary sequences, like Alu elements [726], [727], [742], [747], [753], [757], [774]–[776]. Alu elements constitute with over one million copy numbers roughly 10 % of the human genome, wherefore it might not surprise that ~90 % of human circRNAs contains Alu repeats in their flanking introns, with an average of three elements each upstream and downstream [757], [774], [777]. Nevertheless, the enrichment of Alu elements is significant and reverse complementary sequences in general are not only a conserved feature of circRNA flanking introns, they can even be used to predict circRNA formation [757], [774]. Using minigene vectors, short inverted repeats of only ~30-40 nt are sufficient to induce circularization but longer (~300-500 nts) imperfect repeats are more efficient in circularization of long exons and yield higher circRNA to linear RNA ratios [775], [776]. In vivo, the presence of 105 several inverted repeats within introns can also negatively impact circRNA formation as complementary sequences compete in RNA pairing: intra-intron pairing within an individual intron supports conventional linear splicing, whereas inter-intron pairing across flanking introns supports circularization through back-splicing [757]. Such intron pairing-driven circularization alone does not explain the dynamic circRNA expression that is developmental stage or tissue specific and several trans-acting factors like RBPs have been identified to regulate circRNA biogenesis. For instance, Alu elements are heavily edited and ADAR1, a RNA editing enzyme that converts adenosines to inosines, antagonizes circRNA formation by introducing U:I mismatches in paired sequences, which destabilizes RNA interactions leading to lower circRNA production [742], [774]. Also, DHX9 binds inverted repeat Alu elements, interacts with a specific ADAR1 isoform and interferes with circRNA biogenesis [778]. Another example is QKI, a RBP involved in mRNA splicing, stability and translation. Many circRNAs are upregulated during human epithelial-mesenchymal transition and QKI induces the expression of ~1/3 of the most abundant circRNAs by binding introns upstream and downstream of circRNA-forming exons [738]. Disruption of QKI binding sites on either the upstream or downstream site markedly reduced circRNA formation and introduction of QKI binding sites into adjacent introns of usually exclusively linear exons was sufficient to facilitate circRNA formation [738]. As QKI forms dimers it is hypothesized that QKI binding connects flanking introns to promote back-splicing of in between exons. Also Mbl, a well-established alternative splicing factor, regulates circRNA formation. Similar to QKI, Mbl binds to intronic regions upstream and downstream of circularized exons to promote back-splicing [773]. Interestingly, the second exon and flanking introns of Drosophila mbl are enriched for Mbl binding sites, so that a regulatory feed-back loop promotes increased circMbl formation upon Mbl binding which results in decreased Mbl linear splicing and Mbl protein production [773]. Further, Fus, a RNA and DNA binding protein playing a role in splicing, was found to regulate circRNA formation in murine motor neurons. In detail, Fus binds flanking introns within ~1000 nts of the back-splicing site, causing an increase or decrease in circRNA formation, although the vast majority of back-splicing events observed were stimulated by Fus binding [754]. Contrary to Mbl-mediated back-splicing, Fus-mediated circularization does not compete with linear splicing and cognate linear RNA expression is stable [754], [773]. A screen of ~20 common factors functioning in transcriptional elongation, RNA splicing or RNA processing in Drosophila revealed the combinatorial regulation of circRNA formation by intronic complementary repeats and multiple hnRNP and SR proteins for laccase2 as well as seven other gene loci [776]. Some of the hnRNP and SR proteins were acting in an additive manner, while others were acting redundantly. The effects of combinatorial regulation were unique for 106 each circRNA, consistent with each gene exhibiting a unique set of protein interaction sites [776]. Notably, circMbl expression was independent from hnRNP and SR proteins and vice versa expression of laccase2 and other circRNAs was independent of Mbl. Moreover, the lariat-driven circularization model proposes that circRNA formation takes place at skipped exons that are contained in close proximity within a lariat. A few single-gene and genome-wide studies could show that exon skipping and circRNA formation correlates for single-exon and multi-exon circRNAs and a screen in yeast suggests that circRNAs might be stabilized in the absence of Dbr1 which hydrolyzes 2’,5’-phosphodiester bonds to convert lariats into linear RNAs prior to degradation [723], [741], [779]–[781]. However, exon skipping alone is not sufficient to explain circRNA formation and the analysis of exon skipping events is prone to misconception as linear skipped transcripts might be unstable and circRNAs generated from annotated skipped exons might likewise be generated by direct back-splicing without previous exon skipping [779], [782]. Whether circRNA biogenesis happens co- or post-transcriptionally remains controversial as indications for both biogenesis modes have been described [766], [773], [776]. Within minigene vectors the length of intronic repeats of flanking introns seems to determine whether circularization happens independently of 3’end processing or not: long repeat sequences support co-transcriptional back-splicing while short repeat sequences promote back-splicing only post- transcriptionally after successful 3’end processing [775], [776]. Anyhow, introns flanking circRNAs are less efficiently spliced than all introns and back-splicing is usually less efficient than linear splicing but when key factors of the spliceosome or transcription termination are inactivated, back-splicing is favored over linear splicing [766], [773], [783]. It is hypothesized that circRNAs are less sensitive to spliceosome inhibition because cross-exon interactions might be relatively less impaired than cross-intron interactions during exon and intron definition which should permit back-splicing but prevent linear splicing [783]. Altogether, prevalence, evolutionary conservation as well as multifactorial and dynamic regulation of circRNA biogenesis suggest that this novel class of RNA contains functional capacity. The first evidence of such regulatory function was found for the circRNA of CDR1as [728]. CDR1as is the dominant antisense RNA product of the CDR1 gene locus, which can function as a miRNA sponge due to 73 binding sites for miR-7 [763]. The miR-7 binding sites are complementary to the miR-7 seed region and promote not only the interaction with miR-7 but also sequestration of AGO [728], [784]. However, CDR1as bound by miR-7 and AGO is stable and degradation can only be induced by the binding of miR-671 that is almost entirely complementary to CDR1as thus leading to circRNA slicing. Hence it was found in HeLa cells that co-transfection of miR-7 with CDR1as resulted in a dose-dependent derepression of 107 endogenous miR-7 targets, indicating that CDR1as alleviates miR-7 effects on target genes [785]. When miR-671 was introduced beforehand, transfection of miR-7 and CDR1as resulted in dose- dependent repression of endogenous miR-7 targets, indicating that CDR1as concentrations were not sufficient any more to buffer miR-7 effects [785]. Both CDR1as and miR-7 are highly expressed in the brain and it was found that sequestration of miR-7 by CDR1as reduces midbrain size in zebrafish embryos similar to miR-7 inhibition [728]. Furthermore, a recent analysis in Cdr1as KO mice revealed anatomically normal brain development but deregulation of miR-7 and miR-671 as well as target gene expression levels [784]. In detail, miR-7 was downregulated, while miR-671 was upregulated in Cdr1as KO brain. Accordingly, miR-7 target genes were upregulated, including immediate early and circadian clock genes [784]. In line with this Cdr1as KO mice exhibited a dysfunction of excitatory synaptic transmission that is likely triggered by the overexpression of immediate early genes. This dysfunction manifested in a behavioral prepulse inhibition deficiency, which is linked to a number of neuropsychiatric disorders [784]. It is hypothesized that conditional and constitutive depletion of Cdr1as might have different effects. While miR-7 and AGO may be released after conditional Cdr1as KD leading to target gene repression, miR-7 may be destabilized after constitutive KO leading to target gene derepression [784]. Apart from CDR1as, circSry (16 binding sites for miR-138), circHECTD1 (>10 AGO binding sites) and circZNF91 (24 binding sites for miR-23, 39 binding sites for miR-296) have been suggested to have the ability to function as miRNA sponges but comprehensive analyses are missing [731], [751], [785]. Other circRNAs were not found to be particularly enriched for miRNA binding nor for AGO crosslinking sites [729], [731], [737], [738], [768], [784], [786]. To exert a substantial effect on ceRNAs, an individual circRNA would require a number of binding sites that is comparable to the total number of binding sites of all transcripts expressed in the cell [401], [402]. Such a high number of binding sites or circRNA expression is outside the regular range, wherefore a general function of circRNAs as miRNA sponges is unlikely. In addition, circRNA expression in Plasmodium falciparum and Saccharomyces cerevisiae, two organisms that lack the siRNA machinery, indicates that the predominant function of circRNAs can be independent from miRNA interaction [730], [787], [788]. However, several studies on individual circRNAs claim a miRNA sponging function. One example of such a reported miRNA sponge is circHIPK3, which comprises the second exon of HIPK3 mRNA that spans ~ 1.1 kb and which is the predominant transcript produced from the HIPK3 gene locus in many tissues [789]. Even though circHIPK3 is enriched for synonymous constraint elements (protein coding sequences reduced in synonymous nucleotide substitution mutations likely being under additional selection for an overlapping function) and miRNA 108 binding sites, no enriched binding of an individual miRNA to circHIPK3 could be detected [768]. Anyhow, several other studies reported it to sponge a specific miRNA – either miR-124, miR- 558, miR-30a-3p or miR-7 – by means of a few miRNA binding sites – ranging in number from one to three active sites – thereby regulating cell proliferation [789]–[793]. The data suggests that circHIPK3 directly interacts with AGO and certain miRNAs and that conditional KD of circHIPK3 has similar effects on cell proliferation as overexpression of the associated miRNA. Further, some of the studies show that cell proliferation effects are specific for circHIPK3 and independent of linear or total RNA output of HIPK3 gene locus [768], [789], [792]. Although circHIPK3 is generally highly expressed compared to its linear counterpart, the presence of no more than three functional binding sites of an individual miRNA argues against a potent sponging effect. Moreover, miRNA targets vary in between cell types and studies (one study fails to detect previously identified targets in a similar tissue) and circHIPK3 is differentially deregulated in cancer cell types [768], [793]. Together this suggests, that circHIPK3 interactors and the underlying regulatory gene networks are diverse so that primary and secondary effects might be hard to distinguish. In line with this, the network of interacting partners linking circHIPK3 and miRNA regulatory effects were often not elucidated. Only Zheng et al. show that two miR-124 target genes are similarly repressed by miR-124 overexpression as by circHIPK3 KD and that target repression is rescued after combined overexpression of circHIPK3 and miR-124, which suggests that the circRNA buffers miRNA-target interaction [789]. In addition, Zeng et al. shows that miR-7 expression levels either decrease after circHIPK3 overexpression or increase after circHIPK3 KD suggesting as well that the circRNA buffers the freely available levels of this miRNA [793]. As both analyses lack a positive or negative control, the specificity of the results remains unclear. In sum, it is still uncertain if circRNAs act as miRNA regulators despite displaying only few miRNA binding sites. In specific cases miRNAs may constitute key elements governing robustness to a biological process by regulating important, miRNA-dosage sensitive components of the underlying network [395], [794]. Feedback loops between a miRNA and the targeted important network component might amplify effects to such an extent that circRNAs displaying a few miRNA binding sites might have an impact on miRNA regulation even though the term “sponge” would be misleading in this context. Anyhow, the many numbers of studies reporting a circRNA to function as a sponge need to be reviewed carefully. It is tempting to speculate that circRNAs could stabilize or store miRNAs due to their lack of free ends and their subsequent resistance to miRNA mediated exonucleolytic decay, however, the underlying mechanism of such a function has yet to be uncovered.

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As circRNAs are largely composed of CDS and predominantly located in the cytoplasm they were examined for their potential to encode proteins. It has been found previously that circular introns in 23S rRNA genes of hyperthermophile archaea contain ORFs that can be translated into DNA endonucleases in vitro [795], [796]. Also, artificial circular RNAs that were constructed to contain the EMCV IRES were shown to be translated in vitro; ribosomes traversed the circRNA even multiple times after elimination of stop codons in all reading frames [797]. Moreover, artificial circRNAs were found to be translated in vivo by transformed E. coli and transfected HEK 293 or HeLa cells [798]–[800]. Together these studies provide evidence that translation of circRNA molecules is feasible even in the absence of a cap and a poly(A) tail, which usually support ribosome recruitment. Although initial analysis of ribosome protected and polysome associated RNA failed to detect signs of circRNA translation, a few examples of endogenously translated circRNAs were discovered recently [727], [731], [737], [801]. Re-analysis of previously published ribosome profiling datasets in fly and ribosome profiling of Drosophila heads detected 37 and 122 circRNAs respectively with ribosome footprinting reads across the circular splice junction [1]. The ORF encoded by 40 % of these circRNAs shares the start codon with the host gene and ~ 70 % of these circRNAs contain UTR sequences. Similar features were identified by re-analyzing ribosome profiling datasets in rat and mice, where circRNAs with ribosome protected fragments (RPFs) spanning the backsplice junction were also enriched to contain 5’UTR and CDS sequence as well as the start codon of the host gene [1]. Further, a combination of minigene experiments, polysome profiling, targeted MS, in vitro translation and western blots could show that circMbl, the fly circRNA with the most RPFs (42 RPFs in total from 4 fly head Ribo-Seq libraries), can be translated in vivo [1]. Enrichment of 5’UTR sequences and the start codon of the host gene suggests that translation initiation on circRNA requires regulatory sequence elements like probably the Kozak sequence which are usually depleted within CDS. Moreover, translation initiation on circRNAs is likely less efficient than cap-dependent translation as circMbl is translated with ~10 % of the efficiency of capped circMbl sequence in in vitro assays [1]. The data also suggests that circRNAs in fly heads are often translated by membrane- associated ribosomes or in membrane-rich subcellular areas like e.g. the synapse. Already 20 years ago, a circRNA from the SLC8A1 gene locus was identified that may be translated at the ER. CircSLC8A1 spans the unusually long second exon (1,832 bp), including the start codon, the signal peptide and CDS of the first hydrophobic transmembrane segments and parts of the central cytoplasmic regulatory loop (602 aa) [106], [802]. When transfected into HEK 293 cells, the linearized circSLC8A1 sequence is translated into a 70 kDa protein that has calcium transport activity, although at roughly half the efficiency of the full-length protein [802]. When microsomes 110 of the rhesus macaque kidney cell line LLC-MK2, rabbit heart, rat heart und rat kidney were analyzed by western blot for presence of an endogenous circRNA encoded protein, no band at the exact height of 70 kDa but a strong band at ~ 60 kDa was detected, which might result from circRNA translation and subsequent proteolytic processing of the protein product [802]. More recent analyses of circSLC8A1 in differentiating cardiomyocytes suggest that the circular transcript is enriched at ribosomes in mice and AGO2 in rat compared to the linear transcript [803]. Furthermore, circSLC8A1 is upregulated in human dilated cardiomyopathy when compared to host gene expression [803]. Together this suggest that circSLC8A1 might be involved in pathogenesis of heart tissue and that a circRNA-encoded truncated protein may play a role in this process. Deeper analyses are needed to unambiguously clarify the potential function of circSLC8A1. Like circMbl, a human circRNA identified in myoblasts, circZNF609, contains parts of the 5’UTR, shares the start codon with its host gene and encodes a peptide which terminates shortly after the backsplice junction [743]. Polysome profiling, expression vector experiments, CRISPR/Cas9-mediated gene editing and MS could show that circZNF609 can be translated in vivo. Its translation efficiency is ~1-1.5 % of the linear circZNF609 sequence in transfection assays, but translation activity increases markedly after splicing [743]. This finding is in line with previous studies of mRNA and suggests that the exon junction complex may increase translation of circRNAs through interaction with initiation factors or ribosomal proteins as seen with linear transcripts [804]–[806]. In addition, circZNF609 is highly methylated, which was also found to promote translation of circRNAs [743], [807]. Moreover, circZNF609 plays a role in myogenesis and regulates myoblast proliferation; however, whether the circRNA or the circZNF609 encoded peptide function in myoblast proliferation is unknown [743]. As mentioned above, m6A is a RNA modification that can promote circRNA translation. Yang et al. found that, consensus m6A motifs are enriched in circRNAs compared to all coding mRNAs and that circRNA regions have a higher m6A peak density than all mRNAs identified by methylated RNA immunoprecipitation sequencing (MeRIP-Seq) [807]. By combining m6A IP, RNase R treatment and RNA-Seq they estimate that ~13 % of all circRNAs identified carry m6A modifications [807]. Using GFP minigene reporters in HEK 293 cells they show that a single m6A site is sufficient to promote translation initiation and that eIF4G2, a non-canonical initiation factor; eIF3A, a subunit of eIF3 previously shown to promote cap-independent translation; and YTHDF3, a m6A reader protein; are required for m6A-mediated circRNA translation [807]. Like mRNAs, circRNAs are methylated by METTL3/14 and demethylated by FTO, which enhances or diminishes their translation efficiency, respectively. Apart from identifying 99 circRNAs containing m6A peaks in human foreskin Hs68 fibroblasts and potential translation initiation sites 111 in silico, 250 circRNAs were detected to be associated with polysomes in HeLa cells and 33 peptides were found to be encoded from backsplice junctions of 19 circRNAs by MS in HEK 293 cells [807]. Together the data suggests, that a subset of circRNAs with m6A and translation initiation sites is translated and that translation initiation may be regulated by common m6A readers and erasers in response to physiological conditions like for instance heat shock stress. Indications for another endogenously translated circRNA come from a study in human glioblastomas. CircSHPRH, spanning exons 26-29 from the SHPRH gene, is downregulated in glioblastoma compared to healthy brain tissue and was suggested to suppress tumorigenesis by stabilizing SHPRH protein [808]. CircSHPRH contains a 440 nt ORF (which starts and terminates with overlapping initiation and termination codons “UGAUGA”) encoding a ~ 17 kDa peptide including an unique 14 aa long protein sequence [808]. The peptide shares two ubiquitination sites with the full-length protein potentially protecting the full-length protein against ubiquitin-induced degradation by competing for DTL ligase binding. Stabilized SHPRH, itself a well-known ubiquitin ligase, may subsequently target proliferating cell nuclear antigen (PCNA) which may result in decreased cell proliferation. As the study misses some control experiments, future analyses are required to sufficiently proof the circSHPRH-regulated mechanism in glioblastoma tumorigenesis. Nonetheless, this example provides an indication for the potential function of circRNA encoded peptides and suggests that such peptides may act as dominant negatives of corresponding full-length proteins or protein domains. CircRNAs have further been proposed to function in protein synthesis not only by being translated themselves but also by regulating translation of their host genes. For example, circPABPN1, which consists of exon 6 and is 152 nt long, was suggested to suppress PABPN1 translation by competing for HuR binding. Apart from the 3’UTR, circPABPN1 contains the only nucleotide stretch within PABPN1 transcripts that was identified to interact with HuR in PAR-CLIP analyses (data accessible at NCBI GEO database [809], GEO accession GSE35585). Using HuR RNA immunoprecipitation (RIP), circPABPN1 was found to be the most abundant circRNA associated with HuR [810]. Further, circPABPN1 overexpression resulted in a downregulation of PABPN1 mRNA association with HuR and downregulation of PABPN1 protein levels, while HuR protein und PABPN1 mRNA levels were unchanged [810]. Thus, it was hypothesized that circPABPN1 represses PABPN1 expression by HuR sequestration. Anyhow, it is unclear how circPABPN1, which is present in ~ 12 copies per HeLa cell, selectively inhibits HuR, which is present in ~ 1,300 copies in the cytoplasm of HeLa cells, from PABPN1 mRNA binding [810]. Potentially, the circRNA captures HuR proteins co-transcriptionally when splicing is in progress and both circRNA and mRNA are in close proximity.

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In addition to translational regulation of host genes, circRNAs were also suggested to regulate ribosome biogenesis. For instance, circANRIL that encompasses exons 5-7 of the long non-coding antisense RNA ANRIL was proposed to bind PES1 resulting in impaired pre-rRNA maturation [811]. RIP of BoxB-containing circANRIL identified ribosomes, nucleolar proteins and components of the PES1-BOP1-WDR12 (PeBoW) complex as predominant circRNA interactors [811]. The PeBoW complex controls pre-rRNA processing and is a key regulator of 60S maturation. Consistently, circANRIL overexpression led to pre-rRNA accumulation whereas transient transfection of PES1 reversed pre-rRNA accumulation. Moreover, circANRIL overexpression induced nucleolar stress, indicated by nucleoli fragmentation and p53 activation which promoted apoptosis and impaired proliferation [811]. In samples of patients with coronary artery disease circANRIL expression levels correlated with pre-rRNA abundance and were suggested to have a protective function against atherosclerosis [811]. Earlier studies already showed that patients homozygous for the atherosclerosis risk allele displayed decreased circANRIL expression levels [812]. The ANRIL gene locus at 9p21 is complex and in addition to vascular diseases, linear ANRIL isoform expression is associated with several cancers where ANRIL promotes cell proliferation and cell adhesion while reducing apoptosis [813]–[815]. As circANRIL seems to antagonize linear ANRIL function, it is tempting to speculate that circANRIL may have the potential to act as therapeutic agent in pathogenic conditions linked to ANRIL expression. But apart from this protective function in atherosclerosis, circRNAs have also been implicated in pathogenesis of several other diseases of cardiometabolic origin, cancer or neurological disorders [745], [816], [817]. Often the underlying mechanisms of circRNA function were not yet investigated and remain to be uncovered. As an example, the role of circFoxo3 was evaluated in cancer and cardiomyopathy. It was found that in MEFs circFoxo3 forms a complex with p21 and Cdk2 which abolishes Cdk2 interaction with cycline E and cycline A and enhances Cdk2 interaction with p21 both leading to cell cycle repression at G1/S transition [818]. Further, circFoxo3 was shown to interact with Id1, E2f1, Fak and Hif1α in a doxorubicin-induced cardiomyopathy mouse model, where Foxo3 promoted senescence through relocation of interacting proteins to the cytoplasm during cell stress [819]. Both studies used MEFs for protein-circRNA interaction assays including RIP, Co-IPs and circRNA pull downs by use of biotinylated DNA probes. Still the specificity of reported protein-circRNA interactions is uncertain as also an unspecific control probe was able to pull down p21 and Cdk2 [818]. Further it remains unaddressed if cell cycle regulators also interact with circFoxo3 in the cardiomyopathy model, if circ-RNA-protein interactions increase/decrease during cell stress, if the interactions

113 are of direct or indirect nature for the reported six proteins and how potential protein binding sites look like. Moreover, the role of circFoxo3 was studied in tumors and cancer cell lines where circFoxo3 expression reduced tumor growth, repressed cell proliferation and promoted stress- induced apoptosis [820], [821]. In one study circFoxo3 and a Foxo3 pseudogene were hypothesized to protect Foxo3 mRNA from miRNA-mediated degradation by competing for miRNA binding (both share a sequence stretch with Foxo3 mRNA that contains 25 miRNA binding sites for eight miRNAs) resulting in increased expression of the Foxo3 tumor suppressor protein [820]. Luciferase reporter assays and overexpression experiments of Foxo3 transcripts combined with biotinylated miRNA overexpression and pull-down showed that Foxo3 mRNA, pseudogene and circRNA are bound by miRNAs and that miRNA binding to one transcript isoform reduced miRNA binding to the other transcript isoforms [820]. Further, miRNA overexpression caused Foxo3 protein destabilization and increased cell survival but specificity of miRNA-Foxo3 interaction lacks validation as miRNA overexpression might have triggered global effects on target genes leading to cell survival. Also, the analysis lacks a rescue experiment in which Foxo3 protein would be stabilized by circRNA overexpression in the context of miRNA- mediated Foxo3 mRNA degradation. In a follow-up study in breast cancer cells the tumor suppressive function of circFoxo3 was attributed to circFoxo3 binding to MDM2 and p53. Complex formation of circFoxo3, MDM2 and p53 triggered p53 ubiquitination by MDM2 and subsequent p53 degradation whereas MDM2-induced ubiquitination of FOXO3 decreased [821]. The functional analyses were performed in the same cell line (human mammary/gland MDA- MB-231) as the previous cancer study, but the previously reported miRNA-circFoxo3 protective mechanism remained unaddressed. Also, cell cycle regulators and other previously reported circRNA interactors of stress response and senescence remain to be evaluated in the cancer context. Hence, it is undetermined how both proposed circFoxo3 tumor suppressive mechanism act in concert and if circFoxo3 interacting proteins are indeed dynamically/differentially regulated in cellular context. Besides their potential function in pathogenesis, circRNAs have also been investigated in the context of diseases due to their intrinsically high stability and their cell-type and developmental stage specific expression, for what reason they are well suited to serve as a novel class of biomarkers. CircRNAs have been identified to be particularly enriched in blood as well as extracellular vesicles and exosomes [744], [822]–[824]. They have been found as well in another easily accessible body fluid, saliva, and their enriched expression from disease-relevant genes makes them potential biomarkers for diagnosis of cancer, cardiometabolic diseases and neurological disorders [732], [745], [823], [825], [826]. 114

Taken together, the function of the clear majority of circRNAs remains elusive. Keeping in mind that circRNAs are usually identified computationally, some may turn out to be false negatives arising from transsplicing, tandem duplication or template switching during reverse transcription [786], [827], [828]. However, all the rest is expected to serve some form of biological function, which is likely assigned to various cellular processes, as already yet circRNAs were detected to regulate gene expression on transcriptional, post-transcriptional and translational levels in diverse gene regulatory networks. circRNA expression is especially high in brain in terms of both ratio of circular to linear transcripts as well as isoform diversity. Further, circRNA expression peaks during synaptogenesis, particularly in genes with synaptic function and circRNAs are regulated according to synaptic plasticity. Hence, circRNAs are assumed to function in synaptic processes probably retaining modifications, proteins or RNAs representing the local history of the synapse, where they might play a role in memory retention due to their high stability [829], [830]. But in addition to circular back-splicing, also alternative splicing is particularly widespread in the brain and the nervous system, where a higher complexity of splicing events is achieved by microexons, cryptic exons and retained introns [831]. The function of these neural alternative splicing patterns is also mainly unknown. Hence, the challenge is to decipher how the complexity of the neural transcriptome reflects neuronal functions eventually shaping behavior, learning, and other complex processes of the brain. As circRNAs might function in various cellular processes their misregulation is expected to be involved in various diseases. Evidences already emerged that circRNAs are differentially regulated in pathogenic conditions. In cancer cells for instance, fusion-circRNAs were even identified to originate from chromosomal translocations and reported to promote tumorigenesis and therapy resistance [832]. In this regard it will be important to not only uncover circRNA functions but also the mechanisms of circRNA degradation, nuclear export and localization to develop strategies to control misregulated circRNA expression. The vision of using circRNAs themselves as therapeutic agents to treat patients with a stable, biological molecule are appealing but so far it was found that foreign circRNAs, which were not generated by the cellular spliceosome, triggered an immune response and immune regulators were also implicated in endogenous circRNA expression [833]–[835]. Hence, the interaction of circRNAs with the immune system needs to be clarified in advance of therapeutic developments.

