Poster Session 10: 21:00 - 22:00 Friday, 29th May, 2020 Poster

66 Translational fidelity is maintained through precise aminoacyl-tRNA accommodation dynamics gated by Tu

Dylan Girodat1, Scott Blanchard2, Hans-Joachim Wieden3, Karissa Sanbonmatsu1 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA. 2Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. 3Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada

Abstract

The fidelity of translation is enigmatic, as the efficiency of cognate aminoacyl(aa)-tRNA selection by the is greater than what can be predicted from Watson-Crick base-pairing between the codon in the mRNA and the anticodon in the tRNA. The complexity of this process arises from the fact that aa-tRNA selection is a multistep process aided by auxiliary such as the GTPase elongation factor (EF)-Tu, responsible for delivery of aa-tRNA to the ribosome. As such, the precise structural mechanism of how the ribosome in complex with EF-Tu selects for cognate aa-tRNA remains to be fully resolved.

Here, using all-atom molecular dynamics (MD) simulations, we identify subtle differences between cognate and near-cognate aa-tRNA movement into the ribosome and how conformational rearrangements of EF-Tu aid in tRNA selection. Near-cognate aa-tRNA accommodation follows an alternative trajectory, compared to cognate aa-tRNA, leading to a misaligned position within the A-site. The origins of the alternative trajectory originate from the perturbed base-pairing between the codon and anticodon of the mRNA and tRNA, respectively. The resulting position is ultimately not suitable for peptide bond formation. As aa-tRNA accommodation is initiated EF-Tu undergoes a conformational change involving the rapid conversion of the switch I element from an α- helix to a β-hairpin. The newly formed β-hairpin moves to interact with the acceptor stem of the aa-tRNA and in doing so gates the movement of the aa-tRNA during accommodation through steric and electrostatic interactions. Furthermore, switch I of EF-Tu traverses along either the acceptor stem or near the 3’-CCA end for cognate or near-cognate aa-tRNA, respectively. Ultimately, this is a result of near-cognate accommodating through a misaligned trajectory. Altogether, this project provides evidence for how the ribosome in complex with EF-Tu can sense the identity of the accommodating aa-tRNA and provides a structural description of tRNA selection.

Presenting author email [email protected]

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Translation Mechanism 156 Co-translational translocon insertion and topogenesis of bacterial membrane proteins

Evan Mercier, Marina Rodnina, Wolfgang Wintermeyer Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

Abstract

Integral membrane proteins insert into the bacterial inner membrane co-translationally via the translocon. Transmembrane segments (TM) of nascent proteins adopt their native topological arrangement with the N- terminus of TM1 oriented to the outside (type I) or the inside (type II) of the cell. Here we study TM1 topogenesis during ongoing mRNA translation in a bacterial in-vitro system, applying real-time FRET and protease protection assays. We find that TM1 of the type I LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel early on in translation. Looping presumably is mediated by interactions between positive charges at the N-terminus of TM1 and negative charges in the wall of the peptide-exit tunnel. Early TM1 inversion is abrogated by charge reversal at the N-terminus. Kinetic analysis also shows that co-translational membrane insertion of TM1 is intrinsically rapid and rate-limited by mRNA translation. Thus, the ribosome and translation play vital roles in the insertion and topogenesis of newly synthesized membrane proteins in bacteria.

Presenting author email [email protected]

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Translation Mechanism 185 How ArfB Rescues Stalled

Christine Carbone, Gabriel Demo, Rohini Madireddy, Egor Svidritskiy, Andrei Korostelev University of Massachusetts Medical School, Worcester, MA, USA

Abstract

A translating ribosome stalls when it encounters the end of a non-stop mRNA, truncated during cellular stress or other conditions (Hayes and Keiler, 2010; Keiler, 2015). Alternative rescue factor B (ArfB) rescues stalled ribosomes by catalyzing peptide release from peptidyl-tRNA (Chadani et al., 2011). Crystallographic work (Gagnon et al., 2012) showed that the C-terminal α-helix of ArfB binds in the mRNA entry channel, allowing the catalytic N-terminal domain to reach the A site of the peptidyl transferase center. Thus, binding of ArfB should be incompatible with the mRNAs extending beyond the A-site. However, previous work showed that ArfB can act on ribosomes stalled on a rare codon cluster, and it remains unclear how the ribosome can accommodate both ArfB and mRNA in this case (Handa et al., 2011). In this work, we sought to clarify the mechanism for ArfB action on a broad range of mRNA substrates, using in vitro kinetics and cryogenic electron microscopy (cryo- EM). Surprisingly, ArfB remains highly efficient on mRNAs extending beyond the A site. Using single particle cryo-EM, we identified multiple states that suggest different mRNA-length-dependent structural mechanisms for ArfB-mediated ribosome rescue.