1.6 Aim of the study The aim of this study was to identify non-canonically translated transcripts to gain insights into alternative mechanisms of translation initiation in eukaryotes. Since the discovery of viral IRESs, a growing number of eukaryotic mRNAs was reported to be translated by a cellular IRES- 115 dependent mechanism. As eukaryotic mRNAs are capped, putative IRES elements were hypothesized to constitute a failsafe mechanism by which translation can be maintained or stimulated under conditions in which cap-dependent translation is compromised as for example in stress, mitosis, development or tumorigenesis [40], [505], [836]. Despite great scientific efforts and new technological developments, the existence of cellular IRESs is under constant debate, as well-controlled and extensive characterization of biochemical, structural and mechanistic features of potential cellular IRESs is not achieved yet [27], [64], [72], [837]–[839]. Thus, eukaryotic cap- independent translation was systematically analyzed in this study to elucidate its contribution to global gene expression during early neuronal differentiation. Generating a solid experimental dataset, has the potential to provide insights into the regulation of cellular cap-independent translation initiation as well as to enable the identification of functional sequence elements and trans-acting factors. This could add valuable scientific evidence to the research field to elucidate the status of cellular IRESs. As circRNAs have lately been identified as a new functional class of cellular RNAs, another aim of this study was to investigate potential IRES-mediated translation of these molecules. circRNAs mainly consist of CDS and they are predominantly located in the cytoplasm. Hence, it was hypothesized that circRNAs can be translated like their linear counterparts. It was shown previously that ribosomes are in principle feasible to initiate internal translation on artificial circularized RNAs in vitro and recent data on ectopically expressed GFP- coding circRNAs suggests that internal translation initiation also functions in vivo (although control experiments were missing to exclude protein synthesis from minigene-derived linear concatemers) [797], [800]. As a collaborating research group found evidence for the translation of endogenously expressed circRNAs in fly, it was the aim of this study to investigate IRES- mediated translation of one candidate circRNA in vitro to provide the proof of principle that an endogenous circRNA can be translated. Further, it was the aim to evaluate the contribution of cap-dependent, 5’-end-dependent and IRES-dependent translation to the overall translation potential of the circRNA producing transcript.

2 MATERIAL AND METHODS

2.1 Material 2.1.1 Chemicals and enzymes Reagent Manufacturer Catalogue # 2-Mercaptoethanol (50 mM) Thermo Fisher Scientific 31350010 3’-O-Me-m7G(5’)ppp(5’)G RNA Cap New England Biolabs GmbH S1411L Structure Analog Acetic acid Sigma-Aldrich 537020 116

Acetonitrile FUJIFILM Wako Pure Chemical Corporation Acrylamide/Bis Solution, 37.5:1 Serva Electrophoresis GmbH 10688 Acrylamide/Bis Solution (40 %) Bio-Rad Laboratories, Inc. 1610144 Agarose basic AppliChem GmbH A8963,0500 Albumin Fraktion V Carl Roth GmbH + Co. KG 8076.4 Amino acid mixture Promega Corporation L446A Ammonium bicarbonate Sigma-Aldrich 09830 Ammonium persulfate Sigma-Aldrich A3678 Ampicillin Sigma-Aldrich 10835242001 Antarctic Phosphatase New England Biolabs GmbH M0289 ATP Solution (100 mM) Thermo Fisher Scientific R0441 Creatine Kinase Sigma-Aldrich 10127566001 Creatine Phosphatase Sigma-Aldrich 10621714001 DNase I AppliChem GmbH A3778,0010 dNTP Mix, 10 mM each Thermo Fisher Scientific 11853933 Doxycycline (hyclate) Cayman Chemical Cay14422-5 DpnI New England Biolabs GmbH R0176S DreamTaq DNA Polymerase Thermo Fisher Scientific EP0702 Ethanol undenatured absolute Serva Electrophoresis GmbH 39556 Ethidium bromide 1 % (w/v) Carl Roth GmbH + Co. KG 2218.1 FastAP Thermosensitive Alkaline Thermo Fisher Scientific EF0651 Phosphatase Formic acid FUJIFILM Wako Pure Chemical Corporation G(5’)ppp(5’)A RNA Cap Structure Analog New England Biolabs GmbH S1406S Geneticin (G418) Genaxxon bioscience GmbH M3118.0050 GlycoBlue Coprecipitant (15 mg/mL) Thermo Fisher Scientific AM9516 HEPES Sigma-Aldrich H3375 Immobilized Trypsin Applied Biosystems Iodoacetamide Sigma-Aldrich I1149 LB-Agar (Luria/Miller) Carl Roth GmbH + Co. KG X969.3 Lipofectamine 2000 Reagent Life Technologies 11668-027 Lithium chloride Thermo Fisher Scientific 13207069 Lysyl endopeptidase FUJIFILM Wako Pure 125-05061 Chemical Corporation Nonfat dried milk powder AppliChem GmbH A0830 Novex TBE-Urea Sample Buffer (2x) Thermo Fisher Scientific LC6876 OmniPur DTT Merck Millipore 3870-25 PageRuler Plus Prestained Protein Ladder Thermo Fisher Scientific 26620 peqGOLD TriFast VWR International, LLC 30-2010 peqGOLD TriFast FL VWR International, LLC 732-3314 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F-530S Ponceau S Sigma-Aldrich P3504 Potassium acetate Sigma-Aldrich P1190 Potassium hydroxide Sigma-Aldrich 757551 Ribonuclease R Epicentre RNR07250 Roti-Phenol/Chloroform/Isoamyl alcohol Carl Roth GmbH + Co. KG A156.2 RQ1 RNase-Free DNase Promega Corporation M6101 SDS for molecular biology AppliChem GmbH A2263

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SequaGel Urea Gel Concentrate Biozym Scientific GmbH 900001 SequaGel Urea Gel Diluate Biozym Scientific GmbH 900002 SequaGel Urea Gel Buffer Biozym Scientific GmbH 900003 Sodium Acetate (3 M, pH 5.5, RNase-free) Thermo Fisher Scientific AM9740 Spermidine Sigma-Aldrich 85558 SYBR Gold Nucleic Acid Gel Stain Thermo Fisher Scientific S11494 (10,000 x) T4 DNA Ligase Thermo Fisher Scientific EL0014 T4 Polynucleotide Kinase Thermo Fisher Scientific EK0032 T4 RNA Ligase 2, truncated K227Q New England Biolabs GmbH M0351L TEMED 99 %, p.a. Th. Geyer GmbH & Co. KG 2367.3 tRNA from bovine liver Sigma-Aldrich R4752 Tween 20 Sigma-Aldrich P9416 Trifluoroacetic acid Sigma-Aldrich 302031 TrypLE Express Enzyme (1x) Thermo Fisher Scientific 12605036 UltraPure 0.5 M EDTA, pH 8.0 Thermo Fisher Scientific 15575020 Urea Sigma-Aldrich 666122

2.1.2 Buffers solutions and media Medium Composition Manufacturer Catalogue # 80/20 80 % 2i for mESCs medium 20 % mESC medium 2i for mESCs 50 % Advanced DMEM/F-12 10x Thermo Fisher Scientific 12634028 50 % Neurobasal medium Thermo Fisher Scientific 21103049 1x N-2 Supplement (100X) Thermo Fisher Scientific 17502048 1x B-27 Supplement (50X) Thermo Fisher Scientific 17504044 2 mM L-Glutamine Sigma-Aldrich G7513 0.1 mM β-Mercaptoethanol, Thermo Fisher Scientific 31350010 103 U/mL Leukemia Inhibitory Amsbio AMS-263- Factor (LIF) 100 3 µM CHIR99021 (GSK-3 Sigma-Aldrich inhibitor) Miltenyi Biotec GmbH 361559 1 µM PD0325901 (MEK 130-104-170 inhibitor) AK 45 % Advanced DMEM/F12 Thermo Fisher Scientific 12634028 differentiation 45 % Neurobasal medium Thermo Fisher Scientific 21103049 medium 10 % KnockOUT Serum Thermo Fisher Scientific 10828028 Replacement 0.1 mM β-Mercaptoethanol Thermo Fisher Scientific 31350010 2 mM L-Glutamine Sigma-Aldrich G7513 Feeders KnockOut DMEM Thermo Fisher Scientific 10829018 medium 10 % ESC FBS, qualified Thermo Fisher Scientific 16141079 2 mM L-GLutamine Sigma-Aldrich M3148 Freezing 50 % ESC FBS, qualified Thermo Fisher Scientific 16141079 medium 40 % 80/20 medium 10 % dimethyl sulfoxide Genaxxon Bioscience M6323.0100 HEK medium DMEM, high glucose, GlutaMAX Thermo Fisher Scientific 61965026 10 % Fetal Bovine Serum Thermo Fisher Scientific 10270-106 LB medium 25 g/L LB-Medium (Luria/Miller) Carl Roth GmbH + Co. X968.3 Millipore water KG 118 mESC KnockOut DMEM Thermo Fisher Scientific 10829018 medium 12 % ESC FBS, qualified Thermo Fisher Scientific 16141079 0.1 mM β-Mercaptoethanol Thermo Fisher Scientific 31350010 2 mM L-Glutamine Sigma-Aldrich G7513 1x MEM Non-Essential Amino Sigma-Aldrich M7145 Acids Solution (100X) 1x Nucleosides (100X) Sigma-Aldrich ES-008-D 103 U/mL LIF Amsbio AMS-263- 100 Monolayer Advanced DMEM/F-12 10x Thermo Fisher Scientific 12634028 differentiation 1x N-2 Supplement (100X) Thermo Fisher Scientific 17502048 medium 1x B-27 Supplement (50X) Thermo Fisher Scientific 17504044 Transfection Opti-MEM Reduced Serum Thermo Fisher Scientific 31985062 assembly Medium medium

Buffer/Solution Composition Manufacturer Catalogue # Annealing buffer 20 mM TrisHCl pH 7.5 Carl Roth GmbH + Co. 9090 25 mM NaCl KG A2942 10 mM MgCl2 AppliChem GmbH M2670 Sigma-Aldrich Antarctic 50 mM Bis-Tris-Propane- New England Biolabs B0289S Phosphatase HCl GmbH Reaction Buffer 1 mM MgCl2 0.1 mM ZnCl2 CutSmart Buffer 50 mM Potassium Acetate New England Biolabs B7204S 20 mM Tris-acetate GmbH 10 mM Magnesium Acetate 100 µg/ml BSA DNA gel-loading 10 % glycerol Sigma-Aldrich G5516 dye (10x) 20 % SDS AppliChem GmbH A2263 0.5 M Na2EDTA Sigma-Aldrich E5134 1 % (w/v) OmniPur Sigma-Aldrich 2830-OP bromophenol blue 1 % xylene cyanol FF Sigma-Aldrich X4126 Firefly Luciferase 75 mM Hepes (pH 8.0) Sigma-Aldrich H3375 Reagent 0.1 mM EDTA 4 mM MgSO4 530 µM ATP Sigma-Aldrich A2383 270 µM Coenzyme A PJK GmbH 102211 470 µM DTT Merck Millipore 3870-25 470 µM D-Luciferin PJK GmbH 102212 Gelatine solution 0.1 % (w/v) gelatine in H2O Sigma-Aldrich G1393 HF buffer Thermo Fisher Scientific F-518 Renilla Luciferase 2.2 mM Na2EDTA Sigma-Aldrich E5134 Reagent 220 mM K3PO4 (pH 5.1) Sigma-Aldrich P5629 0.44 mg/mL BSA Sigma-Aldrich A9418 1.1 M NaCl AppliChem GmbH A2942 1.3 mM NaN3 0.6 µg/mL Coelenterazine PJK GmbH 102171 (in methanol) 119

RNase R Reaction 20 mM Tris-HCl (pH 8.0) Epicentre Buffer 100 mM KCl 0.1 mM MgCl2 RQ1 DNase 40 mM Tris-HCl (pH 8.0) Promega Corporation M198A reaction buffer 10 mM MgSO4 10 mM CaCl2 RQ1 DNase Stop 20 mM EGTA (pH 8.0) Promega Corporation M199A Solution SDS loading 300 mM Tris-HCl (pH 6.8) Carl Roth GmbH + Co. 9090 buffer 50 % glycerol KG G5516 10 % (w/v) SDS Sigma-Aldrich A2263 5 % (v/v) 2- AppliChem GmbH 31350010 mercaptoethanol Thermo Fisher Scientific SDS-Page running 25 mM Trizma base Th. Geyer GmbH & Co. 93362 buffer 190 mM glycine KG 23391 0.1 % (w/v) SDS Serva Electrophoresis A2263 GmbH AppliChem GmbH T4 DNA Ligase 400 mM Tris-HCl Thermo Fisher Scientific B69 Buffer 100 mM MgCl2 100 mM DTT 5 mM ATP T4 RNA Ligase 50 mM Tris-HCl New England Biolabs B0216L Reaction Buffer 10 mM MgCl2 GmbH 1 mM DTT TBE buffer (pH 445 mM Serva Electrophoresis 37186.04 8.3) Tris(hydroxymethyl)- GmbH aminomethane 15166.02 445 mM boric acid Serva Electrophoresis E5134 10 mM Na2EDTA (pH 8.0) GmbH Sigma-Aldrich TBS (pH 7.5) 25 mM Tris Serva Electrophoresis 37186.04 150 mM NaCl GmbH A2942 AppliChem GmbH Towbin buffer 25 mM Tris Serva Electrophoresis 37186.04 192 mM glycine (pH 8.3) GmbH 23391 20 % (v/v) MeOH Serva Electrophoresis 4627 GmbH Carl Roth GmbH + Co. KG Urea Buffer 8 M Urea Sigma-Aldrich Sigma- 666122 0.1 M Tris (pH 8.5) Aldrich 37186.04 Serva Electrophoresis GmbH

2.1.3 Bacterial strains and eukaryotic cell lines Strain Species Genotype DH5α E. coli F- Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 - + - hsdR17(rK ,mK ) glnV44 deoR nupG purB20 thi-1 gyrA96 relA1 λ XL1-blue E. coli endA1 gyrA96(nalR) thi-1 recA relA1 glnV44 lac [F’ proAB

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q r - + lacl ZΔ-M15 Tn10(Tet )] hsdR17(rK ,mK )

Cell line Species Source HEK 293 H. sapiens Selbach Lab, MDC Berlin A17 iCre mESC M. musculus Mazzoni Lab, New York University

2.1.4 Antibodies Target Host Manufacturer Catalogue # anti-α-Tubulin Rabbit New England Biolabs GmbH 2125S anti-mouse IgG (HRP conjugated) anti-rabbit IgG (HRP conjugated anti-V5 Tag Mouse Thermo Fisher Scientific R960-25

2.1.5 Plasmids Name Backbone Insert Purpose pciRS-RLuc- pcDNA3 CDR1as upstream, expression vector giving rise to a IRES-wt RLuc, EMCV-R IRES, linear and a circular RNA reverse complement transcript with IRES-driven RLuc CDR1as upstream expression from the circular transcript pGEX-6p-1- pGEX-6P-1 dominant negative cloning of constitutively active h4E-BP1-T37A- human 4E-BP1 4E-BP1 into mammalian T46A-S65A- expression vectors T70A p2lox-GFP-C- P2lox GFP GFP positive control vector His-V5 during generation of stable mESCs with dox-inducible expression, using master line from Mazzoni p2lox-Ascl1-C- P2lox Ascl1 vector for generation of stable His-V5 mESCs with dox-inducible expression, using master line from Mazzoni pS2f-IMCg-F3- p2lox-GFP- dominant negative plasmid to generate stable mESC 4EBP1-4Ala C-His-V5 human 4E-BP1 with inducible expression of Ascl1 and const. active 4E-BP1 pSF2.GFPLuc S2f-(lMCg) pSF2.GFPLuc S2f-(lMCg) [2] [2] pEbg-BioID pEBG BioID Used as backbone to integrate 4E-BP1 for co-transfection assays pciNeo-Fluc- pCiNeo FLuc, HCV IRES Bicistronic reporter assays HCV-Rluc-boxB pciNeo-Fluc- pCiNeo FLuc, EMCV IRES Bicistronic reporter assays (used EMCV-Rluc- as positive control) boxB pciNeo-Fluc- pCiNeo FLuc, EMCV IRES Bicistronic reporter assays (used EMCVmut- mutated (TCCA tetra as negative control) 121

Rluc-boxB loop) pFluc-high pRL-3Xb FLuc Used as negative control in expression bicistronic reporter assays with pRL-5boxB RLuc CDS, 5xboxB in siRNA co-transfection 3'UTR pEbg-4EBP pEBG dominant negative 4E- high expression vector for BP1 dominant negative 4E-BP to be used to decrease global cap- dependent translation, used for co-transfection in bicistronic reporter assays pEBG-GST pEBG Used as negative control in bicistronic reporter assays with 4E-BP1 co-transfection

2.1.6 Primer Name Sequence Purpose EMCV ACCTGTCCACAGGTGCCTCT primers for site directed mutagenesis to IRES GCGGC mutate the GCGA tetraloop at 547-550 nt of TCCA 547- EMCV IRES of the ciRS-eGFP-IRES-wt 550-fwd plasmid into TCCA EMCV ACCTGTGGACAGGTGGGG IRES GGTTCCG TCCA 547- 550-rev SalI-4E- ATA GTC GAC ATG TCC to amplify 4E-BP1 from pGEX-6p1-h4E- BP1-fwd GGG GGC AGC BP1-T36A-T46A-S65A-T70A and insert it BamHI-4E- ccggcgGGATCCTTAAATGTCC into SalI-BamHI-cut pSF2.GFPLuc BP1-rev ATCTC TTACTCAAACTGGCTGGGG used in Gibson assembly: 1) p2lox-GFP-C- F2A-SbfI- ATGTAGAAAGCAATCCAGG His-V5 digested with EcorI and 4EBP1- TCCAcctgcaggtATGTCCGGGG dephosphorylated 2) Ascl1 amplified from 4Ala-fwd GCAGC p2lox-Ascl1-C-His-V5 with oligos F2A-GST- p2lox- cgggctgcaggaattgctagcAATGTCC fw & F2A-Ascl1-rev (GC buffer, 3% DMSO, NheI- ATCTCAAACTGTGACTCT Annealing Temp 72°C) 3) 4EB-P1-4Ala 4EBP1- amplified from pGEX-6p1-h4E-BP1-T36A- 4Ala-rev T46A-S65A-T70A with oligos F2A-SbfI- F2A-Ascl1- AGCCAGTTTGAGTAAATCA 4EBP1-4Ala-fwd & p2lox-NheI-4EBP1- rev AAGTTAAGAGTTTGTTTGA 4Ala-rev 4) assemble via Gibson CAGGgaaccagttggtaaagtcc F2A-GST- TTACTCAAACTGGCTGGGG fw ATGTAGAAAGCAATCCAGG TCCAatggcccctatactagg ATA CTT TCT CGG CAG to be used for genotyping of stable A17iCre Loxin fwd GAG CA mESCs clones generated with p2Lox plasmid CTA GAT CTC GAA GGA [840] Loxin rev TCT GGA G CCAGTGTCTGCAATATCCA oligos to produce PCR template for T7 GGGTTTCCGATGGCACCTG transcription to produce cirRNAs in vitro, cirC-3'ss-fw TGTC pciRS plasmids used as template with primers

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CACAGGTGCCATCGGAAAC cirC-3'ss-fw & cirS-rev and T7-cirS-fw & cirC-5'ss- CCTGGATATTGCAGACACT cirC-5'ss-rev; PCR products were purified rev GGAAGACC from gel, mixed and used as templates in the TAATACGACTCACTATAGG next PCR reaction with T7-cirS-fw & cirS-rev GGTCGACATAAGCTTGATA as primers T7-cirS-fw TCG cirS-rev TCActgctcgttcttcag T7-MBL1 TAATACGACTCACTATAGG PCR primers to produce DNA template for (fly)-utr-fw GACTCAGCTTACACACAAAA in vitro transcription of circMBL-RLuc AAGCAGTAAAAT reporter. Use T7-MBL1 (fly)-utr-fw & MBL1 MBL1 (fly)- GGGTCGTACACCTTGGAAG (fly)-UTR-Rluc-rev to amplify UTR of UTR-Rluc- CCATCGTATTTTGGGGTTA circMBL1 (Ch2R:13162914-13163568). Mix rev TAGATATATAACTATTTGCT PCR product with the product of Rluc-fw & Rluc-fw ATGGCTTCCAAGGTGTACG STOP-NheI-TEV-bamHi-Rluc-rev (pciRS- ACCC RLuc-IRES-wt as template) and use as template with T7-MBL1 (fly)-utr-fw & STOP-NheI-TEV-bamHi-Rluc-rev as primers. cirS- tgctgaagaacgagcagTGAgggGTCG bridging oligo for circularization of RNA bridging ACATAAGCTTGATATC products produced with T7-cirS-fw & cirS- oligo rev T7- GAAATTAATACGACTCACTA primers to be used with pciRS-RLuc-IRES-wt mbl20ntUT TAGGGATCTATAACCCCAA as template, to generate a version of RLuc R-fw AATACG reporter with the shortened UTR (=20nt) STOP- tcaGCTAGCTCCTTGAAAATA upstream of mbl AUG NheI-TEV- CAAATTTTCggatccctgctcgttcttc bamHi- agcacgcg Rluc-rev bridging ATTTTGGGGTTATAGATCC bridging oligo for circularization of Mbl1- oligo CTTAGCTAGCTCCTTGAAAA 20ntUTR-RLuc RNA, amplified with primers mbl20ntUT T T7-mbl20ntUTR-fw & STOP-NheI-TEV- R bamHi-Rluc rev T7-MBL1 TAATACGACTCACTATAGG PCR primers to produce template for in vitro (fly)- GCGTATTTTGGGGTTATAG transcription of negative control circMBL- reverseUTR ATATATAACTATTTGC RLuc reporter (with mbl UTR in reverse -fw orientation. T7-MBL1 (fly)-reverseUTR-fw & GGGTCGTACACCTTGGAAG MBL1 (fly)-reverseUTR-Rluc-rev used to MBL1 (fly)- CCATCGTATTACTCAGCTTA amplify UTR of circMbl. PCR product mixed reverseUTR CACACAAAAAAGCAGTAAA with the product of Rluc-fw & STOP-NheI- -Rluc-rev AT TEV-bamHi-Rluc-rev (pciRS-RLuc-IRES-wt Rluc-fw ATGGCTTCCAAGGTGTACG as template) and used as template with T7- ACCC MBL1 (fly)-reverseUTR-fw & STOP-NheI- TEV-bamHi-Rluc-rev as primers. bridging TATAACCCCAAAATACGCCC bridging oligo for circularization of Mbl1- oligo MBL1 TTAGCTAGCTCCTTGAAAAT reversedUTR-RLuc RNA, amplified with (fly)- primers T7-MBL1 (fly)-reverseUTR-fw & reverseUTR stop-TEV-2'-methoxyl-rev stop-TEV- TmTmAGCTAGCTCCTTGAA for in vitro transcription template: used with 2'- AATACAAATT T7-MBL1 (fly)-utr-fw or T7-mbl20ntUTR-fw methoxyl- (instead of STOP-NheI-TEV-bamHi-Rluc- rev rev) to produce 2'-methoxyl- template for in vitro transcription, to avoid non-template G 123

addition EcoRI- tatatagaattcACG CAT TCT oligos to clone bicistronic luciferase reporter: Ccdc47 fwd ACG TAA CGG ACG G primers to amplify 5'UTR of Ccd47 tatagtcgacTGC ACC TTA AAA (ENSMUST00000172702), digested with SalI-Ccdc47 ACA AGC TGC ACC EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcGCGCGGGGGTG oligos to clone bicistronic luciferase reporter: Cdkn1c fwd GG primers to amplify 5'UTR of Cdkn1 tatagtcgacCGC TGT TCT GCT (ENSMUST00000037287.7), digested with SalI-Cdkn1c GGC TGA TTG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcCCG GGG TTC oligos to clone bicistronic luciferase reporter: Check1 fwd GCA TGC TTT C primers to amplify 5'UTR of Check1 tatagtcgacGAC TCC AAG CAC (ENSMUST00000172702), digested with SalI-Check1 AGC GAC TGT AAA ATG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcTCC CTC TCG GTG oligos to clone bicistronic luciferase reporter: Dclk2 fwd TGG TTT GTG primers to amplify 5'UTR of Dclk2 tatagtcgacCGC GGC TGC TCG (ENSMUST00000029719), digested with SalI-Dclk2 GGA G EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcCGG GGG CGC oligos to clone bicistronic luciferase reporter: Ero1lb fwd GCA G primers to amplify 5'UTR of Ero1lb tatagtcgacGGT GAC CTC GGC (ENSMUST00000071973), digested with SalI-Ero1lb CGC T EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcCGC AGC TCG TTG oligos to clone bicistronic luciferase reporter: Gtf2a1 fwd GCT CGC primers to amplify 5'UTR of Gtf2a tatagtcgacTTC CAC ACA CAA (ENSMUST00000021345), digested with SalI-Gtf2a1 CAC AAA CAA GAG GG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI-Palld tatatagaattcGGA CAG GCA oligos to clone bicistronic luciferase reporter: fwd AGA CCG ACC TC primers to amplify 5'UTR of Palld tatagtcgacATT TGA GCA ATC (ENSMUST00000121785), digested with SalI-Palld TCC ACA CAC TCG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcGGC GCA CAC ACT oligos to clone bicistronic luciferase reporter: Pcdh1 fwd AGG CC primers to amplify 5'UTR of Pcdh1 tatagtcgacCCT GGC AGG CTC (ENSMUST00000057185), digested with SalI-Pcdh1 CAG AAT CAG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI-Pex5 tatatagaattcTCC CTT CCC CCA oligos to clone bicistronic luciferase reporter: fwd GCC C primers to amplify 5'UTR of Pex5 tatagtcgacGGT GAC CAG CAG (ENSMUST00000112532), digested with SalI-Pex5 CTT CCC G EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcGGG TCC AGA oligos to clone bicistronic luciferase reporter: Riok1 fwd GGA AGA AGG TCA G primers to amplify 5'UTR of Riok1 tatagtcgacGGC TAG GGG TGT (ENSMUST00000021866), digested with SalI-Riok1 GAG CCT TC EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI-Tfe3 tatatagaattcGAG GAG GGG oligos to clone bicistronic luciferase reporter: 124 fwd GAA GAG GAC GAG primers to amplify 5'UTR of Tfe3 tatagtcgacGGC AGG GGT CCG (ENSMUST00000137467), digested with SalI-Tfe3 GGA G EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcCTG CTC TCT GTT oligos to clone bicistronic luciferase reporter: Zfp808 fwd ACT CTC CGT TGC primers to amplify 5'UTR of Zfp808 tatagtcgacGTC GCC ACC TTG (ENSMUST00000099449), digested with SalI-Zfp808 TAG ATC GCT G EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcCGG TGT TGG CTT oligos to clone bicistronic luciferase reporter: Fam96b CCT GCG ATC primers to amplify 5'UTR of Fam96b fwd (ENSMUST00000164884), digested with SalI- tatagtcgacCCC GGA ACC GCG EcoRI & SalI, cloned into pciNeo-Fluc- Fam96b rev GTT CC HCV-Rluc-boxB (EcoRI & SalI cut) aattGGTTCCGAGACATCGCC annealed oligos for oligo cloning and ligated Hsbp1 1 GCCAAGCTGGGCACCGGG into pciNeo-Fluc-HCV-Rluc-boxB (EcoRI & GAG SalI cut) tcgaCTCCCCGGTGCCCAGCT Hsbp1 2 TGGCGGCGATGTCTCGGAA CC EcoRI- tatatagaattcACT CGC GCA GTG oligos to clone bicistronic luciferase reporter: Lama1 fwd CCT GTG primers to amplify 5'UTR of Lama1 tatagtcgacGTT GTC GCC GCC (ENSMUST00000035471), digested with SalI-Lama1 GCT CC EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcGAG GGC GGG oligos to clone bicistronic luciferase reporter: Sh3glb2 CGC AGG primers to amplify 5'UTR of Sh3glb2 fwd (ENSMUST00000028214), digested with SalI- tatagtcgacGGC GCG CCC GTA EcoRI & SalI, cloned into pciNeo-Fluc- Sh3glb2 rev CGC HCV-Rluc-boxB (EcoRI & SalI cut) ACG GAA AAG CAG AAC primers used for nested PCR to amplify the Dclk2 Fwd CTC GT region around Dclk2 5'UTR AAG AGG AAA ACG TGT (ENSMUST00000029719) Dclk2 Rev TAC GGT TCT Sh3glb2 GCAGTGGGCAAACTTGTCA primers used for nested PCR to amplify the Fwd G region around Sh3glb2 5'UTR Sh3glb2 AGT GTT ACA GTG CAT (ENSMUST00000028214) Rev CCG CA CTCGTGGTCTACAGGCCCG used with oligo EcoRI-Cdkn1c fwd to amplify Cdkn1c 5'UTR (ENSMUST00000037287.7) and parts of 1st Cdkn1c Rev exon CCTGGAAAGAAGTCAGCAC used with oligo EcoRI-Palld fwd to amplify TTTTG Palld 5'UTR (ENSMUST00000121785) and Palld Rev parts of 1st exon CACAGAGATGGCTGGTGAG used with oligo EcoRI-Tfe3 fwd to amplify GC Tfe3 5'UTR (ENSMUST00000137467) and Tfe3 Rev parts of 1st exon EcoRI- tatatagaattcGTA CCC CAC CCC oligos to clone bicistronic luciferase reporter: Atp5e fwd TTC CGC primers to amplify 5'UTR of Atp5e SalI-Atp5e tatagtcgacGGT GTC TCG AGC (ENSMUSG00000016252), digested with

125 rev GGG CG EcoRI & SalI, cloned into pciNeo-Fluc- HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcTTG CTC TCC TTC oligos to clone bicistronic luciferase reporter: Brwd3 fwd CTC CTC CGC primers to amplify 5'UTR of Brwd3 tatagtcgacCCT TTT CCC GAG (ENSMUST00000150434), digested with SalI-Brwd3 GGG GTT TGG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcGAG ATG GGA oligos to clone bicistronic luciferase reporter: Lsm6 fwd AAG CCG CTG CCG primers to amplify 5'UTR of Lsm6 tatagtcgacGTT TAA CAA CCT (ENSMUST00000130325), digested with SalI-Lsm6 TGG GAG GCA GGA AAC C EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) aattcGCAGAGCGACGTGAGG annealed oligos for oligo cloning and ligated Lsm7 1 CGACAAG into p1581 (EcoRI & SalI cut) tcgacCTTGTCGCCTCACGTCG Lsm7 2 CTCTGC EcoRI- tatatagaattcGTC AGT CTG oligos to clone bicistronic luciferase reporter: Luzp1 fwd GGG GAG CCT G primers to amplify 5'UTR of Luzp1 tatagtcgacGTC TAC AGC CAG (ENSMUST00000170102), digested with SalI-Luzp1 TCA CGA TGG G EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcTGT GCT TTA AAC oligos to clone bicistronic luciferase reporter: Pqbp1 fwd CAA GAG CCC TTC GG primers to amplify 5'UTR of Pqbp1 tatagtcgacAGT GGG TAG CTG (ENSMUST00000115654), digested with SalI-Pqbp1 ACC AGA CCT AGA AAC EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcAGG AGC GTG oligos to clone bicistronic luciferase reporter: Rad51 fwd AGG ATT TGG CG primers to amplify 5'UTR of Rad51 tatagtcgacGAC TGT CCC GCG (ENSMUST00000028795), digested with SalI-Rad51 GCC AC EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcATT GGC CGA TCA oligos to clone bicistronic luciferase reporter: Stmn2 fwd ATA TTT AAT GCT TGG AG primers to amplify 5'UTR of Stmn2 tatagtcgacTGT AGG GAT GTG (ENSMUST00000029002), digested with SalI-Stmn2 CAC GCA CG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) EcoRI- tatatagaattcAGG CGT GCA oligos to clone bicistronic luciferase reporter: Znhit6 fwd GAG CTT GCC primers to amplify 5'UTR of Znhit6 tatagtcgacGAG CTC ACG AGC (ENSMUST00000098534), digested with SalI-Znhit6 CTA GGC CG EcoRI & SalI, cloned into pciNeo-Fluc- rev HCV-Rluc-boxB (EcoRI & SalI cut) tatatagaattcAGG CCG AGT oligo to clone bicistronic luciferase reporter: GCG CTG TG amplify shorter 5'UTR isoform of Cdkn1c (ENSMUSG00000037664) together with EcoRI-Cdkn1c fwd, digested with EcoRI & EcoRI- SalI, cloned into pciNeo-Fluc-HCV-Rluc- Cdkn1c fwd boxB (EcoRI & SalI cut) EcoRI- tatatagaattcTGG AGC AAG used with SalI-Riok1 rev to generate deletion Riok1del1- AAC GAA AGG CTG GG mutants of Riok1 5'UTR: digested PCR fwd product with EcoRI+SalI and ligated into EcoRI- tatatagaattcCTT TCC CGG CCA EcoRI+SalI cut dephosphorylated pciNeo-

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Riok1del2- CCG TCA C Fluc-HCV-Rluc-boxB fwd EcoRI- tatatagaattcTGGCCGCGTTTGC Riok1del3- CGAAAC fwd EcoRI- tatatagaattcGGC GGG AAT Riok1del4- CGC GGA GAC G fwd EcoRI- tatatagaattcAGGGACGCCCCG used with SalI-Fam96b rev to generate Fam96bdel1 GCGAGA deletion mutants of Fam96b 5'UTR: digested -fwd PCR product with EcoRI+SalI and ligated EcoRI- tatatagaattcCGCACGACAAATA into EcoRI+SalI cut dephosphorylated Fam96bdel2 CACCGCTTCTAGC pciNeo-Fluc-HCV-Rluc-boxB -fwd EcoRI- tatatagaattcTCC CTT GCA CGA Fam96bdel3 CCC CGT AGC -fwd EcoRI- tatatagaattcGCG GAA GGA Fam96bdel4 GCG GAA GTA CAC ACC -fwd NheI-4Ebp tatgctagcATG TCC GGG GGC primers to clone 4E-BP1(4Ala) into pciNeo fwd AGC vector. Amplified 4E-BP1(4Ala) from pS2f- tatatgcggccgcAA TGT CCA TCT IMCg-F3-4EBP1-4Ala, digested with NheI & NotI-4Ebp CAA ACT G NotI and cloned into pEbg-BioID NheI & rev NotI cut T7- GAAATTAATACGACTCACTA primers to generate monocistronic sequence- bicistronic- TAaattcgctagc of-interest-RLuc DNA amplicons for in vitro reporters- transcription universal- Fwd bicistronic- CCTCGAGATAATATCCTCGA reporters- TAGGG universal- Rev GAAATTAATACGACTCACTA T7-EMCV- TACCGCCCCCCCCCCCTAAC IRES-Fwd G GAAATTAATACGACTCACTA T7-Pqbp1- TATGTGCTTTAAACCAAGA Fwd GC bicistronic- CCTCGAGATAATATCCTCGA reporters- TAGGGCCC Rev mmActb- GGCTGTATTCCCCTCCATCG qPCR fwd mmActb- CCAGTTGGTAACAATGCCA rev TGT mmPou5f1 CACCATCTGTCGCTTCGAG qPCR fwd G mmPou5f1 AGGGTCTCCGATTTGCATA rev TCT

127 mmSox1 CACAACTCGGAGATCAGCA qPCR fwd A mmSox1 TGTAATCCGGGTGTTCCTTC rev mmTubb3 TAGACCCCAGCGGCAACTA qPCR fwd T mmTubb3 GTTCCAGGTTCCAAGTCCA rev CC mm4E-BP1 AGCGAGTGGACTCAGAGG qPCR fwd AG mm4E-BP1 AAGTGTGGCTTTCCCAGGT rev A AttB2 fwd CGCGCCGACCCAGCTTTC qPCR GAG ACC GAG GAG AGG V5-tag rev GTT A

2.1.7 Kits Kit Manufacturer Catalogue # GeneJET Gel Extraction Kit Thermo Fisher Scientific K0692 GeneJET PCR Purification Thermo Fisher Scientific K0702 Kit Maxima First Strand cDNA Thermo Fischer Scientific K1641 Synthesis Kit NucleoBond Xtra Midi Plus Macherey-Nagel GmbH & Co. 740412.50 KG Pierce Coomassie (Bradford) Thermo Fisher Scientific 23200 Protein Assay Kit Plasmid Miniprep DNA EURx Molecular Biology E3500-02 Purificatoin Kit Products Qubit RNA HS Assay Kit Thermo Fisher Scientific Q32852 RNeasy Mini Kit Qiagen 74104 SensiFAST SYBR No-ROX Bioline BIO-98020 Kit TranscriptAid T7 High Yield Thermo Fisher Scientific K0441 Transcription Kit

2.1.8 Devices and consumables Name Manufacturer Catalogue # AlphaImager HP System ProteinSimple 92-13824-00 Bioruptor Plus sonication Diagenode s.a. B01020001 device Biosphere Filter Tips Sarstedt AG & Co. KG 70.762.211, 70.1130.210, 70.760.211 BRAND microcentrifuge Sigma-Aldrich Z334006-1PAK, BR780546- tube 500EA BRAND PCR tubes Sigma-Aldrich BR781305-1000EA C1000 Touch Thermal Cycler Bio-Rad Laboratories, Inc. CELLSTAR cell culture Greiner Bio-One 664160, 639160 dishes International GmbH 128

Centrifuge 5424 Eppendorf AG 5405000514 Centrifuge 5424 R Eppendorf AG 5404000010 Centrifuge 5804 Eppendorf AG 5804000320 Centrifuge 5804 R Eppendorf AG 5804000320 Centro LB 960 Microplate Berthold Technologies 5811000325 Luminometer GmbH & Co. KG CFX96 Touch Real-Time Bio-Rad Laboratories, Inc. 1855196 PCR Detection System CO2 Incubator CB 220 BINDER GmbH Corning gel-loading pipet tips Sigma-Aldrich CLS4853-400EA Eksigent ekspert nanoLC 415 AB Sciex Eppendorf BioSpectrometer Eppendorf AG 6135000009 basic Eppendorf Combitips Eppendorf AG E-6406, E-6403 advanced Eppendorf DNA LoBind Eppendorf AG 0030108051 Tubes Eppendorf Research plus Eppendorf AG 3125000044 multichannel pipette Eppendorf Research plus Eppendorf AG 3123000020, 3123000039, single channel pipette 3123000055, 3123000063 Eppendorf Thermomixer Sigma-Aldrich T1317-1EA Compact Filter Top cell culture flasks Greiner Bio-One 690175, 658175 International GmbH Greiner centrifuge tubes Sigma-Aldrich T2318-500EA, T1943- 1000EA Immobilon-P Membrane, Merck Millipore IPVH00010 PVDF, 0.45 µM Innova 42 Incubator Shaker VWR International, LLC M1335-0010 Leica DM IL LED Leica Microsystems LUNA Automated Cell Logos Biosystems L10001 Counter LUNA Cell Counting Slides Logos Biosystems L20001 Mini-PROTEAN Tetra Cell Bio-Rad Laboratories, Inc. 1658000EDU Mini Trans-Blot Cell and Bio-Rad Laboratories, Inc. 1703989 PowerPac Basic Power Supply Multipette M4 Eppendorf AG 4982000012 Multi-well cell culture plates Greiner Bio-One 657160, 665180, 662160 International GmbH P-2000 Laser-Based Sutter Instrument Company P-2000/F Micropipette Puller System PCR tubes neoLab Migge GmbH 294981340, 294981320 Petri dishes Greiner Bio-One 632102 International GmbH PIPETBOY acu 2 INTEGRA Biosciences AG 612-0926 Q Exactive Plus Thermo Fisher Scientific Quali – “Low Retention” – Kisker Biotech GmbH & Co. G018 Microcentrifuge tubes KG Qubit 2.0 Fluorometer Thermo Fisher Scientific 1104004846 129

Qubit Assay Tubes Thermo Fisher Scientific Q32856 Reprosil Saphir 100 C18, 1.8 Dr. Maisch GmbH ra118.9e µm S1000 Thermal Cycler Bio-Rad Laboratories, Inc. 1852196 SafeSeal SurPhob Filter Tips Biozym Scientific GmbH VT0270, VT0230, VT0200 Serological pipettes Sarstedt AG & Co. KG 86.1252.025, 86.1253.025, 86.1254.025, 86.1685.020 Trans-Blot Turbo Transfer Bio-Rad Laboratories, Inc. 1704150 System Vacuum Filter 0.22 µm TPP Techno Plastic Products 99505 “rapid” Filtermax AG

2.1.9 Software and online tools Name Manufacturer Version AlphaView ProteinSimple CFX Manager Bio-Rad Laboratories, Inc. DinoXcope AnMo Electronics Corporation Fiji (ImageJ) Johannes Schindelin, Albert Cardona, Mark Longair, Benjamin Schmid, and others GOrilla [841] GraphPad Prism GraphPad Software Inc. 5 MaxQuant Cox & Mann [842] 1.5.3.30 Mendeley Elsevier 1.17.9 MikroWin Labsis Laborsysteme GmbH 2000 Perseus [843] 1.5.8.5 R 3.4.0 R Studio RStudio, Inc. 1.0.143 RNAfold Institute for Theoretical Chemistry, University Vienna Serial Cloner SerialBasics 2.6.1

2.2. Methods 2.2.1 Cell culture 2.2.1.1 Cell culture, passaging, freezing and thawing A17iCre mESCs and mESC clones with stable integration of dominant negative 4E-BP were grown in 80/20 medium as adherent monolayers at 37 °C, 5 % CO2. Medium was changed on a daily basis and cells were subcultered once or twice a week depending on the level of confluency. For passaging, cells were briefly washed in PBS and treated with TrypLE until cell detachment. Trypsinization was stopped with feeders medium and cells were pelleted by centrifugation at 900 rpm for 3 min. Resuspension in fresh 80/20 medium and separation into single cells was achieved by thorough up and down pipetting with a p1000 micropipette. Afterwards cells were seeded in 0.1 % gelatine coated cell culture containers or on 10 µg/mL laminin coated cover slips.

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To freeze cells, 70 % confluent T75 flasks were trypsinized and centrifuged as described above. Resuspension was carried out with freezing medium and cells were divided into four aliquots à 1 mL. Cells were transferred into cryotubes, stored at -80 °C over night and moved to liquid nitrogen the next day. Cryotubes with frozen cell aliquots were quickly thawed in a waterbath at 37 °C. Feeders medium was added and cells were collected at 900 rpm for 3 min. Resuspension took place in 80/20 medium and cells were seeded in 0.1 % gelatine coated cell culture containers.

2.2.1.2 Transient transfection Transfection of bicistronic reporter plasmids was performed in 96-well plates using Lipofectamine 2000. For 4E-BP co-transfection, 50 ng reporter plasmid were mixed with 50 ng of 4E-BP-encoding plasmid. For siRNA co-transfection, 75 ng of reporter plasmid and 40 nM siRNA were used. Transfection mixes were assembled in two steps. First, 0.2 µL Lipofectamine 2000 was mixed with 25 µL Opti-MEM per well of a 96-well plate and incubated for 5 min. Second, the Lipofectamine mix was added to the DNA or DNA/siRNA mix which was diluted in 25 µL Opti-MEM per well of a 96-well plate. The combined DNA- or DNA/siRNA- transfection reagent mix was incubated for 20 min before it was added to the cells. Cells were lysed 24h post transfection. AllStar siRNA was used as a negative control in the siRNA co-transfection assays and GST-encoding plasmid was used as a negative control in the DNA co-transfection assays. All transfection reactions were performed in technical triplicates.

2.2.1.3 Stable transfection For stable integration of dominant negative 4E-BP into A17iCre mESC line, cells of a low passage number were thawed and amplified for two days. On the third day medium was supplemented with 1 µg/mL doxycycline to induce recombinase expression. Transfection took place the next day. For that, a transfection mix containing 8 µL Lipofectamine 2000 in 250 µL Opti-MEM was incubated for 5 min before it was added to 3 µg of plasmid DNA in 250 µL Opti-MEM. The DNA-transfection reagent mix was incubated for additional 20 min and then added to 700,000 cells in a well of a 6-well plate. Antibiotic selection was initiated the next day by adding fresh media supplemented with 400 µg/mL G418. The selection took place for nine days in which the media supplemented with G418 was changed daily. During the selection period, all untransfected negative control cells died and the vast majority of positive control cells which were transfected with a GFP-encoding plasmid were positive for GFP expression signal. The surviving 4E-BP-transfected cells were trypsinized and single cells from the polyclonal cell line 131 were seeded in two 96-well plates. Clones were expanded for a week and ten monoclonal lines were frozen and tested for V5 expression by immunofluorescent staining.

2.2.1.4 Induction and neuronal differentiation For neuronal differentiation, mESCs in culture were detached by TrypLE, washed, centrifuged and resuspended in AK medium. mESC clots were separated into single cells by thorough up and down pipetting and 2x106 cells per uncoated 10 cm dish were cultured in suspension for a day. The cells did form embryoid bodies which were collected by centrifugation for 3 min at 700 rpm. Cells were then evenly distributed into a 6-well plate (3.33x105 cells per well) in fresh AK medium supplemented with 3 µg/mL doxycycline. 24h after induction the cells were harvested by withdrawing the medium followed by two washing steps with PBS and storage at -80 °C.

2.2.2 Cloning and plasmid preparation 2.2.2.1 Polymerase chain reaction (PCR) DNA was in vitro amplified with either DreamTaq DNA polymerase (for screening) or Phusion high-fidelity DNA polymerase (for cloning). PCR reaction mixes were assembled on ice like this:

DreamTaq reaction mix Amount

10x Dream taq buffer 5 µL

dNTP mix (10 mM) 1 µL

Forward primer (10 µM) 1.5 µL

Reverse primer (10 µM) 1.5 µL

Dream taq polymerase 0.2 µL

(5 U/µL)

gDNA/cDNA/plasmid 10-50 ng

DNA

H2O ad 50 µL

Final volume 50 µL

Phusion reaction mix Amount 5x HF buffer 10 µL dNTP mix (10 mM) 1 µL Forward primer (10 µM) 2.5 µL

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Reverse primer (10 µM) 2.5 µL Phusion polymerase 0.5 µL (2 U/µL) gDNA/cDNA/plasmid 10-50 ng DNA

H2O ad 50 µL Final volume 50 µL

Amplification was carried out in a thermal cycler using the following PCR programs:

Step Purpose DreamTaq Polymerase Phusion Polymerase 1 Initial denaturation & 95°C for 3 min 98°C for 3 min Polymerase activation 2 Denaturation 95°C for 30 sec 98°C for 10 sec

3 Annealing ta for 30 sec ta for 30 sec

4 Elongation 72°C for Te 72°C for Te Cycles (steps 2-4) 30 30 5 Final extension 72°C for 5 min 72°C for 5 min 6 Storage 8°C for ∞ 8°C for ∞

The annealing temperature (ta) was adjusted to the melting temperature of the primer pair and the

MgCl2 concentration of the reaction mix. For amplification with DreamTaq DNA polymerase the annealing temperature was set 3-4°C below the melting temperature of the primers. The annealing temperature for amplification with Phusion DNA polymerase was calculated using the Tm Calculator tool from ThermoFisher Scientific (https://www.thermofisher.com/de/de/home/ brands/thermo-scientific/molecular- biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific- web-tools/tm-calculator.html).

Further, the Elongation time (Te) was adjusted to the size of the DNA fragment to be amplified. For DreamTaq DNA polymerase the elongation time was 1 min per kb of DNA and for Phusion DNA polymerase the elongation time was 30 sec per kb of DNA. PCR products were analyzed by agarose gel electrophoresis. If PCR fragments were used for cloning, they were either directly purified by GeneJET PCR purification kit or extracted from agarose gels with GeneJET gel extraction kit. DNA was eluted in 20-50 µL H2O.

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2.2.2.2 Agarose gel electrophoresis DNA fragments were separated by agarose gel electrophoresis. Therefore, 1-2 % of agarose in TE buffer was supplemented with ethidium bromide (0.04 µL/mL). An electric field of ~100 V was applied to gels containing DNA in 1x DNA loading dye and images were acquired under UV light by a UV-Gel-Imager.

2.2.2.3 Restriction digest and dephosphorylation Plasmid backbones used for cloning were linearized with the appropriate restriction enzyme and coding sequences needed for cloning were cut from the desired plasmid by digest with two appropriate restriction enzymes. Standard restriction digest reaction mixes were assembled like this:

Restriction digest mix Amount 10x CutSmart buffer 5 µL Restriction enzyme I (20 U/µL) 0.5 µL Restriction enzyme II (20 U/µL) 0.5 µL Plasmid DNA 2-5 µg

H2O ad 50 µL Final volume 50 µL

Reaction mixes were incubated for at least 2 h up to O/N at 37 °C. Subsequent dephosphorylation of 5’ ends was carried out with either Antarctic phosphatase (5 U) or FastAP phosphatase (5 U) which was directly added to the restriction mix. In case of Antarctic phosphatase, 5 µL of 10x Antarctic phosphatase reaction buffer were also added to the restriction mix. Dephosphorylation took place at 37 °C for 30 min up to two hours. DNA fragments were purified on 0.8-2 % agarose gels, cut and extracted with GeneJET gel extraction kit. DNA was eluted in 20-50 µL of H2O.

2.2.2.4 Ligation Ligation of vector backbones with PCR products or inserts from other plasmids was carried out with T4 DNA ligase like this:

Ligation mix Amount 10x T4 DNA ligase buffer 1 µL

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Vector backbone ~ 100 ng DNA insert 2-3fold (mol) more than vector T4 DNA ligase (5 U/µL) 0.5 µL

H2O ad 10 µL Final volume 10 µL

As negative control, a ligation mix of the same components lacking the DNA insert was assembled. Ligation took place at RT for at least 1h up to O/N.

2.2.2.5 Oligo cloning Custom designed short oligos were used for cloning into linearized plasmid backbones. Therefore, forward and reverse oligos were annealed to short dsDNA fragments like this:

Annealing mix Amount Oligo I (100 µM) 2 µL Oligo II (100 µM) 2 µL 10x annealing buffer 2 µL

H2O 14 µL Final volume 20 µL

The annealing reaction mix was incubated on a thermal cycler set to the following program for heating and slow cooling down:

Step Purpose Time & Temperature 1 Denaturation 95°C for 5 min 2 Annealing 95°C to 25°C Cycles 72 (-1°C/cycle) 3 Storage 25°C for ∞

In case oligos needed to be concatemerized, oligos were phosphorylated with T4 Polynucleotide kinase prior to annealing in a reaction mix like this:

Phosphorylation mix Amount oligo in annealing buffer 20 µL

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DTT (100 mM) 1.25 µL ATP (10 mM) 2.5 µL T4 PNK (10 U/µL) 1 µL Final volume ~25 µL

Phosphorylation of 5’ ends took place at 37 °C for 30 min. Afterwards, the kinase was heat inactivated at 65 °C for 20 min. Phosphorylated oligos were then annealed to their complementary counterpart as described above. Double stranded phosphorylated oligos were mixed and ligated like this:

Ligation mix Amount Phosphorylated ds oligos I 25 µL Phosphorylated ds oligos II 25 µL ATP (10 mM) 5 µL T4 DNA ligase (5 U/µL) 1 µL Final volume 56 µL

The ligation mix was incubated at RT for 30 min and ligated duplexes were purified by agarose gel electrophoresis afterwards, Bands of the appropriate size were extracted with GeneJET gel extraction kit and eluted in 20 µL H2O. Finally, ligation into vector backbone was performed by setting up the following reaction:

Ligation mix Amount Oligo mix 0.5 µL Plasmid backbone (cut, non-dephosphorylated) ~ 100 ng 10x T4 ligation buffer 1 µL T4 DNA ligase (5 U/µL) 0.5 µL

H2O 6 µL Final volume 10 µL

As negative control, a ligation mix of the same components lacking the oligo was assembled. Ligation took place at RT for at least 1h up to O/N.

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2.2.2.6 Site-directed mutagenesis In order to introduce specific nucleotide changes in a target sequence, PCR based site-directed mutagenesis was performed. Therefore, partially overlapping primers containing the mutated sequence were designed and used in a PCR like this:

Phusion reaction mix Amount 5x HF buffer 4 µL dNTP mix (10 mM) 0,4 µL Forward primer (10 µM) 2 µL Reverse primer (10 µM) 2 µL Phusion polymerase 0.2 µL (2 U/µL) plasmid DNA 10-50 ng

H2O ad 20 µL Final volume 20 µL

The same reaction mix without primers was assembled and used as a negative control. DNA amplification was carried out in a thermal cycler using the following protocol:

Step Purpose Time & Temperature 1 Initial denaturation 98°C for 30 sec 2 Denaturation, 98°C for 10 sec 3 Annealing & elongation 72°C for 6 min Cycles (steps 2-3) 25 4 Storage 8°C for ∞

Afterwards, the PCR reaction mix was treated with 0.5 µL DpnI (20 U/µL) for at least 30 min in order to digest the plasmid template. For transformation, 5 µL of digested PCR mix were added to 50 µL of competent cells as described in the next section.

2.2.2.7 Bacterial transformation and overnight culture For transformation, 50 µL of competent E. coli cells (DH5α or XL-blue) were thawed on ice and 5 µL ligation mix was added and mixed by flicking the tube. Cells were kept on ice for further 10 min. Heat shock treatment was performed by incubating bacteria in a water bath at 42 °C for 137

~45 sec. Afterwards cells were immediately transferred back to ice for another 5 min. Then, 200 µL of LB medium was added and cells were grown at 37 °C shaking (300 rpm) for 30 min to one hour before the cell suspension was plated on LB agar supplemented with ampicillin (0.1 mg/mL) and incubated at 37 °C O/N. Next day, colonies were picked and transferred to 4 mL (mini culture) or 100 mL (midi culture) of LB medium with ampicillin (0.1 mg/mL) and grown for ~14 h at 37 °C, 300 rpm. Subsequent plasmid preparation was performed by use of either plasmid miniprep DNA kit or NucleoBond Xtra midi kit.

2.2.3 Molecular biology 2.2.3.1 RNA extractions RNA extraction was carried out with peqGOLD TriFast (adherent cells) or peqGOLD TriFast FL (cells in suspension) according to the manufacturer’s instructions starting with 100 µL TriFast per sample instead of 750 µL. All steps of the protocol were scaled down accordingly. In case RNA amount of the sample was expected to be low, 1 µL of GlycoBlue coprecipitant (15 mg/mL) was added to the sample prior to addition of TriFast reagent. Purified RNA was dissolved in DEPC water and concentrations were determined with a photo spectrometer (> 100 ng/µL) or with Qubit RNA HS assay kit (5 -100 ng/µL).

2.2.3.2 cDNA synthesis Purified RNA was treated with DNase prior to cDNA synthesis. A reaction mix for DNA digest was assembled like this:

DNA digest mix Amount RNA 1 µg RQ1 10x RNase-free DNase buffer 1 µL RQ1 RNase-free DNase (1 U/µL) 1 µL

DEPC H2O ad 10 µL Final volume 10 µL

The DNA digest mix was incubated at 37 °C for 30 min. Afterwards, 1 µL of RQ1 DNase stop solution was added and DNase inactivation was performed at 65 °C for 10 min. The following cDNA synthesis was carried out by use of Maxima first strand cDNA synthesis kit. The synthesis reaction mix was assembled like this:

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cDNA synthesis mix Amount RNA (in DNase digest mix) 11 µL 5x reaction mix 4 µL Maxima enzyme mix 2 µL

DEPC H2O 3 µL Final volume 20 µL

Reverse transcription took place in a thermal cycler using the following incubation protocol:

Step Purpose Time & Temperature 1 Annealing random 25°C for 10 min hexamer primers 2 Annealing oligo(dT) primers 50°C for 15 min & reverse transcription 3 Termination 85°C for 5 min 4 Storage 8°C for ∞

Afterwards cDNA was diluted in H2O (1:50) and stored at -20 °C.

2.2.3.3 Quantitative real-time polymerase chain reaction (qRT-PCR) Quantitative real time PCR (qPCR) was performed by use of the SensiFAST SYBR No-ROX kit. Reaction mixes were assembled like this:

qPCR mix Amount cDNA (1 ng/µL) 5 µL Forward primer (10 µM) 0.8 µL Reverse primer (10 µM) 0.8 µL 2x SensiFAST SYBR No-ROX mix 10 µL

DEPC H2O 3.4 µL Final volume 20 µL

Reaction mixes lacking cDNA were prepared as well and used as a negative control. qPCR reactions were carried out in technical duplicates in a quantitative PCR instrument using the following protocol:

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Step Purpose Time & Temperature 1 Initial denaturation 95 °C for 2 min 2 Denaturation 95 °C for 5 sec 3 Annealing 58°C for 20 sec 4 Elongation 72°C for 20 sec Cycles (step 2-4) 39 5 Melting curve 60 – 95 °C for 10 sec (+1 °C/10 sec)

2.2.3.4 In vitro transcription In vitro transcription reactions to generate capped linear RNA reporters or (non-capped) circular RNA reporters were carried out by using the TranscriptAid T7 High Yield Transcriptions Kit with some modifications. Reaction mixes were assembled like this:

In vitro transcription capped Amount T3 RNA Polymerase (20 U/µL) 0.5 µL Template DNA (20-25 ng/µL) 1 µL m7GpppG or ApppG (40 mM) 0.85 µL ATP (10 mM) 0.5 µL UTP (10 mM) 0.5 µL CTP (10 mM) 0.5 µL 5x reaction buffer 2.0 µL

MgCl2 (50 mM) 0.4 µL NaCl (600 mM) 0.25 µL DTT (400 mM) 0.25 µL

DEPC H2O 2.75 µL Final volume 10 µL

In vitro transcription circRNA Amount T7 RNA Polymerase (200 U/µL) 1.25 µL Template DNA (20-25 ng/µL) 10 µL GMP (100 mM) 12.5 µL ATP (100 mM) 2.5 µL

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UTP (100 mM) 2.5 µL CTP (100 mM) 2.5 µL 5x reaction buffer 10.0 µL

MgCl2 (1 mM) 1.0 µL DTT (400 mM) 1.25 µL

DEPC H2O 4 µL Final volume 50 µL

Transcription took place at 37 °C for two hours. After 5 min of incubation, either 0.5 µL or 2.5 µL GTP was added to the capped or circRNA reaction mix, respectively. The reaction was stopped by adding either 2 U or 5 U DNase I to the capped or circRNA reaction mix, respectively. After 30 min of DNase I treatment at 37 °C the capped RNA was extracted by use of the RNeasy Mini Kit following the manufacturers protocol while the non-capped RNA for circularization was extracted manually. Therefore, 3 µL of EDTA (0.5 M) were added. The mix was filled up with DEPC treated H2O to 200 µL. Then 200 µL phenol:chloroform:isoamyl alcohol (25:24:1) were added, the mix was vortexed and centrifuged at maximum speed for 5 min. The upper aqueous phase was transferred to a fresh tube and 125 µL LiCl (8 M) were added. After incubation for 30 min on ice, the mix was centrifuged at maximum speed for 10 min and the pellet was resolved in 100 µL of RNase-free water. Subsequently, 10 µL of NaOAc (3 M, pH 5.2) and 330 µL EtOH (absolute) were added. After 30 min of incubation at -20 °C, the mix was centrifuged another 30 min at maximum speed at 4 °C. Finally, the RNA pellet was washed with 70 % EtOH, dried and diluted in 100 µL RNase-free water.

2.2.3.5 RNA circularization For circularization, a strategy was chosen in which a bridging oligo is annealed to the RNA to be circularized to bring the 5’- and the 3’-ends in close proximity before a RNA ligase synthesizes the bond between the 5’-guanosine monophosphate and the 3’-hydroxyl terminal end, which are in the middle of a double stranded DNA/RNA hybrid due to the oligo. For oligo annealing the following reaction mixes were assembled:

Oligo annealing Amount In vitro transcribed RNA (400 pmol) xx µL Bridging oligo (20 µM) 5 µL EDTA (50 mM) 4 µL

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Tris-HCl (500 mM, pH 7.5) 4 µL

DEPC H2O up to 200 µL Final volume 200 µL

The reaction was incubated for 3 min at 95 °C and afterwards continuously cooled down using the following protocol:

Step Purpose Time & Temperature 1 Initial denaturation 95 °C for 3 min 2 Annealing curve 95 °C to 25 °C with -0.1°C/1 sec

RNA was afterwards extracted by adding 20 µL NaOAc (3 M, pH 5.2) and 660 µL EtOH (absolute). The mix was incubated for at least 30 min at -20 °C followed by centrifugation at maximum speed for at least 30 min. The resulting RNA pellet was dried and eluted in 20.5 µL of DEPC treated water. Then circularization was performed by assembling the following reaction mixes:

Circularization Amount In vitro transcribed RNA with bridging 20.5 µL oligo annealed 10x T4 RNA ligase buffer 2.5 µL T4 RNA ligase 2 (10 U/µL) 2.0 µL Final volume 25 µL

The ligation was incubated for 4 h at 37 °C. Afterwards the RNA was extracted by EtOH precipitation and a small aliquot of it was used for RNase R digestion to validate circularization.

RNase R treatment Amount circRNA (5 ng/µL) 2.0 µL 10x reaction buffer 1.0 µL RNase R (20 U/µL) 0.2 µL

DEPC H2O 6.8 µL Final volume 10 µL

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After RNase R treatment, the digested aliquot and a small undigested aliquot of the circRNA were run next to each other on an urea gel. The vast majority of the circRNA was also run on the same gel but a few lanes apart from the others so that the gel could be cut in two halves. One half was stained later to approximate at which height linear and circular RNA species were running. The other half of the gel was used to cut the band with the circularized RNA without previous staining. The 4 % TBE-urea gels (1 mm thick, 20 x 20 cm) prepared according to one of the following recipes:

Reagent Amount 40 % Acrylamide:N,N’-Methylene bisacrylamide (40:0.6) 3.5 mL 10 M Urea 24.5 mL 5x TBE (pH 8.3) 3.5 mL 100 % TEMED 17.5 µL 10 % APS 263 µL

H2O 3.22 mL Final volume 35 mL

Reagent Amount UreaGel concentrate 5.6 mL UreaGel buffer 3.5 mL UreaGel diluent 25.9 mL 100 % TEMED 14 µL 20 % APS 140 µL Final volume 35 mL

The empty gel was pre-run for 15 min at 300 V. In the meantime, 2x TBE-urea sample buffer was added to the RNA samples which were then heated at 70 °C for 3 min and placed on ice afterwards. Samples were loaded and the gel was run for at least 1.5 h at 300 V or until the xylene cyanol of the loading dye reached at least the middle of the gel. Then the gel was cut in two halves and the part with the RNase R digested aliquot next the undigested aliquot was stained with 1x SYBR gold in 1x TBE for a few minutes. RNA bands were visualized by UV transillumination. The circRNA band of the preparative unstained part of the gel was cut blindly but by estimating the appropriate height from the other stained part of the gel using a printout in original size. A gel piece and 500 µL elution buffer were added into a microcentrifuge tube and 143 incubated shaking or on a rotating wheel over night at room temperature. The next day, RNA was extracted by EtOH precipitation and dissolved in 10 µL DEPC treated water. Concentration was measured by Qubit and eluted RNA samples were re-run on a TBE-urea gel to check for integrity. In case of contamination with linear RNA species samples were cleaned by RNase R digestion and run again on TBE-urea gel.

2.2.3.6 In vitro translation For in vitro translation assays, Drosophila embryo extracts were prepared as previously described [844]–[846] and reaction mixes were assembled like this:

In vitro translation Amount Drosophila embryo extract 4.0 µL Creatine kinase (10 µg/µL) 0.16 µL tRNA calf liver (4 µg/µL) 0.25 µL Amino acid mixture (1 mM) 0.25 µL Creatine phosphate (600 mM) 0.28 µL Spermidine (4 mM) 0.25 µL DTT (50 mM) 0.24 µL

Mg(OAc)2 (15 mM) 0.40 µL KOAc (2 M) 0.30 µL HEPES-KOH (1 M, pH 7.4) 0.24 µL FLuc RNA reporter (10 ng/µL) 0.625 µL RLuc RNA reporter (5 ng/µL) 1.0 µL

RNase free H2O 2.0 µL Final volume 10 µL

In order to inhibit cap-dependent translation, 1 µL of cap analogue (10 mM) was added to the reaction mix and the amount of water reduced accordingly. The reaction was carried out at for 1 h at 25 °C. Afterwards 9 µL were used for reporter assay readout.

2.2.4 Methods of protein biochemistry 2.2.4.1 Protein extraction Protein extraction was performed in UREA buffer. Therefore, cells were places on ice and 300 µL buffer were added to each well of a 6 well plate. Cells were detached with a cell scraper and the cell suspension was collected in a protein low binding tube. Next, the cell suspension was 144 sonicated in a Bioruptor for four high power sonication cycles (15 sec ON, 45 sec OFF) at 4 °C. Afterwards, cell debris was spun down at 14,000 g for 3 min at 4 °C. The supernatant was recovered and an aliquot was taken to determine protein concentration by Bradford assay.

2.2.4.2 Bradford protein assay

Protein extracts obtained as described above, were first diluted 1:4 in H2O. Then, protein concentration was measured with Pierce Coomassie (Bradford) protein assay kit. Therefore, 20 µL of albumin protein standards with defined concentrations (100 ng/µL, 250 ng/µL, 500 ng/µL, 750 ng/µL, 1 µg/µL) were mixed with 1 mL of 1x Bradford reagent, incubated at RT for 5 min and analyzed in a spectrophotometer to obtain a standard curve. Afterwards, 20 µL of protein sample were mixed with 1 mL of 1x Bradford reagent and protein concentration was determined with a spectrophotometer by use of the previously obtained standard curve.

2.2.4.3 SDS-PAGE Samples containing 3-20 µg of protein in 1x SDS loading buffer were boiled at 95 °C for 5 min before they were loaded on self-casted 15 % SDS gels which were prepared according to this recipe:

Reagent 15% SDS 5% SDS separating gel stacking gel

H2O 1.4 mL 2.2 mL 30 % Acrylamide/Bis (37.5:1) 3 mL 0.5 mL Tris (1.5 M, pH 8.8) 1.5 mL 0.25 mL 20 % SDS 0.03 mL 0.015 mL 10 % APS 0.06 mL 0.03 mL TEMED 0.005 mL 0.003 mL Final volume 6 mL 3 mL

Protein samples were run at 90 V in the stacking and the separating part of the gel until the bromophenol blue dye band reached the bottom of the SDS gel.

2.2.4.4 Western blot Right after SDS-PAGE the stacking part of the SDS gel was removed and semidry protein transfer to a PVDF membrane was performed in Towbin buffer with a western blotting transfer system at 25 V, 1 A for 30 min. Afterwards, the efficiency of the transfer was determined by 145

Ponceau staining of the membrane for 5 min at RT and/or by coomassie staining of the gel for 5 min at RT. In case a Ponceau staining was performed, the staining was rinsed off the membrane with TBS before it was blocked in TBS, 3 % milk, 0.05 % Tween 20 for one hour at RT, 35 rpm. After blocking, the membrane was cut in two parts at the band of 35 kDa of the PageRluer Plus Prestained ladder. The lower part of the membrane (10-35 kDa) was treated with anti-V5 antibody (1:5000) in blocking solution and the upper part of the membrane (35-250 kDa) was treated with anti-α-Tubulin antibody (1:4000) in blocking solution O/N at 4 °C, 35 rpm. Next day, membranes were washed three times 10 min in TBS, 0.05 % Tween 20 at RT, 35 rpm. Afterwards, TBS, 0.05 % Tween 20 was removed and the secondary antibody was added in TBS, 3 % milk, 0.05 % Tween 20 for two hours at RT, 35 rpm. Anti-V5 stained lower part of the membrane was treated with anti-rabbit HRP (1:5000) and anti-α-Tubulin stained upper part of the membrane was treated with anti-mouse HRP (1: 10,000). Then, membranes were washed three times 10 min in TBS, 0.05 % Tween 20 at RT, 35 rpm. In the meantime, homemade ELC reagent was prepared like this:

ELC reagent Amount Tris (1.5 M, pH 8.5) 10 mL

H2O2 2.6 µL Coumaric acid 25 µL Luminol 50 µL Final volume ~10 mL

The membrane was covered with ELC reagent for 1 min and chemiluminescence was analyzed with a chemiluminescence imager.

2.2.4.5 Mass spectrometry First, 100 µg of proteins were reduced in DTT (2 mM) for 30 min at 25 °C and subsequently free cysteines were alkylated in iodoacetamide (11 mM) for 20 min. Digestion with Lys-C (1:40 w/w) was carried out at 30 °C for 18 h under gentle shaking. Afterwards, the sample was diluted three times with ammonium bicarbonate solution (50 mM) and 7 µL of immobilized trypsin were added before the sample was incubated at 30 °C for 4 h under rotation. Then, 10 µL trifluoroacetic acid were added and trypsin beads were removed by centrifugation. 15 µg of digested protein were desalted on STAGE Tips, dried and dissolved in 20 µL acetic acid in water (0.5 %) [847]. 146

For the analysis, 5 µL of digested sample were injected in duplicates on a LC-MS/MS system using a 240 min gradient from 5 % to 45 % solvent B (80 % acetonitrile, 0.1 % formic acid) into solvent A (5 % acetonitrile, 0.1 % formic acid). A 30 cm capillary with 75 µm inner diameter was packed with 1.8 µm C18 beads for chromatographic separation. At one end, a nanospray tip was generated with a laser puller, allowing fretless packing. The nanospray sources was operated with a spray voltage of 2.1 kV and an ion transfer tube temperature of 260 °C. A data dependent mode with a top10 method was chosen for data acquisition on the Q Exactive Plus: a survey MS scan with a resolution of 70,000 at m/z 200 was followed by up to ten MS/MS scans on the most intense ions with an intensity threshold of 5,000. When selected once for fragmentation, ions were excluded from selection for 30 sec to increase new sequencing events. The MaxQuant proteomics pipeline with the Andromeda search engine and Uniprot database built in was used for data analysis [848]. Carbamidomethylation of cysteines was selected as fixed modification, while oxidation of methionine and acetylation of the N-terminus were selected as variable modifications. Parameters were left as default except for the search engine peptide assignments, which were filtered at 1 % FDR and the feature match between runs, which was enabled.

2.2.4.6 Luciferase reporter gene assay Homemade luciferase reporter assay reagents were used to detect luciferase activities with Centro LB 960 Microplate luminometer (Berthold) and MikroWin software package (Mikrotek Laborsysteme). Per sample, 10 µL of lysate or 9 µL of in vitro translation reaction mix were treated with 45 µL of FLuc reagent (75 mM Hepes pH 8.0, 0.1 mM EDTA, 4 mM MgSO4, 530 µM ATP, 270 µM Coenzyme A, 470 µM DTT, 470 µM luciferin) and 45 µl of RLuc reagent (2.2 mM Na2EDTA, 220 mM K3PO4 pH 5.1, 0.44 mg/mL BSA, 1.1 M NaCl, 1.3 mM NaN3, 0.6 µg/mL colenterazine).

3 RESULTS

3.1. Generation and characterization of mouse embryonic stem cells expressing inducible dominant negative 4E-BP1 As the aim of this study was to elucidate alternative mechanisms of translation initiation that are independent of the cap, we were looking for a model system which allows us to reliably create a condition that favors non-canonical translation initiation. Further, we were interested in a system that also naturally switches to physiological conditions in which repression of cap-dependent

147 translation is thought to be of importance. Hence, we generated clonal mESC lines with stable integration of a known suppressor of cap-dependent translation, dominant negative 4E-BP1, and stable integration of a proneuronal factor, ASCL1, strongly promoting neuronal differentiation.

3.1.1 Working principle of dominant negative 4E-BP1 in cap-dependent translation inhibition To investigate cap-independent translation mechanisms, a model system with inducible expression of dominant negative 4E-BP1 was established. 4E-BP1 is a translational repressor whose activity is regulated via phosphorylation by the mTOR signaling pathway. Human 4E-BP1 contains in total seven phosphorylation sites: Thr37, Thr46, Ser65, Thr70, Ser83, Ser101 and Ser112 [249]. The first five of these are evolutionary conserved and phosphorylation of the two most N-terminal threonine residues is required for subsequent phosphorylation of the residual C- terminal sites [238], [239]. The phosphorylation state of 4E-BP1 determines whether 4E-BP1 is bound to eIF4E. In the hypophosphorylated form 4E-BP1 interacts with eIF4E through a similar site as eIF4G, thereby inhibiting eIF4F complex assembly and preventing translation initiation. Hyperphosphorylation, especially phosphorylation of Thr70, Ser65 and Ser112, causes a release of 4E-BP1 from eIF4E [240]. This in turn permits the association of eIF4E with eIF4G which is needed for eIF4F assembly at the mRNA 5’end to recruit ribosomes. The 4E-BP1(4Ala) mutant constitutively binds eIF4E, as four of its phosphorylation sites (Thr37, Thr46, Ser65, Thr70) were replaced with alanine [849]. Like this, phosphorylation of 4E- BP1(4Ala) is abrogated and cap-dependent translation is constantly repressed by 4E-BP1(4Ala) binding eIF4E. To generate a model system with inducible inhibition of cap-dependent translation, 4E-BP1(4Ala) was stably integrated into the genome of mESCs.

3.1.2 ASCL1 induces differentiation of mouse embryonic stem cells into neurons To study cap-independent translation mechanisms in steady state mESCs and during the process of differentiation into neurons, the transcription factor ASCL1 was, in addition to 4E-BP1(4Ala), stably integrated into the mESC genome. ASCL1 is a proneural transcription factor which functions in proliferation and differentiation of neural stem and progenitor cells. It further regulates cell migration, axon guidance and synapse formation during neurogenesis [850]. ASCL1 is exceptional among proneural factors, because it acts as a pioneer factor that alone is sufficient to reprogram mouse and human fibroblasts or ESCs into induced neuronal cells [851], [852]. The induction of ASCL1 expression in our model system leads to differentiation of mESCs into induced neurons (iNs) within only one week. After 24 h of ASCL1 induction cells are phenotypically still close to mESCs (Fig. S1) but it was shown that already after 12 h differentiating cells can express neuronal Tubb3 and that about 780 genes are upregulated on 148 transcriptional level with nervous system development being the most significantly enriched GO biological process term [853]. At 48 h, cells already express genes that are linked to specific neuronal cell types, especially noradrenergic and cortical interneuron markers, but these are heterogeneously expressed across the cell population [853]. Hence, ASCL1 induction leads to a rapid transition from an embryonic towards a neuronal cell identity already at early time points of the differentiation process.

3.1.3 Recombinase mediated cassette exchange to generate stable cell lines expressing dominant negative 4E-BP1 Both, dominant negative 4E-BP1(4Ala) and ASCL1 were integrated into the genome of mESCs via inducible cassette exchange (ICE). In detail, the male A17 mESC line which carries the ICE locus upstream of the HPRT gene on the X chromosome was used for transgenesis [854]. The ICE locus contains the tetracycline response element upstream of the Cre transgene which is floxed by heterologous loxP sites and which is followed by a Δneo gene that misses a promoter and the start codon (Fig. 4). Upon doxycycline induction, Cre is expressed and after p2Lox plasmid transfection, ASCL1 and 4E-BP1(4Ala) are inserted by Cre-Lox recombination in between the two loxP sites, exchanging Cre for the two new transgenes (Fig. 4). Further, the recombination introduces a promoter and a start codon upstream of the Δneo gene so that successfully modified recombinants are G418 resistant. As a control, another mESC line was generated with stable integration of ASCL1 only. In the following, this mESC cell line is referred to as ‘wt’, although it carries the inducible ASCL1 transgene in the ICE locus.

Fig. 4: Inducible cassette exchange stably integrates dominant negative 4E-BP1 and ASCL1 into mESCs. Doxycycline induces tetracycline response element (TRE) driven expression of Cre recombinase, which after transfection of p2Lox plasmid, mediates recombination at heterologous LoxP sites (green & purple triangles) such that PGK promoter and ATG start codon are integrated upstream of the neomycin resistance gene that lacks a promoter and a start codon (Δneo) and that ASCL1, F2A cleavage peptide and 4E-BP1(4Ala) are integrated downstream of TRE at the ICE locus on the X chromosome. Adopted from Iacovino et al, 2014 [840].

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3.1.4 Expression of dominant negative 4E-BP1 reduces cap-dependent translation and promotes cap-independent translation After antibiotic selection, successful integration of 4E-BP1(4Ala) and ASCL1 into the genome of mESCs was validated by western blot and qPCR for the V5 peptide tag, which is C-terminally attached to 4E-BP1(4Ala). Three different monoclonal cell lines were selected and 4E-BP1(4Ala) protein expression could be confirmed in all clones tested after 24 h of doxycycline induction (Fig. 5A). 4E-BP1(4Ala) expression was also readily detectable by qPCR after 7 d of doxycycline induction and stem cell differentiation into iNs. Like wt mESCs, monoclonal 4E-BP1(4Ala)- expressing cell lines exhibited a severe decrease in expression of stem cell markers Oct4 and Sox1, while expression of the neuronal marker Tubb3 was increased (Fig. 5B). Hence, stable integration and inducible expression of 4E-BP1(4Ala) could be demonstrated on RNA and protein level. Further, the overexpression of 4E-BP1(4Ala) in synergy with ASCL1 drove neuronal differentiation and similar marker gene expression as in wt cells expressing ASCL1 alone, suggesting that monoclonal cell lines can differentiate into iNs in the presence of dominant negative 4E-BP1. To determine the effects on cap-dependent and cap-independent translation by 4E- BP1(4Ala) overexpression, we performed dual luciferase reporter assays using bicistronic reporter constructs. These bicistronic reporters contained an EMCV IRES in between the 5’-terminally located FLuc and 3’-terminally located RLuc ORFs. That way, cap-dependent FLuc expression and IRES-mediated RLuc expression are derived from the same transcript (Fig. 5C). By taking the ratio of RLuc/FLuc expression, one can quantify the ability of a certain sequence element to mediate IRES driven translation. To do so, reporter plasmids were transfected into wt and monoclonal mESCs that were subsequently induced with doxycycline for 24 h before luciferase values were analyzed. All selected transgenic clones displayed an increase in RLuc/FLuc expression ratios upon 4E-BP1(4Ala) overexpression (Fig. 5D). Likewise, wt mESC showed a slight increase in RLuc/FLuc expression after doxycycline induction; however, the changes of RLuc/FLuc ratios after overexpression of ASCL1 alone were small and not significant. A mutated non-functional EMCV* IRES reporter was used as negative control and displayed only very low levels of RLuc/FLuc expression (~3 % of EMCV). Doxycycline induction did not significantly alter RLuc/FLuc ratio of EMCV* reporters in wt and clonal cell lines (Fig. 5D). In sum, results of the bicistronic reporter assays indicate that overexpression of 4E- BP1(4Ala) promotes EMCV IRES-mediated translation over cap-dependent translation. Overexpression of ASCL1 also slightly stimulates cap-independent translation initiation, although too much lesser and not significant extend. Hence, the generated model system can be used to 150 investigate cap-independent translation mechanisms after 4E-BP1(4Ala) induced inhibition of cap-dependent translation.

3.2 Identification of upregulated genes upon dominant negative 4E-BP1 expression in mouse embryonic stem cells and induced neurons using proteomic analysis To identify cap-independently translated transcripts genome wide, we performed LC-MS/MS analyses in three 4E-BP1(4Ala) expressing mESC clones after 24 h of induction. The proteomic analysis detected ~6,400 proteins and 13 out of them were significantly (p <0.05) enriched by more than 4-fold (log2(wt/mut)≤-2) in at least one individual 4E-BP1(4Ala) expressing clone when

Fig. 5: Inducible expression of dominant negative 4E-BP1 and ASCL1 promotes cap- independent translation and neuronal differentiation. (A) Lysates of untreated or 24 h induced monoclonal mESC lines A6, B4 and C6 were analyzed for 4E-BP1(4Ala) protein expression by Western blotting using anti-V5 (C-terminally attached to 4E-BP1(4Ala)) and anti-α-tubulin antibodies. (B) Marker gene expression of wt mESCs and monoclonal lines A6 and C6 was analyzed by quantitative real-time PCR in undifferentiated cells and induced neurons after seven days of induction. Expression levels were calculated by the comparative ΔΔCT method. Actin expression was used for normalization of Oct4, Sox1, V5 and Tubb3. V5-tag is attached to ASCL1 in wt cells and to 4E-BP1(4Ala) in clones A6, C6. Error bars represent standard deviation of two technical replicates. (C) Schematic representation of bicistronic reporter structure. Firefly luciferase (FLuc) open reading frame (ORF) is located upstream of Renilla luciferase (RLuc) ORF on bicistronic reporter mRNAs. ORFs are separated by an intermediate sequence of interest which is tested for its ability to mediate internal translation initiation. (D) Dual luciferase reporter assays of wt mESCs and 4E-BP1(4Ala) expressing clones A6, B4 and C6 after transfection with bicistronic EMCV IRES and functionally inactive EMCV* IRES reporters. Luciferase levels were determined 48 h after transfection including 24 h of doxycycline (Dox) induction when indicated. IRES mediated RLuc translation was normalized to cap-dependent FLuc translation. Error bars represent standard error of the mean of three biological replicates.

151 compared to wt stem cells (Fig. 6, Fig. 7). The clones displayed some differences in protein expression levels with the result that some of the most significantly upregulated genes in one clone were not significantly enriched in another. Such phenotypic heterogeneity within clonal cell populations can emerge from a combination of intrinsic and extrinsic factors and is often observed in mammalian cellular in vitro systems [855], [856]. In mESCs heterogenous gene expression can be caused by inherent expression noise, stochastic switching between two correlated gene expression states (Nanog-high and Nanog-low state) as well as stochastic expression bursts [857]. Epigenetic regulation, as well as the supplementation of media with “2i”, two commonly used inhibitors in stem cell culture targeting MEK and GSK3β, greatly influence heterogenous gene expression [857], [858]. Throughout the 24 h of 4E-BP1(4Ala) induction, cells were cultured in the absence of 2i, which likely enhanced the cell heterogeneity prior to MS analysis. Besides, it is possible that transgenic clones are not genetically identical due to individual accumulation of numerical and/or structural chromosomal aberrations during passaging. It was already reported earlier that the E14TG2 cell line, which is a parental cell line of A17 used in this study, shows certain chromosomal instability and is aneuploid at passage 29 (p29) with a modal karyotype of 42 that can easily increase to 52 chromosomes within four passages (p33) [859]. These cells are specifically prone to acquire trisomy 8 and partial or total duplication of chromosome 11 which are associated with higher proliferation rates constituting a selective advantage of aneuploid cells over euploid cells under long-term culture conditions [859]–[861].

Fig. 6: Upregulate genes after dominant negative 4E-BP1 induction in mESCs were selected as candidates for putative cap-independent translation. Differentially expressed proteins between wt mESCs and 4E-BP1(4Ala) expressing mESCs were analyzed individually for each clone (A6, B4 and C6) and were examined in a pooled analysis in which data of all clones was combined.

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The first subset of candidate genes was selected based on either significant (p ≤ 0.05) enrichment of ≥ 4- fold (log2≤-2) in individual or pooled clones’ analyses (17 candidate genes). A second subset of candidate genes was selected based on persistent enrichment of ≥ 2.8-fold (log2≤-1.5) in all clones (nine candidate genes).

However, taking all clones together as a whole and comparing the combined clonal cell population against wt, variations of single clones are equalized and significantly upregulated genes in individual clones are indeed found to be upregulated in the combined cell population. In addition, the combined analysis discovered four more candidate genes which were significantly (p ≤ 0.05) enriched by more than 4-fold that were not found to be significantly enriched when looking at single clones only (Fig. 6, Fig. 7). According to these findings, we draw our attention to proteins which were consistently enriched in all the three clonal cell lines to minimize unspecific effects and clone-specific alterations in the candidate selection. In this manner, we could identify nine more candidate genes which were reliably enriched more than 2.8-fold in all 4E-BP1(4Ala) exrepssing cells (Fig. 7). In total, the MS analysis identified a set of 26 proteins which were more than 2.8-fold upregulated after 24 h of 4E-BP1(4Ala) overexpression (Tab. S1). These 26 candidate genes of cap-independent/ 4E-BP1-resistant translation initiation were selected for further analysis.

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Fig. 7: Proteomic analysis identified upregulated genes after dominant negative 4E-BP1 induction in mESCs. Differentially expressed proteins between wt mESCs and 4E-BP1(4Ala) expressing mESC clones A6, B4 and C6 were determined 24 h after induction by LC-MS/MS. Analysis was performed in three biological replicates and 6,409 proteins were detected. Volcano plots depict Log2(LFQ value wt/LFQ value clone) on the x-axis and –log10(p-value) on the y-axis (two sample t-test). Upregulated candidate genes are listed and highlighted according to different selection criteria. See candidate legend and color code panels for details. LC-MS/MS performed by Guido Mastrobuoni

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3.3 Dominant negative 4E-BP1 resistant genes are linked to DNA repair and neuron projection development The selected candidate genes of 4E-BP1-independent translation initiation were subjected to GO term analysis using the GOrilla software tool by applying standard settings and the two unranked lists of genes running mode [841]. No significant GO term enrichment in molecular function and cellular component could be found for the candidate gene set. However, an enrichment in the regulation of double-strand break repair via homologous recombination (p < 0.001) and neuron projection development (p < 0.001) could be detected for biological processes (Tab. 4). Notably, both enrichments show low statistical significance, implying that the trend of cap-independent translation regulation of genes involved in DNA repair and neurite outgrowth is rather weak in our cell system. Indeed, it was previously reported that cap-independent translation initiation plays a role in protein expression of localized mRNA in dendrites and axons in response to receptor activity and eIF4G2 phosphorylation, indicating that cap-independent translational control in neurite outgrowth can be found in other neuronal cell systems [862]–[866].

Tab. 4: Enriched GO terms for candidate genes of cap-independent translation. GO term enrichment of selected candidate genes was analyzed with the GOrilla software tool comparing the candidate gene set to the background set of all proteins identified by mass spectrometry. For details on statistical analysis see http://cbl-gorilla.cs.technion.ac.il/ and Eden et al., 2009 [841]. GO term Enrichment P-value FDR Genes q-value Regulation of double-strand 47.92 6.85x10-4 1 Rad51, Chek1 break repair via homologous recombination (GO:0010569) Neuron projection 9.13 8.23x10-4 1 Lama1, Palld, development (GO:0031175) Pqbp1, Stmn2

Also cap-independent translation control of genes involved in DNA repair have previously been described in several other systems [867]–[869]. Importantly, Chek1 and Rad51, two candidate genes identified in this study, were found to exhibit reduced dependency for eIF4E in breast cancer cells, where elevated levels of eIF4G1 contribute to the selective translation of both mRNAs in response to ionizing radiation [870]. Taken together, GO term analysis revealed an association of the identified candidate genes of cap-independent translation initiation with DNA repair and neurite development which is in agreement with previously published data.

3.4 Validation of selected candidates of cap-independent translation in bicistronic reporter assays After identifying candidate genes for cap-independent translation initiation by MS analysis in 4E- BP1(4Ala) overexpressing cell clones, we went on to validate candidate gene expression in 156 bicistronic reporter assays. To investigate if selected genes were indeed enriched due to active cap- independent translation and not due to any secondary effects, we cloned their 5’UTR sequences into luciferase reporter vectors. In case a gene exhibits several expression isoforms, we selected the 5’UTR sequence of the longest protein coding isoform for our analysis. Bicistronic reporter assays are a standard method in the field to investigate translation initiation mechanisms. Consequently, candidate sequences were cloned in between FLuc and RLuc ORFs to generate bicistronic reporter constructs which allow for simultaneous analysis of both cap-dependent and IRES-mediated translation (Fig. 5C). The bicistronic vectors were transfected into mESCs and expression of the second cistron (RLuc) was quantified and compared to EMCV IRES to determine the ability of a candidate sequence to drive internal translation initiation. Expression of the first, cap-dependently regulated cistron (FLuc) was used for normalization. Note that bicistronic reporters can be used to identify bona fide IRES elements only, while other sequence elements that mediate cap-independent but 5’-end dependent translation initiation can be missed by the bicistronic reporter design [61], [64], [67]. Mechanisms of translation initiation which have a relaxed requirement for the cap structure but still require active ribosome scanning might be more prevalent in mammals, which rarely express tandem bicistronic transcripts like the ones used in reporter assays but rather express bicistronic transcripts with overlapping ORFs [14], [871]. Cap-independent but 5’-end dependent translation events can be evaluated using a set of monocistronic reporters carrying either a regular m7G-cap, a cap analogue or a cap analogue which is followed by a stable hairpin

3.4.1 Bicistronic reporter assays in human embryonic kidney and mouse embryonic stem cells in context of dominant negative 4E-BP1 expression Bicistronic reporters were cloned for 17 of the 26 selected candidate genes. For the remaining eight candidate genes amplification of the 5’UTR was unsuccessful. The candidate reporters were tested in mESCs as well as in HEK293 cells to determine if potential IRES elements are conserved in different cell types. Further, candidate reporter plasmids were co-transfected with 4E-BP1(4Ala) to test candidate’s IRES activity as well as their stimulation under conditions where cap-dependent translation is inhibited. In mESCs, dual luciferase reporter assays revealed high translational activity for two tested candidates, Fam96b and Riok1, which was ~3-fold or ~12-fold higher than translational activity of EMCV (Fig. 8A). However, after 4E-BP1(4Ala) overexpression, ratios of candidate reporter expression decreased by half whereas the ratio of EMCV reporter expression increased by half. In general, translation initiation on IRES elements is triggered in the context of 157 diminished cap-dependent translation. Since IRESs are unaffected by the mechanism of inhibition they have an advantage over the cap structure in recruiting the translational machinery. Components of the translational machinery are in turn readily accessible, as ribosomes are less frequently engaged in translation. Hence, less of them are bound to mRNA and more are freely available in the cytoplasm. The decrease in Fam96b- and Riok1-mediated translation in this setting is indicating that their mode of translation initiation is rather unlikely to be an internal one. The other 15 candidates showed lower translational activity ranging from the same order of magnitude to an order of magnitude below the translational activity of EMCV (Fig. 8A). Further, the ratio of RLuc/FLuc translation from candidate reporters slightly decreased or did not change upon 4E-BP1(4Ala) induced inhibition of cap-dependent translation except for Pqbp1, whose RLuc/FLuc translation ratio slightly but insignificantly increased (Fig. 8A). To gain better insights into translational abilities of the tested candidates, absolute values of RLuc and FLuc reporter expression were compared to each other. The absolute values confirmed that an unchanged ratio of most candidate reporters was the result of an equal decrease in cap-dependent FLuc translation and candidate-mediated RLuc translation after 4E- BP1(4Ala) overexpression (Fig. 9A). This indicates that most of the candidates don’t own IRES activity. Strikingly, absolute values also revealed that Pqbp1 does not only promote efficient translation of the downstream RLuc ORF but also stimulates translation of the upstream FLuc ORF of bicistronic reporters (Fig. 9A). Further, the slight increase in RLuc/FLuc expression ratio was caused by a relatively less decrease in RLuc expression compared to FLuc expression and did not result from stimulated internal translation initiation. Hence, Pqbp1 is rather behaving like a CITE in the bicistronic setting where it enhances translation of both cap-dependent and potentially internally mediated translation under physiological conditions but does not possess strong IRES activity when cap-dependent translation is repressed. The bicistronic reporters were tested as well in HEK293 cells. In this cell line all reporters except for one showed very low translational activity ranging from one to two orders of magnitude below EMCV-mediated reporter expression (Fig. 8B). All of these lowly translated candidate reporters displayed a decrease in RLuc/FLuc expression ratios after 4E-BP1(4Ala) co- transfection, suggesting that the tested candidate sequences are unable to mediate internal translation initiation in HEK293 cells. This was also confirmed by absolute reporter expression levels (Fig. 9B).

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Fig. 8: Candidate genes show activity in bicistronic reporter assays. (A, B) Dual luciferase reporter assays in wt mESCs (A) and HEK293 cells (B) transfected with bicistronic reporters containing 5’UTR sequences of candidate genes in between two luciferase cistrons. Cap-dependent Firefly luciferase (FLuc) translation and IRES mediated Renilla luciferase (RLuc) translation levels were determined 24 h after transfection with and without 4E-BP1(4Ala) co-transfection. Error bars represent standard deviation of three biological replicates. EMCV IRES reporter and non-functional EMCV* IRES reporter were used as controls. (C, D) RNAi based validation of bicistronic candidate reporters in dual luciferase assays in wt mESCs (C) and HEK293 cells (D). Cells were co-transfected with bicistronic reporter vectors and siRNAs targeting FLuc (+ siRNA) or non-targeting control siRNAs (- siRNA). FLuc and RLuc activities were determined 24 h post transfection. Error bars represent standard deviation of three biological replicates. Bicistronic EMCV IRES reporter and monocistronic FLuc and RLuc reporters were used as controls.

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However, one of the candidates performed better in the bicistronic assay than the others: Pqbp1 displayed high translational activity that was ~2-fold as high as the translational activity of EMCV (Fig. 8B). Upon 4E-BP1(4Ala) co-expression, Pqbp1-mediated RLuc/FLuc expression level remained high, but did not increase, which contrasts with EMCV-mediated reporter expression that increased 1.8-fold. This suggests, that the translational activity of Pqbp1 could be of physiological relevance in HEK291 cells. In contrast to mESC assays, Pqbp1 did not enhance translation of the upstream FLuc reporter in bicistronic reporters in HEK293 cells (Fig. 9B). This indicates that Pqbp1 was not acting as a CITE in HEK293 cells and although Pqbp1 was not acting like a bona fide IRES element either, it mediated potential internal translation initiation with similar efficiency as EMCV IRES. In summary, bicistronic reporter assays could detect only low levels of potential internal translational activity that was around background expression levels for most tested candidates in both mESC cells and HEK293 cells. As EMCV IRES was also just weakly translated in mESCs (an order of magnitude lower than in HEK293 cells), candidates performed comparably better in mESCs than in HEK293 cells although their ability to mediate potential internal translation initiation was similarly low in both cell types. In order to confirm that potential internal translation initiation was mediated by an IRES and not caused by artefacts of bicistronic reporters that can arise during transcription, we performed siRNA-based control experiments. Thereby the validation of Fam96b, Pqbp1 and Riok1 was of special interest, as the three of them promoted notable levels of potential internal translation initiation that were higher or in the range of EMCV IRES-mediated translation.

3.4.2 siRNA-based validation of bicistronic reporter constructs in human embryonic kidney and mouse embryonic stem cells We performed a control experiment to validate the functionality of bicistronic candidate reporters, because it is known that DNA transfection of bicistronic expression vectors is prone to produce false positive hits in IRES identification [64]. One of the critical steps of this assay is the transcription of the expression vector. In case the candidate sequence of interest contains a weak splice site or a weak promoter, aberrant reporter transcripts could be generated from which the first cistron is spliced out or transcription is just initiated upstream of the second cistron (Fig. 10). Both cases would yield monocistronic reporters that miss the first cistron and contain only the

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A 25,000,000 mESC FLuc ctrl FLuc +4E-BP1 20,000,000 RLuc ctrl RLuc +4E-BP1

15,000,000

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luciferase value 5,000,000

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Lsm6 Pex5 Riok1 Ero1lb Gtf2a1Hsbp1Lama1 Luzp1Pcdh1 Pqbp1Rad51 Stmn2Zfp808Znhit6EMCV Ccdc47Check1 Fam96b EMCV*

B HEK293 07 8.0×10 FLuc ctrl FLuc +4E-BP1 RLuc ctrl RLuc +4E-BP1 6.0×10 07

4.0×10 07

07 luciferase value 2.0×10

0.0

Pex5 Lsm6Luzp1Pcdh1 Riok1Stmn2 Znhit6EMCV Ccdc47Check1Ero1lb Gtf2a1Hsbp1Lama1 Pqbp1Rad51 Zfp808 EMCV* Fam96b Fig. 9: High translational activity of Pqbp1 in bicistronic reporter assays. (A, B) Dual luciferase reporter assays in wt mESCs (A) and HEK293 cells (B) transfected with bicistronic reporters containing 5’UTR sequences of candidate genes in between two luciferase cistrons. Cap-dependent Firefly luciferase (FLuc) translation and IRES mediated Renilla luciferase (RLuc) translation levels were determined 24 h after transfection with and without 4E-BP1(4Ala) co-transfection. Error bars represent standard deviation of three biological replicates. EMCV IRES reporter and non-functional EMCV* IRES reporter were used as controls. second cistron, which would be cap-dependently translated in these scenarios. To avoid that after spurious transcription truncated monocistronic reporter expression is falsely identified as a candidate’s ability to mediate IRES-driven translation, we applied an RNAi control experiment. Thereby the integrity of bicistronic reporters is validated by co-transfection of a siRNA targeting the first cistron [65]. This leads to degradation of bona fide bicistronic transcripts, while unanticipated monocistronic transcripts would be unaffected by the knockdown. Thus, expression levels of the first cistron will decrease to similar extents as expression levels of the second cistron unless both are expressed from separate transcripts.

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Fig. 10: Working principle of siRNA-based validation of bicistronic luciferase reporters. Schematic representation of a RNAi control assay to test bicistronic reporters used in internal ribosome entry site (IRES) identification. Co-transfection of bicistronic expression vectors and a siRNA targeting the first cistron (FLuc) of the reporter allows to distinguish bona fide IRES elements (1st indicated transcript) from splice sites (2nd + 3rd transcript) and cryptic promoters (4th + 5th transcript) that generate capped monocistronic reporters containing the second cistron (RLuc) only. Adapted from Terenin et al., 2017 [64].

We performed the siRNA validation experiments in mESCs and HEK293 cells. A bicistronic EMCV reporter served as positive control in this assay and exhibited a severe decrease in FLuc and RLuc expression after co-transfection of FLuc-targeting siRNA demonstrating the expected reliability of the bicistronic reporter. Contrary, the negative control, composed of two individual vectors encoding FLuc and RLuc separately, displayed a decrease in FLuc expression only while RLuc expression was stable or even slightly increased upon FLuc siRNA co-transfection. The validation assay in mESCs revealed for almost all candidate reporters high levels of RLuc expression after siRNA-mediated FLuc knockdown as it was seen in the negative control (Fig. 8C). RLuc expression of candidate reporters was barely depleted and ranged from ~100 % to 50 % of the expression in the untreated control in all tested candidates except for one. This indicates that a substantial amount of RLuc expression of these candidate reporters is generated from aberrant transcripts which are not of bicistronic nature. It is still possible that candidate sequences mediate RLuc translation by internal translation initiation; however, these results suggest that the contribution of internal translation initiation to RLuc reporter expression in this assay is rather minor. To determine potential IRES activity of candidates containing cryptic splice sites or promoters resulting in aberrant monocistronic transcripts, the reporters could be transcribed in vitro and transfected into mESCs in the form of RNA. The only candidate reporter which showed a decrease in RLuc expression after siRNA- mediated knockdown of FLuc that was similar to the decrease in EMCV-RLuc reporter was 162

Pqbp1 (Fig. 8C). In both, EMCV and Pqbp1 reporters, RLuc expression was reduced to ~15 % of the untreated control suggesting that RLuc was primarily expressed from bicistronic transcripts i.e. by internal translation initiation. The siRNA-based validation of candidate reporters was also performed in HEK293 cells (Fig. 8D). Here, co-transfection of FLuc-targeting siRNA efficiently depleted RLuc expression of the bicistronic EMCV control reporter as expected. In the second control where FLuc and RLuc were expressed from two separate monocistronic transcripts, siRNA-mediated knockdown of FLuc also decreased RLuc expression to ~75 % when compared to the untreated control. Thus, siRNA-mediated knockdown of FLuc had some unspecific side effects on RLuc expression in HEK293 cells. The validation of candidate reporters inHEK293 cells showed a considerable decrease in RLuc expression after siRNA-mediated knockdown of FLuc for most of the tested candidates (Fig. 8D). Overall, the decrease in RLuc expression was in between ~60 % to ~8 % of the expression in untreated controls. Considering that in the negative control assay RLuc expression was also reduced to 75 %, a reduction of candidate driven RLuc expression to ~60 % is less meaningful. Only two candidate reporters, Pqbp1 and Zfp808, passed the validation as they displayed severely reduced RLuc expression after FLuc knockdown which was in the same range as the EMCV positive control and in the same range as FLuc repression (Fig. 8D). Taken together, the validation assays in mESCs and HEK293 cells indicate that substantial amounts of RLuc were not produced from bicistronic reporter transcripts so that for the majority of tested candidates no clear conclusion can be drawn on their abilities to promote internal translation initiation. Considering that translational activity of candidates was mostly very low and that candidate-driven translation initiation could generally not be triggered by 4E- BP1(4Ala)-mediated inhibition of cap-dependent translation, the results suggest that the majority of tested candidates is unlikely to promote meaningful levels of internal translation initiation. The only candidate sequence, which passed the validation assay in both cell lines was Pqbp1 (Fig. 8C, D). One further candidate, Zfp808 passed the test inHEK293 cells but its translational activity was low (26 % of EMCV IRES) and significantly reduced upon 4E- BP1(4Ala) co-expression (9 % of EMCV IRES), suggesting that potential internal Zfp808- mediated translation initiation is of minor relevance compared to cap-dependent Zfp808 translation (Fig. 8D). Pqbp1 instead exhibited high translational activity (110 % of EMCV IRES in mESCs, 250 % of EMCV IRES in HEK293 cells) that was stable upon 4E-BP1(4Ala) co- expression (120 % of EMCV IRES in mESCs, 230 % of EMCV IRES in HEK293 cells). It further substantially enhanced cap-dependent translation of bicistronic reporters in mESCs. In order to further evaluate a potential CITE-like mechanism or internal translation initiation on 163

Pqbp1, we in vitro synthesized differentially capped monocistronic reporters and tested them in a more controlled Drosophila cell-free in vitro translation system. The other two candidates Fam96b and Riok1 which displayed very high RLuc/FLuc ratios in mESCs (2.9-fold and 12-fold higher than EMCV IRES) did not pass the siRNA-based validation. This suggests that both candidates harbor sequence elements which are efficiently promoting the generation of aberrant monocistronic transcripts e.g. an alternative promoter or spice site and/or sequence elements which promote high cap-dependent translation efficiency of aberrantly produced transcript. In order to identify functional sequence elements within Fam96b and Riok1, we performed a series of deletion mutations to narrow down the part of the sequence which constitutes high reporter expression activity.

3.4.3 Mapping of functional sequence elements in selected genes Fam96b and Riok1 A series of deletion reporters was cloned to identify functional sequence elements within Fam96b and Riok1 that contribute to RLuc activity in bicistronic reporter assays. Sequential deletions of Fam96b suggested that the functionally active element is located in the second half of the sequence towards the 3’-end (Fig. 11A). While deletion of the first 2/5th of Fam96b sequence had almost no effect on RLuc expression, deletion of 3/5th diminished RLuc expression by 60 % and deletion of 4/5th diminished RLuc expression by 85 %, indicating that sequence elements with the highest impact on RLuc expression are located in a stretch of ~100 nt around the middle towards the 3’-end of Fam96b. Deletion reporters of Riok1 showed a similar step-wise reduction in RLuc activity with the functionally relevant stretch of ~180 nt being located around the middle towards the 5’end of the sequence (Fig. 11A). While deletion of the first 1/5th of Riok1 sequence had almost no effect on RLuc expression, deletion of 2/5th reduced RLuc expression by ~65 % and deletion of 3/5th reduced RLuc expression by ~91 %. Taking the RNAi assay and the deletion studies together, these results suggest that cryptic promoters or cryptic splice sites within Fam96b and Riok1 generate aberrant RNA transcripts and that these cryptic elements are contained within the demonstrated sequence stretches. Indeed, Fam96b contains a canonical splice acceptor site preceded by a polypyrimidine tract and several potential branchpoints close to consensus within the functionally active sequence stretch that was identified by the deletion studies (Fig. 11B). Likewise, Riok1 contains the promoter sequence Cage1_1 within the functionally active stretch identified by the deletion studies (Fig. 11C). Cage1 and Riok1 are divergent genes in very close proximity which even partially overlap in some of their 5’UTR isoforms. This setting suggests that the Cage1_1 promoter might possibly function in a bidirectional way so that it 164 could promote transcription of a truncated Riok1-RLuc transcript in the reporter assays. Interestingly, Cage1_1 indeed exhibits certain features, which are common for bidirectional promoters, like lack of a TATA box, presence of a CCAAT box, enrichment in CGs, localization within a CpG Island, a motif close to NRF-1 consensus binding site (CGCGCATGCTCC) and a motif close to GABPA consensus binding site (CCGGAAGT) [872]. In summary, the reporter assays demonstrate that Fam96b and Riok1 mediate high reporter expression which is rather caused by sequence elements affecting RNA transcription than by elements promoting internal translation initiation. These results demonstrate the need for adequate validation of bicistronic reporter assays, as otherwise results can easily be misinterpreted due to artefacts.

Fig. 11: Deletion series of Fam96b and Riok1 reveal functionally active elements of transcriptional regulation. (A) Dual luciferase reporter assays in wt mESCs transfected with bicistronic candidate reporters carrying deletions of increasing length at the candidates 5’ terminus. Cap- dependent Firefly luciferase (FLuc) and IRES mediated Renilla luciferase (RLuc) translation levels were determined 24 h post transfection. Number of deleted base pairs from 5’end is indicated. Error bars represent standard deviation of three biological replicates. Bicistronic EMCV IRES and non-functional EMCV* IRES reporters were used as control. (B) Fam96b 5’ UTR sequence with functionally active sequence stretch highlighted in red and indicated putative splicing elements (highlighted in varying colors). (C) Riok1 5’ UTR sequence with functionally active sequence stretch highlighted in red and indicated promoter region (green) and CCAAT-box (purple).

3.4.4 Selected candidate gene Pqbp1 is cap-independently translated in vitro Out of 17 candidates that were tested for internal translation initiation in bicistronic reporter assays, Pqbp1 was the only one that passed the siRNA-based validation in both mESCs and HEK293 cells. Further, in mESCs Pqbp1 displayed both potential IRES activity which was as strong as EMCV IRES and potential CITE activity that substantially enhanced translation of the cap-dependent reporter within a bicistronic reporter construct. To further determine the cap- dependency of Pqbp1 we generated differentially capped monocistronic reporters that carry 165 either an ordinary m7Gppp-cap or a non-functional Appp-cap analogue. The cap-analogue itself is not recognized by the translational machinery i.e. it does not recruit ribosomes, but it protects the RNA from degradation. By comparing translational rates of m7Gppp-capped and Appp- capped RNAs one can distinguish the contribution of cap-dependent and cap-independent translation to the overall translational output of the transcript. We performed in vitro translation assays using the differentially capped RNA reporters in a cell-free Drosophila embryo system. While the expression of the RLuc control reporter was widely reduced when m7Gppp-cap was replaced with cap analogue, CrPV IRES-, HCV IRES- and Pqbp1-mediated RLuc expression was unimpaired (Fig. 12A). The CrPV IRES reporter was even 3-fold more efficiently translated when it was Appp-capped, while Pqbp1- and HCV-mediated translation was only slightly increased (Fig. 12B). These results show that the contribution of m7Gppp-cap to Pqbp1 translation is low and that in vitro cap-independent translation of Pqbp1 is as efficient as cap-dependent translation. However, translation levels of Pqbp1 and HCV IRES reporters were an order of magnitude lower than translation levels of the RLuc control reporter missing a 5’UTR (Fig. 12B). This indicates that expression of both sequences is rather inefficient in the Drosophila system, probably due to inadequate concentration, absence or differential evolutionary conservation of specifically required initiation factors compared to mammals. Moreover, it is possible that the in vitro synthesized reporters lack RNA modifications that contribute to translation efficiency. We went on to investigate how cap-independent translation of Pqbp1 behaves in vitro during cap-dependent translation inhibition. Therefore, we added the m7Gppp-cap in trans to the translation system. Excess of the cap-structure increases the competition between capped mRNAs in recruiting the translational machinery so that the m7Gppp-cap itself is of no translational advantage anymore and cap-independent translation initiation is more efficient. For the m7Gppp-capped RLuc control reporter, translation is severely decreased by addition of the cap in trans (Fig. 12C). In contrast, m7Gppp-capped Pqbp1 reporter expression is even slightly increased after addition of the cap (Fig. 12C). While under normal conditions Pqbp1 reporter expression is an order of magnitude lower than RLuc control reporter expression, it slightly exceeds RLuc reporter expression under m7Gppp-induced cap-dependent translation inhibition. These results suggest that under specific conditions in which cap- dependent translation is diminished, cap-independent translation of Pqbp1 is sufficient to compete with repressed cap-dependent mRNAs at least in vitro. The viral m7Gppp-HCV IRES behaves like Pqbp1 as it is expressed at similar levels and as its expression is stable during translation inhibition by excess m7Gppp (Fig. 12C). The m7Gppp- CrPV IRES instead is two orders of magnitude more efficiently translated than Pqbp1, however, 166 after addition of cap in trans, CrPV IRES translation reduced remarkably (Fig. 12C). Anyhow, during inhibition of cap-dependent translation CrPV IRES expression is still the most efficient one in this system. Such a dramatic decrease in CrPV IRES reporter expression seems unexpected, but it was already shown elsewhere that m7Gppp-capped viral IRESs perform worse under m7Gppp-induced translation inhibition than their Appp-capped counterparts in vitro [67].

Gcap A Acap C 100

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Acap RLuc Acap CrPVGcap HCVAcap HCV Gcap RLuc Acap Pqbp1Gcap CrPV Gcap Pqbp1 Fig. 12: Pqbp1 is cap-independently translated in vitro. (A) Drosophila cell-free in vitro translation assay comparing translation of monocistronic Pqbp1 reporters carrying either m7Gppp-cap or Appp-cap analogue. Renilla luciferase activity (RLuc) was normalized to Firefly luciferase activity (FLuc) of a m7Gppp-capped monocistronic FLuc internal control reporter. Error bars represent standard deviation of three biological replicates. CrPV and HCV reporters were used as positive controls and RLuc reporter lacking a 5’ UTR was used as negative control. (B) Individual RLuc and FLuc translation levels of m7Gppp-capped or Appp-capped reporters shown in (A). Error bars represent standard deviation of three biological replicates. (C) Drosophila cell-free in vitro translation assay testing m7Gppp-capped Pqbp1 reporter in the presence or absence of cap-dependent translation inhibitor (m7Gppp added in trans). m7Gppp-capped monocistronic FLuc reporter was used as internal translation control (lower panel). Error bars represent standard deviation of three biological replicates. m7Gppp-capped CrPV and HCV reporters were used as positive controls and m7Gppp-capped RLuc reporter lacking a 5’ UTR was used as negative control.

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In summary, the in vitro translation experiments show that Pqbp1 exhibits low cap- dependency and that cap-independent translation of Pqbp1 yields meaningful levels of protein expression when compared to cap-dependent and HCV IRES translation during inhibition of cap-dependent translation. Nevertheless, Pqbp1 translation is far less efficient as CrPV IRES- mediated translation in the Drosophila system, which might be explained by the fact that CrPV is specialized on insect infection whereas Pqbp1 was originally identified in mouse and HCV only infects humans and chimpanzees [873]. Thus, it is recommended to confirm cap-independent translation of Pqbp1 in a mammalian cell system, either in vitro or even better in vivo by RNA transfection. After demonstrating that Pqbp1 can efficiently promote cap-independent translation, it remains to be clarified how efficiently Pqbp1 mediates 5’end-independent and internal translation initiation. In vivo assays in mESCs suggest that Pqbp1 can boost cap-dependent translation when located downstream of the ORF. This resembles the working mechanism of plant viral CITEs that enhance translation initiation from their location in the 3’UTR but can also function when placed at the 5’end [874]. CITE-like translation is 5’end-dependent with ribosomes scanning the 5’UTR from 5’ to 3’ direction. By inserting stable hairpins at the very 5’end of bicistronic or monocistronic Pqbp1 reporters it can be determined how much Pqbp1 translation initiation relies on a free 5’end. Further, hairpins in bicistronic reporters can reveal whether potential internal translation initiation of Pqbp1 is dependent on translation of the upstream cistron. Ultimately, it is recommended to perform the assays by RNA transfection of reporters to complement the siRNA-based control experiments which already suggested that Pqbp1 is free of cryptic splice sites or cryptic promoters causing artefacts.

3.5 CircMbl can be translated in a cap- and 5’-end-independent manner in vitro Mbl is a RNA binding protein that was initially described in Drosophila. It is conserved in mammals and regulates alternative splicing of pre-mRNAs. The Mbl gene locus is complex and gives rise to 13 protein coding transcripts as well as 30 circRNAs in Drosophila melanogaster (circBase as of 15 December 2015, NCBI Gene as of 9 May 2019) [108], [736]. Eight of these circRNAs harbor the potential to code for a circRNA-specific protein, as they contain ORFs that span the head-to-tail junction [1]. One of these ORFs is an infinite ORF, meaning that the circRNA does contain a start but no stop codon in none of the three reading frames, which would generate a “Möbius protein” [1]. In order to investigate, if circMbl can indeed be translated into peptides, collaborators from the Kadener Lab in Jerusalem performed ribosome-profiling in fly heads. They identified a number of circRNAs with ribosome-footprinting (RFP) reads that span the circular backsplice 168 junction [1]. The most abundant among them was circMbl that also exhibited a high stop codon score, strongly suggesting that the stop codon is used during active circRNA translation. Furthermore, the translation of circMbl was supported by minigene assays in S2 cells and transgenic flies. The minigene experiments revealed that circMbl produces protein and that protein production was abrogated by mutation of the 5’ss and enhanced when cap-dependent translation was repressed by starvation, 4E-BP or FOXO overexpression [1]. Additionally, mass spectrometry of fly heads identified a peptide that can only be synthesized from one of the circMbl isoforms and not from a linear Mbl RNA isoform [1]. However, transcription of the minigene constructs generated also linear concatemers that could potentially have contributed to peptide expression. Further, it was not possible to validate the peptide encoded by the most abundant ribo-circMbl by MS, as it could not be ionized sufficiently. Thus, we performed in vitro translation assays to collect additional evidence for cap- independent translation of circMbl and to determine the characteristics of circMbl translation initiation in greater detail. First, we determined the capacity of circMbl to mediate translation initiation from linear m7Gppp-capped, Appp-capped or m7Gppp-capped bicistronic RNA reporters. Comparing translational rates of all three reporters allows to make distinctions between the circMbl efficiency in mediating cap-dependent, cap-independent and 5’-end independent/internal translation initiation. In vitro translation assays in a cell-free Drosophila system revealed that the m7Gppp- capped circMbl reporter was ~8-fold more efficiently translated than the Appp-capped circMbl reporter and ~10-fold more efficiently translated than the bicistronic circMbl reporter (Fig. 13A). These results suggest that circMbl translation is less powerful than linear Mbl translation and that the prevailing mechanism of protein expression is cap-dependent. However, as circRNAs are in general more stable than mRNAs and cap-independent translation can be predominant under certain cellular conditions, a circMbl translation efficiency of 10 % could still be meaningful. Moreover, circMbl encoded peptides are unique in their domain composition and could have specific regulatory function in RNA metabolism or Mbl protein production through regulatory feedback loops. For that reason, we continued to investigate the translational efficiency of circMbl in comparison to other cap-dependent and cap-independent reporters as well as in the context of cap-dependent translation inhibition. Therefore, we generated circularized reporters containing either full-length circMbl (circMbl-RLuc), a deletion of most of the circMbl sequence (del- circMbl-RLuc) or full-length circMbl in reverse orientation (rev-circMBL-RLuc) (Fig. 13B). In the in vitro translation system, the translational efficiency of circMbl was ~25 % of the translation efficiency of a linear cap-dependent reporter (m7Gppp-RLuc) (Fig. 13C). In comparison, CrPV 169

IRES displayed ~75 % of the translation efficiency of a linear cap-dependent reporter. Deletion of most of the circMbl sequence completely abrogated translation and reverse orientation of circMbl decreased translation by a third. Addition of m7Gppp-cap in trans inhibited cap-dependent translation remarkably so that cap-dependent control reporter expression was reduced by ~85 % (Fig. 13C). In contrast, translation efficiencies of circMbl and CrPV IRES were elevated more than 2-fold. Also, circMbl in reverse orientation displayed a more than 2-fold increase in translation efficiency. Taken together, these results demonstrate that circMbl like CrPV IRES translation is indeed cap-independent and can further be stimulated under conditions of cap-dependent translation repression. In this context circMbl can be translated as efficiently as a cap-dependent reporter under normal conditions. Thus cap-independent translation of circMbl has the potential to make a meaningful contribution to Mbl protein production when cap-dependent translation is reduced. Surprisingly, reversed circMbl was also able to mediate circRNA translation even though the sequence contains multiple uORFs. Some of them even display a strong AUG context (3 out of 5 uORFs with AUG codons matching in -3 and/or +4 position the Kozak sequence [17]) which is associated with reduced translation of the downstream ORF [84], [319]. This suggests that either the effect of translational repression by uORFs in 5’UTRs cannot be transferred to uORFs within circRNAs or that circMbl is functionally active through its secondary structure and not through its primary nucleotide sequence.

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Fig. 13: CircMbl can be cap-independently translated in vitro. (A) Drosophila cell-free in vitro translation assay comparing a linear monocistronic circMbl reporter carrying a m7Gppp-cap to a linear monocistronic circMbl reporter carrying an Appp-cap analogue and a linear bicistronic circMbl reporter carrying a m7Gppp-cap. Renilla luciferase activity (RLuc) was normalized to Firefly luciferase activity (FLuc). Error bars represent standard deviation of three biological replicates. (B) Linear and circular mbl- RLuc RNA reporters synthesized for in vitro translation were analyzed by TBE-Urea gel electrophoresis and SYBR gold staining. To control for circularity, reporters were digested with RNaseR. (C) Drosophila cell-free in vitro translation assay comparing circularized circMbl reporter, a circMbl deletion reporter (del- circMbl, negative control) and a reversed circMbl reporter (rev-circMbl, negative control) to linear m7Gppp-capped RLuc and linear Appp-capped CrPV reporters (positive control). Assay was carried out in the presence or absence of cap-dependent translation inhibitor (m7Gppp added in trans). m7Gppp- capped monocistronic FLuc reporter was used as internal translation control (lower panel). Error bars represent standard deviation of three biological replicates. Some of the data have been published previously in Pamudurti et al., 2017 [1].

4 DISCUSSION

4.1 Potential role of cap-independent translation of Pqbp1 PQBP1 is a rather unpopular (reflected by 87 hits at PubMed for the search term PQBP1 as of 23 May 2019 [875]) polyglutamine-binding protein that was first identified in 1998 [876]. It is localized in the nucleus and the cytoplasm, with dominant nuclear localization and mutations within the gene have been linked to neurodegenerative disorders. In human tissues, Pqbp1 is ubiquitously expressed at largely homogenous expression levels which are higher in ovaries and lower in the pancreas [877]. PQBP1 is a disordered or denatured protein with a unique domain

171 structure. Its N-terminal WW domain binds proline-rich motifs [878]. The subsequent disordered polar amino-acid-rich domain consists of five hepta- and an imperfect stretch of di-amino acid repeats, which are required for interaction with polyglutamine tracts [878], [879]. The third largely disordered C-terminal domain is unique with no homologous sequences found in other proteins [879], [880]. PQBP1 functions in mRNA metabolism by regulating transcription, splicing and translation of its targets. For instance, it can bind to the C-terminal domain of RNA Pol II, which leads to reduced transcription [881]. The binding is enhanced by RNA Pol II phosphorylation suggesting that PQBP1 interacts with actively transcribing RNA Pol II [881]. The interaction is further enhanced by mutant ATXN1 that causes spinocerebellar ataxia type-1 (SCA1), leading to an even greater decrease in transcription, reduced phosphorylation of RNA Pol II and eventually cell death [881]. As PQBP1 can also interact with the polyglutamine tract of HTT with increased affinity to mutant HTT carrying expanded polyglutamine stretches, it is suspected that a similar mechanism of impaired transcription might play a role in the pathology of Huntington’s Disease [878], [880]. Moreover, transgenic mouse expressing the human Pqbp1 gene showed a late-onset, gradually progressing motor neuron disease-like phenotype with reduced amounts of on neurons in the spinal anterior horn and fewer Purkinje and granule cells in the cerebellum [882]. Anterior horn tissue of the spinal cord displayed an upregulated transcription of mitochondrial genes prior to cell death, indicating that PQBP1 induced mitochondrial stress, which is a common pathological defect among human neurodegenerative disorders [883]. Moreover, PQBP1 seems to function at the interface of transcription and splicing as it is not only binding RNA Pol II but also splicing factors like SF3B1, WBP11 and U5-15kD [884]– [887]. In complex with U5-15kD and U5-52K, PQBP1 might even be integrated in the early spliceosome prior to its activation [888], [889]. In HeLa cells PQBP1 was found to bind several members of the SF3B protein complex and in mouse embryonic cortical neurons it was shown to regulate about 1,400 alternative splicing events of 457 target mRNAs [887]. Targets were enriched for GO terms of neuron projection development and morphogenesis, dendrite development and axogenesis and PQBP1 deletion lead to reduced dendritic outgrowth [887]. In a conditional knockout mouse model, Pqbp1 was depleted from neural stem progenitor cells, which resulted in an increased cell cycle length, especially affecting the M phase [890]. The mice displayed microcephaly and a decreased stem cell pool during development [890]. Changes in transcription and splicing patterns were detected in a group of cell cycle genes that control the M phase [890]. Interestingly, Pqbp1 itself was deregulated, showing more pronounced aberrant splicing than transcription [890]. Further, the phenotype could be rescued by in utero gene therapy [890]. In another neuron-specific conditional knockout mouse model, Pqbp1 KD resulted in 172 reduced dendritic spine formation and cognitive decline due to aberrant splicing of genes with synapse-related functions [891]. Remarkably, changes in splicing patterns of PQBP1 conditional KD mice were similar to splicing patterns of Alzheimer’s disease model mice and 40 % of exon skipping and inclusion events in the Alzheimer’s disease model could be restored by PQBP1 overexpression [891]. It should be noted that PQBP1 stability is impaired in Alzheimer’s disease due to SRRM2 which gets phosphorylated and shifts to the cytoplasm at early stages of the disease [891]. However, PQBP1 is also located in the cytoplasm where it is associated with a function in granule formation and translational regulation. In neuronal U373MG cells, PQBP1 was shown to interact with KHSRP, SFPQ/PSF, DDX1, Caprin-1 and two subunits of the intracellular transport-related dynactin complex [892]. This interaction was also found in primary neurons, where PQBP1 and its interaction partners formed RNA-dependent granules at the perikaryon, dendrites and axons, but not at synapses [892]. PQBP1 relocates to stress granules upon stress, where it also interacts with FMR1. This suggests that PQBP1 together with its partner might be involved in cytoplasmic mRNA metabolism including transport, localization and local translation [892]. In accordance with that, the fly homolog of PQBP1 was suggested to play a role in photoreceptor cells, where it might regulate mRNA translation through interaction with dFMR1 [893]. Apart from directing intellectual ability in neuronal cells, PQBP1 was identified to be one out of seven key drivers of a regulatory gene network related to atherosclerosis and coronary artery disease [894]. Silencing of PQBP1 in macrophages activated genes from the network and decreased the cholesterol-ester accumulation in foam cells, indicating that PQBP1 is also playing a role in vascular tissues [894]. Furthermore, PQBP1 is involved in the IRF3-dependent innate immune response to HIV-1 infection [895]. By directly binding reverse-transcribed HIV-1 DNA, associating with and stimulating cGAS activity to initiate signaling in dendritic cells, PQBP1 is acting as an immune regulator [895]. Our analyses revealed that Pqbp1 can be cap-independently translated in vitro and that it mediates internal translation initiation with comparable efficiency as EMCV IRES in bicistronic reporter assays in vivo. Potential internal translation initiation was confirmed by RNAi-based validation, however Pqbp1-driven internal translation was not stimulated during cap-dependent translation inhibition in vivo. Further, Pqbp1 enhanced cap-dependent translation when located downstream of the ORF, which suggests a CITE-like function. Cap-independent translation was as efficient as cap-dependent translation and could be stimulated by inhibition of cap-dependent translation in vitro, although Pqbp1 was overall inefficiently translated in the Drosophila in vitro system. Thus, validation in a mammalian in vitro system or preferably RNA reporter transfection 173 is recommended. Overall it is unclear whether Pqbp1 acts as a bona fide IRES, but it was shown that Pqbp1 has a low cap-dependency and that Pqbp1 remains to be efficiently translated during cap-dependent translation inhibition by 4E-PB1 in vivo. Such resistance to cap-dependent translation inhibition could play a role during stress conditions like for example HIV-1 infection or mitochondrial oxidative stress in neurodegenerative or cardiovascular disease [896], [897]. As PQBP1 was shown to mediate innate immune response by binding reverse transcribed HIV-1 DNA, persistent translation of Pqbp1 mRNA during virus infection might be required for effective immune response [895]. Indeed, infection by HIV-1 was shown to activate GCN2 kinase resulting in phosphorylation of eIF2α [898], [899]. This leads to a decrease of global protein synthesis through sequestration of eIF2α by eIF2B and abrogation of ternary complex formation before GCN2 is eventually cleaved by HIV-1 protease [898], [899]. But also other translation initiation factors like eIF4G, PABP and eIF3d were reported to be cleaved during HIV-1 infection [900]. To test if Pqbp1 continues to be translated during HIV-1 induced cap- dependent translation inhibition, in vitro translation assays with purified HIV-1 protease or reporter assays in co-transfected/infected cells could be performed. In the case of disease-related mitochondrial oxidative stress, sustained translational control of Pqbp1 might be rather harmful. In both, neurodegenerative and cardiovascular, disease contexts PQBP1 overexpression is suggested to promote disease progression [883], [894]. Hence, persistent Pqbp1 translation during oxidative stress might reinforce the devil’s circle and further intensify mitochondrial dysfunction in neurodegeneration or accumulation of cholesteryl esters in atherosclerosis causing more severe outcomes. To test whether Pqbp1 translation is maintained during oxidative stress, reporter assays could be performed in cells treated with arsenite or hydrogen peroxide. Interestingly, both PQBP1 overexpression and under-expression were associated with pathophysiologic conditions [880]. Thus, it seems that tight regulation of Pqbp1 translation is critical at least for specific Pqbp1 functions. For instance, dPQBP1 mutant flies exhibit learning disability and a shortened lifespan [901]. Both can be reversed in a dose-dependent manner but while excessive dPQBP1 expression recovers learning disability, neither insufficient nor excessive dPQBP1 expression is recovering lifespan [901]. Hence, relaxed cap-dependency of Pqbp1 is likely well controlled, potentially involving one or more interaction partners or specific translation initiation factors, whose identity would be of interest in order to uncover Pqbp1s mechanism of cap-independent translation. Analysis of Pqbp1’s 5’UTR for binding sites of those factors or for RNA modification sites like m6A methylation could help to identify factors enabling cap- independent translation. In case no motifs can be found, deletion assays could help to narrow

174 down sequence stretches that are required for cap-independent translation. EMSA or RNA pull- down assays could further help to identify proteins binding to Pqbp1 mRNA. Forward-thinking, after elucidating the mechanism of action of cap-independent translation of Pqbp1, the next step would be to figure out whether interference with this mechanism could help to prevent or slow down disease progression in neurodegenerative disorders or atherosclerosis. As Pqbp1 was identified as a key driver in coronary artery disease, specific interference with cap-independent translation of Pqbp1 might have potential to decrease mitochondrial stress fueling atherosclerosis. However, atherosclerosis as well as neurodegenerative diseases are chronic conditions, wherefore potential medication would be administered over long periods of time. It would be challenging to find a way to interfere with the translational machinery (providing the mechanism would rely on standard eIFs) in the long term without causing substantial side effects. Nevertheless, strategies to interfere with mRNA translation in neuronal cells could be reconsidered in treating neurodegenerative diseases given the lack of efficient treatment options to date. Summarizing, Pqbp1 has the potential to regulate a wide range of target mRNAs through transcriptional, alternative splicing or translational control. Over- and under-expression of Pqbp1 has previously been linked to disease conditions like mental retardation syndromes or atherosclerosis. Tight translational control of Pqbp1 therefore seems warranted. The reported ability of Pqbp1 to undergo cap-independent translation initiation could play a role in Pqbp1 dysfunction in disease contexts and might contribute to development and progression of disease outcomes. Hence, further investigation of Pqbp1 translational regulation will provide insights into pathological mechanisms and might provide new approaches to advance the development of corresponding treatments.

4.2 Role of circRNA translation Our analyses revealed that circMbl mediates cap-independent as well as internal translation initiation in a fly in vitro translation system. Although internal translation initiation of circMbl was rather weak, we hypothesized it might still play a role during conditions in which cap-dependent translation is limited. And indeed, while cap-independent and internal translation initiation of circMbl reporters was only 12 % and 10 % of cap-dependent translation under regular conditions, internal translation initiation was stimulated to increase by a factor of ~3.5 when cap- dependent translation was inhibited. Since the translation of capped linear control mRNAs was repressed by a factor of 2.5 (FLuc reporter) to 6.3 (RLuc reporter) under the same conditions, the results suggest that circMbl-driven translation has the potential to compete with cap-dependent mRNA translation under specific cellular conditions. 175

The circMbl sequence that exhibits cap-independent and internal translation initiation activity spans 456 nt and is located within the 5’UTR of Mbl. More specifically, the sequence is contained within the second exon of Mbl where it is located directly upstream of the start codon and the first 189 protein coding nts. The sequence has a low minimum free energy of - 77.4 kcal/mol and a low GC content of 34 % [902]. Interestingly, it contains several stretches of multiple adenosine nucleotides: two oligo(A)s with the length of five nucleotides, two oligo(A)s with the length of six nucleotides and one oligo(A) with the length of 17 nucleotides. It was shown previously that PABP can bind the consensus motifs AAAAA and ACDAAYM (D = U, A or G; Y = G, U or C; M = A or C) in human and UAUAUA in yeast [903], [904]. Further, the PABP binding affinity increases with increasing oligo(A) length of up to twelve nucleotides [905]. As circMbl contains several oligo(A) stretches and four times the UAUAUA-binding motif it might interact with one or several PABPs. In general, PABP binding to the poly(A) tail of mRNAs is associated with higher mRNA stability and mRNA translation [906]–[908]. Enhanced translation is mediated through the interaction of PABP with eIF4G which is thought to generate a ‘closed-loop’ conformation between the mRNA 5’ and 3’ end [909]. It was previously reported that PABP can also bind 5’UTRs of a small subset of mice mRNAs regulating transcription, DNA binding, nuclear processes and cell cycle control including Mbnl1 (three 5’UTR CLIP tags) and Mbnl3 (one 5’UTR CLIP tag) [910]. Hence, PABP binding to the 5’UTR of Mbl might be conserved in flies. Interestingly, PABP also binds to A-rich sequences in its own 5’UTR, establishing a feed-back loop that suppresses PABP synthesis [911], [912]. Thus, translational regulation by PABP might be determined by interaction partners who either promote or repress translation, although more promoting interactions are known until now. To verify that PABP binding to circMbl is involved in internal translation initiation, deletion experiments could be performed to investigate if circMbl translation is reduced upon removal of the 17 residues long oligo(A) stretch. Further, in vitro translations could be carried out in a system previously depleted of PABP. Also, circMbl RNA pull-down assays could be performed using biotinylated DNA oligo probes against the circMbl backsplice junction, followed by PABP Western blotting. Radiolabeled circMbl in vitro translation reactions could be separated on sucrose density gradients to not only identify potential PABP interaction but also analyze the composition of initiation complexes assembled on the circMbl IRES. To test which factors are required for initiation, the system can be manipulated to lack activity of individual eIFs due to depletion or targeted inhibition. Interestingly, a recent non-peer reviewed publication reported that 10-nt AU-rich motifs are capable of driving translation initiation of circRNAs [913]. A 10 nt long oligo(A) stretch upstream of a circRNA encoding GFP enhanced reporter expression when PABPC1 was co- 176 transfected and tethering of a Puf-PABPC1 fusion protein to circGFP similarly enhanced reporter expression [913]. Unfortunately, the experiments lack appropriate controls ruling out that reporter expression is not driven by linear concatemers, which tend to be generated by circRNA reporter plasmids [1]. It remains uncertain how the short AU-rich motifs mediate circRNA translation and whether the mechanism is active in natural circRNAs. Even though AU- rich hexamers were enriched in circRNAs compared to linear mRNAs and analyses predicted that an AU-rich hexamer will occur by chance every 50 nts, no endogenous circRNAs were investigated for motif-mediated internal translation [913]. Hence, it is unclear whether the identified motifs are active in natural circRNA contexts. Nevertheless, the results indicate a potential positive effect of PABP on circRNA translation. But not only circMbl mediated internal translation, also the inverted circMbl sequence that was used as a negative control was able to promote internal translation with ~60 % of the efficiency of circMbl in forward orientation. Unexpectedly, reverse circMbl was also stimulated by cap-dependent translation inhibition in the same way as circMbl in forward orientation. As nucleotide sequences in the reverse orientation will fold into different secondary structures due to asymmetrical free energies of stacking base pairs (e.g. 5’-GC/GC-3’ -3.4 kcal/mol and

5’CG/CG-3’ -2.4 kcal/mol) it is rather unlikely that circMbl is forming≃ an IRES that drives translation through≃ structural elements in both forward and reverse orientation [914]. As oligo(A) stretches are palindromic they could also function in reverse orientation to recruit PABP. The long 17 nt oligo(A) stretch of circMbl is still located upstream of the authentic Mbl start codon in the reserve sequence, however, several upstream AUGs and upstream ORFs could reduce translation initiation from the authentic start codon or could cause synthesis of an N-terminally extended reporter. Interaction of PABP with reverse circMbl could be investigated as mentioned above and translation of N-terminally extended reporters could be examined by Western blots for RLuc. In Drosophila, the Mbl gene locus gives rise to 30 circRNAs in total (circBase as of May 2019 [736]). Of these, eight contain the second exon and thus have capacity to encode potential C-terminally truncated protein isoforms [1]. Putative circMbl encoded peptides were found in fly synaptosomes and circRNAs have also been found to be enriched in synaptosomes and synaptic genes in several other organisms [737], [742], [747], [749], [915]. Hence, circMbl translation might play a role in synaptic functions under conditions in which cap-dependent translation is diminished or linear mRNAs might be degraded. As circMbl sequence is also present in the 5’UTR of linear Mbl transcripts, effects of circRNA translation might be more pronounced during localized mRNA degradation or destabilization. Growing evidence suggests that local mRNA and protein metabolism are involved in neuronal functions and synaptic plasticity [916], 177

[917]. This regulation is substantially shaped by functional sequence elements within 3’UTRs [918]–[920]. As circMbl lacks 3’UTR sequences it has the potential to be differentially regulated from linear mRNA counterparts. Furthermore, circMbl encoded peptides could act as dominant negatives of the full-length Mbl protein. As previous studies indicated a role of MBNL proteins in mRNA localization, local translation and stability in addition to its well-established function in splicing, circMbl encoded peptides might interfere with such regulatory mechanisms at synapses potentially shaping underlying processes like e.g. memory formation [921]. Few other studies have recently found indications for endogenously translated circRNAs (see section 1.5.1) [743], [807], [808]. Taken together, the process of circRNA translation seems to be a regulated one instead of happening by chance, as circRNAs of varying abundances can be found associated with ribosomes and translational efficiency of different types of circRNAs varies at least when expressed from minigenes [1], [743], [807]. But only a small proportion of circRNAs has translational potential and the vast majority of circRNAs cannot be detected in association with ribosomes or polysomes [1], [727], [731], [737], [801], [807]. circRNAs are biased to originate from the 5’ terminal part of their host gene and 14 % of human circRNAs as well as 17 % of mouse circRNAs deposited at circBase are annotated to contain 5’UTR sequences [736]. Further, about half of the circRNAs that contain overlapping ORFs with their host gene have the potential to encode known protein domains involved in protein-protein interaction or protein- RNA interaction [913]. In addition, there are indications that polysome-associated circRNAs have longer putative ORFs compared to total circRNAs and that ribosome-associated circRNAs often share the start codon and parts of the 5’UTR with their host gene [1], [807]. Together, this suggest that only few circRNAs function in translation and that these circRNAs are likely to originate from the 5’-terminal end of the host transcript likely sharing the same regulatory 5’UTR, start codon and N-terminal protein domains. Moreover, the studies suggest that circRNA translation is inefficient under regular conditions but can be activated when cap-dependent translation is compromised [1], [743]. Evidence exists that upregulation of circRNA translation involves circRNA splicing and/or methylation [743], [807]. The exon-junction complex, eIF4G2 and eIF3A might play a role in regulating circRNA translation but so far there are at most indirect hints [743], [807]. Hence, a couple of open questions remain regarding the mode of action of circRNA translation and involved interaction partners: Which factors are required to drive internal translation from circRNAs? Which cellular conditions must be met to allow for functionally relevant circRNA translation? Do circRNA encoded peptides perform a function or is circRNA translation indirectly affecting the regulation of surrounding transcripts competing for involved factors?

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Future investigations will provide mechanistical insights that will help to elucidate the function of circRNAs in translation helping to further define regulatory roles of this new class of RNA. A better understanding of circRNA-mediated protein synthesis might eventually enable the development of therapeutic applications of circRNAs that might have potential in the treatment of neurological disorders as circRNA might fulfill regulatory functions in neuronal processes also in healthy organisms [737], [742], [784], [922], [923].

4.3 Challenges in validation of internal translation initiation The bicistronic reporter assay is the gold standard among the techniques that can be used to validate internal translation initiation. The unique feature, which discriminates internal initiation from other modes of translation initiation, is by definition that ribosomes are recruited to an internal part within the mRNA to be translated. To prove that the ribosome indeed enters the mRNA internally, one need to rule out potential ribosomal entry at the 5’end. Therefore, one could either use a circularized reporter that lacks any ends, a monocistronic reporter with a stable stem loop inserted at the 5’end or a bicistronic reporter. As generating circularized reporters is technically more challenging than generating linear ones, this method is not considered first choice. Further, a monocistronic reporter whose 5’end is blocked by a stable hairpin is still exposing the sequence under investigation at the 5’end and the stable element could introduce structural constraints that would interfere with the intrinsic initiation mechanism. Thus, the use of bicistronic reporters became the most conventional way to test IRES activity. The working principle of such bicistronic reporter assays is simple: translation of the upstream cistron is cap-dependent, while translation of the downstream cistron is dependent on the intermediate sequence of interest. Due to the bicistronic setup, the reporter comes with its internal control and contribution of IRES-mediated translation can be set in relation to cap- dependent translation. However, the assay is prone to generate artifacts, wherefore additional control experiments are required to achieve unambiguous results. When reporter assays are carried out by DNA transfection of reporter-encoding plasmids, reporter expression might not only be translationally but also transcriptionally controlled. Monocistronic mRNAs might arise due to cryptic splice sites or promoters within the plasmid backbone or within the putative IRES-containing sequence under investigation itself. These aberrantly generated mRNAs will be capped and translationally competent in contrast to monocistronic mRNAs that would be uncapped and likely unstable when generated by breakage [72]. For example, firefly luciferase (sequence from the Photinus genus contained in pGL3-Basic, Promega) has cryptic promoter activity that is 10-16 times weaker than the cytomegalovirus (CMV) immediate-early promoter when expressed in human CCL13 and Huh7 cells [924]. When 179 firefly luciferase is the 5’-reporter of a bicistronic construct, expression of the 3’-reporter will be a combination of IRES-dependent translation and firefly promoter activity generating truncated monocistronic transcripts. Another popular example of cryptic promoter activity is the 5’UTR of eIF4G, which was hypothesized to contain IRES activity. However, in a promoterless vector (SV40 promoter and chimeric intron sequence deleted from pRF (formerly pGL3R, Promega)), the eIF4G sequence yielded ~900-fold higher firefly activity than the promoterless pRF control vector in HeLa cells [53]. A series of deletion experiments mapped the promoter to a region of 168 bases upstream of the translation initiation site (TIS), with the most critical elements located between 69 and 49 bases upstream of the TIS [53]. But eIF4G was also shown to contain a cryptic splice site. Baranick et al. engineered a bicistronic vector eliminating all potential 5’ ss in the transcribed sequence upstream of the putative IRES by silent mutations [57]. That construct contains Gausia luciferase as 5’-reporter and GFP as 3’-reporter within a pGL4.75[hRluc/CMV] backbone (Promega) [57]. Using this vector, eIF4G had similar translational activity as the non- IRES sequences of γ-actin or β-globin, which was about five times lower than EMCV-dependent or cap-dependent translation in Hela cells [57]. Deeper analysis identified a pyrimidine-rich tract and a 3’ss between 65 to 52 bases upstream of the TIS, which were used for splicing between the native murine leukemia virus 5’ss or a cryptic 5’ss of env within the upstream vector backbone [57]. Splicing between the untranslated vector backbone and the bicistronic reporter might happen more often as anticipated. Commonly used pRL vectors (Promega) are designed to contain a chimeric intron downstream of the constitutive promoter region and upstream of the RLuc sequence to increase the expression level of the reporter [925]. Although splice sites of the chimeric intron are modified to reflect consensus sequences that allow for optimal splicing efficiency, the splice donor site of the chimeric intron can unintentionally pair with a cryptic splice acceptor site of the putative IRES [925]. This was shown, for example, for the pRL-XIAP- FL construct in which the majority of the produced transcript contained the FLuc reporter only, while the complete RLuc sequence was spliced out due to splicing between the donor splice site of the chimeric intron and the cryptic acceptor splice site in XIAP 5’UTR [65]. Not only chimeric introns but also other functional elements of the vector backbone can lead to aberrant reporter expression. For instance, the TATA-containing cryptic promoter within pMB1 origin of replication (ori) was shown to cause robust RNA expression when the inserted sequence of interest contained a 3’ss [481]. The authors of that study found a significant correlation between reporter activity and aberrant transcript expression from the ori, indicating that commonly used plasmids like pRF, pGL3 and pGL4 are prone to produce transcript artefacts promoting potentially incorrect interpretation of reporter assay results [481]. 180

Apart from aberrantly generated transcripts, another potential source of misinterpretation of reporter assays is the issue of background translation levels. In general, any kind of bicistronic reporter will yield expression of the downstream cistron [64]. The readout will never be zero. Hence, translational activity of putative IRESs needs to be set in context of positive and negative controls in order to figure out from which value onwards one could consider the expression meaningful. Most of the time the natural context of a sequence under investigation is its 5’UTR. In this setting putative IRES-dependent translation is usually in competition with 5’end or cap- dependent translation and in bicistronic reporters the translational activity of the putative IRES is usually inefficient compared to cap-dependent or viral IRES-dependent translation [27], [66], [73]. Inserting a sequence in the intercistronic space of a bicistronic reporter is typically yielding an expression ratio from first cistron/second cistron of about five- to twofold over the negative no IRES control [27]. As translation efficiency of this negative control should be considered background level, so in principle zero and very small, a stimulation of five to twofold is still small and putative IRES-activity substantially weaker than cap-dependent translation. [27]. It is important to keep this in mind. Further one needs to keep an eye on variations in the range of background expression, which are small but might appear meaningful when not properly controlled for. It was for example described that the design of a bicistronic reporter can affect expression levels of upstream and downstream cistrons. Both in vivo and in vitro, the expression from a downstream cistron increased with increasing lengths of the intercistronic region which lacked IRES activity [551], [926]. The expression level further increased in vitro by overexpression of PABP [926]. Different concentrations of translational regulators might explain as well why also in vivo expression levels from the second cistron of a bicistronic reporter without IRES in the intercistronic sequences can vary in between cell lines [551], [927]. Also, the order and the type of reporter can influence expression levels. A systematic analysis revealed that when inserted into pBS or pBC vectors (Agilent), poliovirus and EMCV IRESs work better when RLuc is in the upstream reporter position, while IRES activity is substantially weaker when FLuc is the upstream reporter [928]. The same was the case when RLuc was replaced by CAT [928]. Furthermore, FMCV IRES can enhance the translation of the upstream reporter, acting as a CITE by recruiting eIF4F, which makes the translation of the downstream reporter appear weaker [929]. Thus, the putative IRES activity of a sequence of interest cannot be compared between studies that used different cell lines, different intercistronic sequences, a different arrangement of reporters or different viral controls. In general, the usage of viral control IRESs can have a huge impact on the interpretation of reporter assay results. HCV IRES for example has relatively weak activity in bicistronic reporter assays, being about ten times less efficient in HEK293T cells than EMCV IRES [67]. 181

The strengths of EMCV IRES can also vary by almost an order of magnitude depending which commercially available EMCV-coding plasmid is used [930]. The EMCV IRES activity depends on the inserted sequence (some sequences carry an additional adenine nucleotide in the bifurcation loop at the junction of J and K stems, some carry a minimal sequence) and how the sequence is modified around and connected with the AUG start codon of the protein coding gene [930]. Further the commonly used viral control IRESs EMCV and HCV have both been demonstrated to exhibit cryptic promoter activity so that proper control experiments are warranted not only for potential cellular IRESs but also for viral IRES reporters [931]–[933]. But not only the choice of the positive control is important, also the choice of the negative control is crucial for interpretation of results. Frequently a reverse complement of the sequence of interest is used. However, as the reversed sequence is not evolutionary selected against start codons, it almost always contains uAUGs that inhibit downstream reporter expression [64]. Hence antisense 5’UTRs are unreliable controls prone to produce aberrant ORFs which make it impossible to directly compare reporter expression between sense and antisense sequences [64]. To avoid all these pitfalls in the interpretation of bicistronic reporter assays a couple of validation experiments must be implemented. First, DNA transfection assays require validation by RNA transfection assays to unequivocally rule out regulation at the transcriptional instead of translational level. Four sets of mRNA reporters are sufficient to dissect the initiation mechanism promoting translation of a particular sequence of interest (Fig. 14) [64]. Cap-dependency of a sequence can be determined by comparing translational activity of a m7G- with A-capped mRNA. The contribution of 5’-end-dependent translation can be determined by adding another A-capped reporter carrying a stable hairpin at the 5’-end. The hairpin inhibits ribosome entry so that the comparison between translation efficiencies of an A- capped reporter with and without hairpin gives information on how efficiently the free 5’-end is recruiting

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Fig. 14: Sets of mRNA reporters required to dissect the contribution of different modes of translation initiation that are active for a sequence of interest. The contribution of cap- dependent, cap-independent and 5’end-independent translation initiation to the overall translational activity of a candidate sequence can be determined by directly comparing differentially capped monocistronic and bicistronic reporter expression levels. Introduction of stable hairpins close to the reporter 5’end can further help to elucidate the dependencies. Adapted from Terenin et al., 2017 [64]. ribosomes. Neither cap- nor 5’-end-dependent but internal translation can be determined by bicistronic reporter assays. Comparing translation efficiency of the downstream cistron of a bicistronic reporter with translation efficiencies of monocistronic m7G- and A-capped reporters identifies the contribution of IRES-dependent translation to the overall translation efficiency. To unequivocally rule out that translation of the second cistron is coupled to translation of the first cistron, by i.e. translation reinitiation or ribosome shunting, another bicistronic reporter carrying a stable hairpin at the 5’end can be included in the analyses.

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When the contribution of the cap to the overall translation is low and expression of the downstream cistron of the bicistronic reporter equals expression of the capped monocistronic reporter, the sequence might indeed function as an IRES. But also, less efficient putative IRESs might still promote meaningful expression levels under specific cellular conditions. This requires reporter analyses in the corresponding physiological context. When the bicistronic translation efficiency is appreciably lower than cap-dependent translation of a monocistronic reporter, the sequence is unlikely to function as an IRES. Instead it might act as a CITE, when 5’-end- dependent translation is high and cap-dependent translation is low. But in most cases the studied sequence is translated two to three orders of magnitude higher in a capped monocistronic mRNA than in a bicistronic setting, which depicts cap-dependency and the absence of IRES activity [64]. In comparison, EMCV IRES can cause a 100 to 250-fold stimulation of the expressed downstream cistron compared to the upstream cistron, which begs the question of whether it is justified to call cellular IRESs, whose activity is not nearly as efficient, bona fide IRESs [27]. Moreover, it is important to provide absolute values of reporter expression instead of fold changes or ratios so that the strengths of cellular IRESs is not masked or artificially boosted by using background expression levels as a reference. As described above the translational activity of a sequence of interest should rather be compared to itself in a monocistronic context instead of an “empty” vector or a viral element. If IRES activity is tested under specific conditions like 4E- BP overexpression, protease-mediated eIF4G cleavage or in context of pharmacological inhibitors, expression levels of upstream and downstream cistrons should be displayed independently, as the calculation of a ratio can hide differential effects on cap-dependent and internal translation initiation. When bicistronic reporter assays are carried out by DNA transfection in advance of RNA transfections, promoterless plasmid and RNAi tests are required to verify candidates. By deleting the promoter region but keeping the enhancer, it can be determined if bicistronic reporter vectors produce aberrant transcripts due to cryptic promoter activity. Usage of a different vector lacking the promoter and sometimes also the enhancer to screen for cryptic promoters is likely creating a source of error, as different plasmid backbones might generate different kinds of aberrant transcripts making it impossible to compare results obtained from different backbones. As aberrant transcripts due to cryptic splicing will be missed by this approach, another RNAi test is required. Lloyd and colleagues invented a convenient control assay in which cells are co-transfected with siRNAs targeting the upstream cistron of a bicistronic reporter [65]. Expression of both cistrons will be reduced to similar extend if expression is indeed driven by the anticipated bicistronic reporter. But monocistronic reporters containing only the downstream

184 cistron due to upstream splicing, won’t be affected by the knockdown and can easily be identified by this approach. Other tests for RNA integrity like RNase protection assay, RT-PCR and northern blot can be applied as well but cannot replace promoterless and RNAi validation experiments. Further, these assays should be designed to identify aberrantly produced shorter transcripts rather than the full-length bicistronic mRNA [64]. In RNase protections assays and RT-PCR experiments probes and primers need to be designed in a way that no potentially relevant incomplete reporter species is missed. Further, overexposed northern blots might still not be sensitive enough to detect monocistronic transcripts, which might be produced at low levels but could substantially contribute to the overall reporter expression [53], [59], [65]. Also, RT-PCR experiments might not provide enough sensitivity to show differential expression between the two cistrons of a reporter. Cellular IRESs are also often studied by in vitro translation assays. However, the commonly used rabbit reticulocyte lysate (RRL) is only poorly reflecting physiological conditions. Many putative IRESs seem to have weak activity in RRL, because weakly translated mRNAs are not inhibited by supplementation with m7G or 4E-BP or are not proportionally stimulated by capping or a poly(A) tail [64], [934]–[937]. To conclude, the gold standard bicistronic reporter assay is only as powerful in describing IRES activity as is the quality of associated validation experiments. Unfortunately, essential controls are often missing, so that to date a high number of reported cellular IRESs still requires conclusive verification, leaving these putative IRESs in an uncertain or not proven state [27], [59]. Some of the previously reported cellular IRESs have also been refuted during later in-depth examination. For future research it is important to not only stick to strict controls but also consider alternative explanations for translational phenomena, like CITE-like mechanisms, which so far seem to be less prominently addressed. It is vital to create greater awareness on the fact that cellular IRESs are not yet as firmly established as widely believed to enable unbiased investigation in all directions especially to researchers who are newly entering the field [64].

4.4 Data of this study in the context of current literature and future directions Findings of this study revealed that Pqbp1 and circMbl can be translated cap-independently or internally. Previous studies have shown that both linear and circular RNA transcripts can in principle be translated in non-canonical manner, but the physiological context, involved trans- acting factors and cis-acting transcript elements are not well understood. This study contributes to the research field by identifying two RNA transcripts with non-canonical translation activity whose future investigations have potential to reveal their underlying mode of action, thus 185 possibly adding scientific evidence to close the gap of knowledge about non-canonical translation initiation. Firsts, investigations of linear mRNA translation could show that inhibition of cap- dependent translation by overexpression of dominant negative 4E-BP1, can induce expression of potentially cap-independently translated mRNAs during early stem cell differentiation in mouse. Our analysis of 17 of such potentially cap-independently translated candidate genes resulted in one verified candidate, while the residual 16 candidates didn’t pass validation experiments. The relatively low hit rate is not unexpected. As we based our studies on differentially expressed proteins that have been identified by MS, we haven’t had a direct readout. Proteins might have been upregulated due to regulatory changes at protein or transcriptional level wherefore the selected candidates were expected to contain false positives. A direct readout at translational level would be possible through ribosome profiling or polysome profiling followed by RNA sequencing. Ribosome profiling at suitable sequencing depth might even hint on sequence stretches within 5’UTRs that might function in attracting ribosome attachment during cap- independent translation initiation. Moreover, pulsed stable isotope labeling by amino acids in cell culture (pSILAC) would be an option to detect de novo protein synthesis instead of steady state protein levels obtained by our LC-MS/MS approach. Further, inhibition of cap-dependent translation by dominant negative 4E-BP1 is limited meaning a certain level of cap-dependent translation will remain, which potentially adds some false positive candidates to the analysis. Transfection of dominant negative 4E-BP1 stimulated ectopically expressed EMCV IRES translation in mESCs about 2-fold and reduced cap- dependent FLuc reporter expression on average about 30 % after 24 h, but the extend of global cap-dependent translation inhibition after 24 h of dominant negative 4E-BP induction in stably transfected mESCs is unclear. Due to the readout by shotgun proteomics, it is unclear by how much percent the global translation was decreased. Importantly, induction of stable mESC clones not only resulted in expression of dominant negative 4E-BP1 but also resulted in expression of Ascl1, which activated Ascl1-driven neuronal differentiation. As differentiation of mESCs towards neuronal progenitor cells is associated with an increase in global translation, Ascl1 expression might have counteracted dominant negative 4E-BP1 leading to diminished inhibition of global cap-dependent translation and/or enhanced translation of Ascl1 target transcripts [647]. Lastly, as discussed in the previous section (see section 4.3), the gold standard assay to validate cap-independent translation initiation is prone to create artefacts that hamper the identification of true non-canonical translation. Unfortunately, a considerable amount of published studies lacks rigorous validation of bicistronic reporter assays [27], [64]. Hence, the number of true non-canonically translated transcripts might be lower than assumed from the 186 literature. Taken together, the identification of one cap-independently translated mRNA out of 17 studied candidates seems reasonable. As just mentioned, the current literature on non-canonical translation initiation in eukaryotes might be biased creating a picture in which cellular IRESs in particular might appear more common and scientifically sound than the body of evidence suggests (see also examples of cellular IRESs discussed in sections 1.4.1 – 1.4.4) [27], [64]. This is because the field is divided into IRES advocates and IRES skeptics with the number of advocates outweighing the number of skeptics. While the group of advocates widely recognizes IRESs as a standard element of cap- independent translation occurring in an estimated ~10 % of mammalian mRNAs [135], the group of skeptics is questioning if these elements constitute such a prevailing role in cap- independent mRNA translation demanding more stringent controls for the majority of putative cellular IRESs reported. Thus, the status of cellular IRESs might appear confusing to scientist approaching the topic. In many early reports of the 1990s IRESs seemed to be well established but have been challenged by more stringent controls developed in the early 2000s which lead to a fundamental debate that is still ongoing. The initial discovery that certain mRNAs remain associated with translating ribosomes during cellular stress conditions like e.g. apoptosis, hypoxia or virus infection were embraced by the community [38], [41], [44]. IRES-mediated translation was thought to enable cells to produce stress response proteins required to adapt and eventually survive specific stress conditions under which global cap-dependent translation is inhibited. However, nowadays the landscape of translational control appears to be more complex and a couple of alternative mechanisms are emerging that could explain sustained mRNA translation during cell stress regardless of IRES activity. These include relaxed cap-dependency of certain transcripts meaning that cap-dependent translation inhibition is differentially affecting the pool of cellular mRNAs with some being more efficiently translated during stress than others due to differential contribution of the m7G-cap to the overall translation activity [67]. This leaves room for some mRNAs to be preferentially translated during reduced eIF4E activity in a 5’ end-dependent but cap-independent manner. For instance, secondary structure of 5’UTRs as well as nucleotide composition close to the cap play a role in affinity to eIF4E and requirement of eIF4A for efficient translation initiation [272], [559], [938]. Cytidine as initiating nucleotide, for example, causes a lower eIF4E affinity which leads to higher sensitivity of C-starting transcripts for the availability of eIF4E than transcripts starting with one of the other three nucleotides [559]. Further, it was found in two transcriptome-wide studies that inhibition of mTOR and subsequent activation of 4E-BP results in a strong suppression of just a few hundred mRNAs (253 or 144 mRNAs with log2 ≤-1.5) containing a 5’ TOP motif (cytidine at position 1 followed by a stretch of 4-14 pyrimidines) or a PRTE motif 187

(uridine at position 6 flanked by pyrimidines) [79], [269]. All other moderately repressed mRNAs maintain a certain level of cap-dependent translation [79], [269]. In this regard, a CITE-like translation mechanism was proposed [61], [186]. CITEs are elements within UTRs that can recruit key translation initiation factors rendering mRNA translation independent from the m7G-cap structure. However, CITE-mediated translation is still dependent on a free 5’end to promote cap-independent translation without utilization of an IRES [179]. The probably best described example is Apaf-1 mRNA, whose 5’-end-dependent but cap- independent translation is relatively resistant to apoptosis [149], [187]. Another mechanism of cap-independent translation during stress conditions, especially during viral infection, ER stress or amino acid starvation is dependent on uORF translation and eIF2α phosphorylation [13], [327]. Phosphorylated eIF2α leads to reduced abundance of the ternary complex wherefore 40S subunits which resume scanning after translating short uORFs need more time to acquire a new ternary complex to initiate translation at a downstream uORF or the start codon of the main ORF [13]. The best characterized examples are ATF4 and ATF5 mRNAs whose translation is restricted under physiological conditions but specifically stimulated under certain cellular stresses due to two uORFs from which the second one overlaps the authentic start codon [328], [939]. Further, RNA modifications like m6A can promote cap-independent but 5’end-dependent translation [189]. Specific stresses like heat shock or UV treatment induce the redistribution of m6A residues resulting in an enrichment of m6A within 5’UTRs [189]. This suggests 5’UTR m6A plays a role in mediating cap-independent translation during stress. One example is Hsp70 mRNA whose translation is upregulated during heat shock und whose methylation of a m6A site within the 5’UTR is also upregulated during heat shock [189], [311]. Interestingly, m6A was reported to also promote translation of a circRNA reporter with a split GFP ORF [807]. Further, m6A was hypothesized to play a role in translational regulation of highly methylated circZNF609 [743]. As m6A is unable to mediate internal translation initiation in linear mRNAs it is unclear how the initiation mechanism at circRNAs would differ to directly mediate positioning of the circRNA within the entry channel of translationally competent ribosomes. Apart from m6A also N1-methyladenosine (m1A) was recently found to be regulated in response to heat shock, glucose starvation, serum starvation and hydrogen peroxide [940], [941]. As it is positively associated with protein synthesis and enriched around start codons it is tempting to speculate that m1A might also function in cap-independent translation regulation. Another regulatory element of mRNA translation are ribosome modifications like heterogenous composition of ribosomal proteins or rRNA methylation and pseudouridylation. rRNA modifications have been shown to selectively affect translational efficiencies of viral IRES 188 as well as a certain subgroup of cellular mRNAs that was claimed to have IRES activity [70], [118]–[122], [942]. Similarly, non-essential ribosomal proteins like RPS25 and RPL38 were reported to be involved in selective translation of viral and certain subsets of cellular IRES- containing transcripts [124]–[128], [604], [705], [943]. In both cases IRES activity of cellular mRNAs was not unambiguously demonstrated so that translational regulation through ribosomal proteins and rRNA modifications could as well be mediated by cap-dependent or cap- independent translational mechanisms. Due to differential protein composition, modified rRNA bases, interacting factors and yet unidentified post-translational modification, the ribosome is likely present in an endless number of configurations allowing for substantial diversity of selective mRNA translation not necessarily restricted to IRES-mediated initiation [129]. Taken together diverse modes of translation initiation are in play that contribute to cap- independent translation beyond IRESs. Within the past years, transcriptome-wide studies generated deeper insights into global translational regulation and helped to identify those subsets of transcripts with selective and potentially non-canonical regulation, uncovering new layers of translational complexity. However, the progress in transcript-specific studies, especially those focusing on IRES containing ones, was rather limited: despite of almost 30 years of research, still little is known on how cellular IRESs are structured, how they work, how they are regulated and which factors they interact with, eventually leading to the question about their existence [111]. For the future, further investigations of cap-independent translation of Pqbp1 should include RNA transfection assays to show cap-independent translation in vivo. As RNA transfection of mESCs was inefficient, this assay might need to be performed in a different cell culture system though. Ideally, RNA reporter transfections would be carried out as a time course experiment to reflect kinetics shortly after transfection and avoid stability issues of RNA. To further evaluate the 5’end-dependency and the efficiency of Pqbp1 to mediate internal translation initiation, stable hairpins should be introduced at the very 5’end of m7Gppp- and Appp-capped monocistronic and bicistronic reporters. This will help to understand if Pqbp1-mediated translation requires a free 5’end and if Pqbp1-mediated internal translation initiation is independent of the first cistron. To assess the underlying mechanism of action, deletion experiments may help to identify relevant sequence elements within Pqbp1 5’UTR and potentially interacting RBPs could be identified by antisense affinity capture approaches or RNA purification methods followed by MS. Contrary to mRNAs, investigations of cap-independent translation of circRNAs is a new area of research. Since it was discovered at the beginning of this decade that circRNAs constitute a large class of eukaryotic RNA, circRNAs were no longer assumed to be missplicing products but considered functionally relevant, potentially also encoding functional peptides. While early 189 polysome and ribosome profiling approaches failed to detect circRNAs in association with translating ribosomes, our and other recent analyses indicate that few endogenous circRNAs can be translated [1], [727], [731], [737], [743], [801], [807], [944]. Such translatable circRNAs like circZNF609 and circMbl have in common that they share parts of the 5’UTR, the canonical start codon and 5’-terminal coding sequence with the host mRNA. Their reading frame spans the backsplice junction and stops shortly thereafter with the extension beyond the backsplice junction translating into a unique C-terminal peptide end. Translation is initiated via IRES elements, although IRES-mediated circRNA translation is inefficient and in case of circMbl about an order of magnitude lower than cap-dependent translation from the same 5’UTR sequence [1]. In case of circZNF609, inefficient translation was stimulated by inserting an intron into circZNF609 reporters but it remains unclear if splicing enhanced internal translation or rather generated aberrantly spliced and subsequently translated capped monocistronic reporters [743]. As in general, mRNA translation is enhanced by splicing through deposition of the exon junction complex on the mRNA, it is tempting to speculate that the exon junction complex might play a similar regulatory role in internal translation initiation of circRNAs [945], [946]. Further, cap-independent translation of circZNF609 and other circRNAs identified by Yang et al. might be stimulated by m6A peaks, potentially enabling translational regulation via m6A writer, reader and eraser proteins [807]. Lately, a non-peer-reviewed systematic analysis of the translational capacities of circRNAs containing the canonical start codon and residual 5’UTR sequence of their host mRNA was published. The analysis included human (378 samples) and mouse (75 samples) RNA sequencing data from the ENCODE consortium as well as available Ribo-Seq data (~500 human samples, ~1,300 mouse samples) to evaluate features of active translation from ~81,000 predicted circRNAs [947]. By assessing phasing, evolutionary conservation of coding sequence and stop codons, P-site positioning of the ribosome on the RNA and quality of sequencing reads the comprehensive analysis revealed that translation of circRNAs is likely a rare and uncommon event [947]. Thus, the analysis adds to the body of evidence collected in the current literature suggesting that either only a small percentage of circRNAs is translated or that translation of circRNAs is inefficient. Interestingly, both analysis by Stagsted et al. and Pamudurti et al. showed that current methods to promote ectopic overexpression of circRNA via plasmids and minigenes can generate artefacts resulting in false positive protein expression from linear concatemers [1], [947]. Even if the protein coding sequence is split by the splice junction and both parts are arranged in reversed order in the vector, concatemers may arise, which contain repeats of the exon that was supposed to circularize, in a linear fashion. Hence, protein production from ectopically expressed 190 circRNAs needs to be verified by northern blot analysis to rule out cap-dependent translation from aberrantly generated linear mRNAs. Our analysis of in vitro circRNA translation omits the concatemer-creating step of transcription; however, a northern blot of the reaction mix is recommended as a follow-up to verify that circRNA reporters are still circular after the in vitro translation and no breakage or degradation caused 5’-end-dependent translation (circRNA reporters don’t contain a cap) during incubation of the assay. Further, future analyses should be conducted to elucidate the function of the endogenously encoded circMbl peptide. Investigations into this direction were already initiated by the Kadener lab and showed that knockdown of circMbl in flies leads to male developmental lethality or a wing-posture and flight defect phenotype in the survivors [948]. Downregulation and overexpression of circMbl resulted in 39 differentially expressed mRNAs related to muscle and brain functions [948]. It is unclear which role the circMbl encoded peptide is playing in this phenotype, as circMbl is also executing a function on the RNA level by impacting the amount of full-length Mbl protein synthesis via competition between circular and linear Mbl splicing [773]. Insertion of a protein tag just upstream of the stop codon of circMbl by Crispr/Cas9 system could be performed to enable tagged peptide expression from the circRNA but not from the mRNA (because for the linear mRNA the tag sequence is located within the 5’UTR upstream of the start codon). Peptide expression could be observed by immunohistochemistry to detect cellular localization and pull-down experiments could reveal interaction partners. Moreover, the dynamics of circMbl peptide expression within muscle and brain tissue could be investigated in response to distinct stimuli or during development to obtain insights in potential functions during myogenesis, neurogenesis, memory formation, behavior or disease context like myotonic dystrophy. Unfortunately raising a specific antibody against the endogenous circMbl peptide might be difficult, as the circMbl peptide shares its entire sequence with the N-terminal part of the full-length Mbl protein except for two amino acids. In the end, the question remains which kind of role circRNAs as a large subgroup of the transcriptome are playing in the cell. The data indicates that only very few circRNAs are protein coding and that only a very limited amount of circRNAs is functioning as microRNA sponges, an activity of the first functional circRNA, CDR1as circRNA, identified. Only very recently, it was reported that circRNAs can act as inhibitors of the dsRNA-dependent serine/threonine-protein kinase PKR (also known as EIF2AK2), which is a key regulator of innate immune response [949]. The suppression of PKR by circRNAs is sequence unspecific but requires a short dsRNA region [949]. Upon viral infection RNAse L is degrading circRNAs which releases PKR inhibition and stimulates its antiviral activity [949]. Intensive research and the use of various new technologies like CRISPR/Cas9, single-molecule or whole-organism techniques will elucidate 191 potential roles of circRNA in neuronal tissue and neurodegenerative diseases, their potential application as biomarkers or potential activity in extracellular space and cell-cell communication [822]–[824], [950].

5 APPENDIX

5.1 Supplementary figure

undifferentiated day 1 day 2 day 3

wt

clone A6

clone B4

clone C6

Fig. S1: mESCs undergo morphological changes during induced neuronal differentiation. wt mESC and monoclonal lines A6, B4 and C6 during early neuronal differentiation. Pictures taken with an optical reflected light microscope.

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5.2 Supplementary table Tab. S1: List of selected candidates for cap-independent translation initiation. Candidates have been identified by proteomic analysis in mESCs after 24 h of inhibition of cap-dependent translation by dominant negative 4E-BP1 overexpression. Fold change compared to mESCs.

A6 B4 C6 combined 24 h 5'UTR splice fold fold fold fold candidates length sites change p-value change p-value change p-value change p-value GO term function Atp5e 109 2,6 0,384 2,6 0,386 2,9 0,340 2,7 0,080 ATP synthase hydrolase Brwd3 219 2,0 0,250 2,0 0,255 1,8 0,276 1,9 0,043 chromatin modifying? osteoblast differentiation, ER Ccdc47 213 1 3,3 0,000 3,1 0,000 1,1 0,316 2,5 0,011 overload response Cdkn1c 208 1 2,2 0,153 1,7 0,032 3,1 0,005 2,3 0,011 cell cycle Chek1 513 -0,1 0,856 1,2 0,351 2,5 0,024 1,2 0,223 protein phosphorylation

phosphorylation, intracellular Dclk2 682 1,9 0,051 2,4 0,003 2,3 0,058 2,2 0,005 signal transduction Ero1lb 194 0,5 0,013 2,4 0,002 -0,2 0,631 0,9 0,250 oxidation-reduction process

chromosome segregation, iron- Fam96b 275 1,9 0,147 1,9 0,149 2,2 0,112 2,0 0,006 sulfur cluster assembly Gtf2a1 445 2,0 0,147 2,4 0,133 2,0 0,170 2,1 0,042 transcription

cellular response to heat, muscle Hsbp1 38 2,6 0,001 1,1 0,220 2,4 0,005 2,0 0,007 contraction, endodermal cell

differentiation Lama1 70 3,3 0,047 3,1 0,057 3,1 0,059 3,2 0,000 cell adhesion

193

Lsm6 203 1 1,5 0,385 1,7 0,343 1,7 0,343 1,6 0,072 mRNA splicing factor Lsm7 23 2,1 0,230 3,4 0,073 1,6 0,381 2,4 0,128 mRNA splicing factor Luzp1 397 3 2,0 0,127 1,7 0,175 2,2 0,106 2,0 0,006 --- cell migration, actin cytoskeleton Palld 203 1 2,8 0,000 0,9 0,112 0,3 0,571 1,3 0,114 organization Pcdh1 230 1 1,7 0,241 3,1 0,003 3,0 0,003 2,6 0,006 cell adhesion Pex5 75 1 2,5 0,014 0,5 0,468 0,6 0,517 1,2 0,136 cell development Pqbp1 582 1,7 0,328 1,6 0,354 1,9 0,294 1,7 0,056 transcription cofactor Rad51 230 2,1 0,353 3,2 0,109 2,0 0,361 2,4 0,081 DNA strand-pairing hydrolase rRNA processing, Riok1 472 2,1 0,125 2,2 0,121 2,0 0,145 2,1 0,005 phosphorylation Sh3glb2 111 0,9 0,314 0,7 0,374 2,1 0,039 1,2 0,070 --- Stmn2 252 2,9 0,352 2,0 0,521 3,1 0,333 2,7 0,112 tubulin binding transcription, osteoclast differentiation, humoral immune Tfe3 718 2 2,5 0,130 2,8 0,101 3,1 0,083 2,8 0,003 response Zfp808 110 2,0 0,020 1,3 0,129 0,3 0,656 1,2 0,071 transcription Znhit6 136 1,6 0,153 2,0 0,109 1,5 0,182 1,7 0,010 transcription factor binding

194

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ACKNOWLEDGEMENTS

First, I would like to thank my supervisor Dr. Marina Chekulaeva and my doctor father Prof. Dr. Nikolaus Rajewsky for providing me with the opportunity to work on this exciting topic, for giving me advice and sharing their knowledge throughout the past years. Further, I would like to thank the Einstein Stiftung Berlin and the Max Delbrück Center for Molecular Medicine for providing the funding for this study, as well as the Integrative Research Institute (IRI) for the Life Sciences graduate school for providing me with a travel grant and even more important for a supportive environment around the core scientific work. I would like to express my gratitude to the members of my PhD and thesis committee, Prof. Dr. Alexander Löwer, Prof. Dr. Markus Landthaler, Prof. Dr. Andreas Herrmann and of course Prof. Dr. Nikolaus Rajewsky and Dr. Marina Chekulaeve for constructive feedback, guidance throughout the past and for reviewing this thesis. A special thanks goes to Guido Mastrobuoni for performing the proteomic analysis which is a key piece of this study, as well as to all collaborators of the circMbl project in which I was

241 happy to participate: Dr. Nagarjuna Reddy Pamudurti, Dr. Osnat Bartok, Dr. Marvin Jens, Dr. Reut Ashwal-Fluss, Christin Sünkel (Stottmeister), Dr. Mor Hanan, Dr. Emanuel Wyler, Dr. Daniel Perez-Hernandez, Evelyn Ramberger, Shlomo Sheniz, Moshe Samson, Dr. Gunnar Dittmar, Prof. Dr. Markus Landthaler, Dr. Marina Chekulaeva, Prof. Dr. Nikolaus Rajewsky and Prof. Dr. Sebastian Kadener. For scientific and non-scientific discussion, moral support and fun times I would like to thank my fellow labmates David van den Bruck, Dr. Alessandra Zappulo, Camilla Ciolli Mattioli and Dr. Marta Mauri. In the end I would like to say a big thank you to many more friends and family for continuous support, help and welcome distraction!

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