Presenting author email

[email protected]

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Translation Mechanism 389 Resurrection of a factorless internal ribosome entry site from Ancient Northwest Territories cripavirus

Xinying Wang, Reid Warsaba, Eric Jan Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada

Abstract

Translation is a key step of in all organisms. The majority of eukaryotic mRNAs use a scanning cap-dependent mechanism that requires upwards of 12 factors to initiate translation. In contrast, the dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome containing the IGR region was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. This "ancient" virus was named ancient Northwest territories cripavirus (aNCV). Structural prediction of the aNCV IGR sequence generated a secondary structure similar to contemporary IGR IRES structures. There are, however, subtle differences that may impact IRES function. There are also 100 nucleotides upstream of the IRES with an unknown function. Using filter binding assays, we showed that the aNCV IGR IRES could bind to purified salt-washed human ribosomes. They could also compete with excess CrPV IGR IRESs for ribosomes. Toeprinting analysis using primer extension pinpointed the putative start site of the aNCV IGR: a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR IRES can direct internal ribosome entry in vitro in rabbit reticulocyte lysates that was dependent on the integrity of the PKI domain. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus points to the importance of this translational mechanism and will contribute to our understanding of viral RNA evolution.

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Translation Mechanism 563 Investigating the role of DDX3X in translation initiation

Kevin Wilkins1,2, Srivats Venkataramanan1, Lorenzo Calviello1, Bao Thai1, Malvika Tejura1, Stephen Floor1,3 1University of California, San Francisco, San Francisco, CA, USA. 2Graduate Division, University of California, San Francisco, San Francisco, CA, USA. 3Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA

Abstract

Translation of mRNA into protein is an essential process in all cells, and its dysregulation is linked to human diseases. During translation initiation, ribosome scanning is influenced by the secondary structure of the 5′ untranslated region (UTR). A family of enzymes known as RNA helicases modulate ribosome scanning and hence protein synthesis by unwinding and resolving secondary RNA structures. The RNA helicase DDX3X is an ATP-dependent DEAD-box RNA helicase that is implicated in the translation of mRNAs that contain long and highly structured 5′ UTRs. Despite DDX3X being altered in several human cancers and developmental diseases, how DDX3X acts on mRNA during scanning and what makes a given transcript responsive to it is still incompletely understood. Using an in vitro translation assay, I determined that missense mutations in DDX3X lead to decreased translation initiation for mRNAs containing select 5′ UTRs. Mechanistic details regarding how RNA sequence and structure impact DDX3X sensitivity will be presented. We previously found that DDX3 interacts with the small ribosomal subunit, but it is unknown whether direct interaction with the ribosome is necessary for DDX3X activity. I will present the results of experiments aimed to identify the region of DDX3 that interacts with the ribosome and the functional consequences of disrupting this interaction. Taken together, the results of these experiments enhance the mechanistic understanding of the role of DDX3 in protein synthesis.

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Translation Mechanism 614 Selective 40S footprinting reveals cap-tethered ribosome scanning in human cells

Jonathan Bohlen DKFZ, Heidelberg, Germany

Abstract

Translation regulation occurs largely during the initiation phase. Here we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act, and where they leave. We discover that in most human cells scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ~12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo. Preprint: https://doi.org/10.1101/806364

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Translation Mechanism 696 TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

Shaobo Liu1, Hua Jin1,2, Michael Rosbash2 1Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing, China. 2Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts, USA

Abstract

4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

Presenting author email

[email protected]

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Translation Mechanism 755 Single-molecule dynamics of eIF4F-mediated mRNA recognition.

Burak Cetin, Seán O'Leary University of California, Riverside, Riverside, California, USA

Abstract

Recognition of the mRNA 5’ cap by the heterotrimeric eIF4F complex is a critical step in initiation. As a final point of control prior to peptide synthesis, regulating this dynamic initiation event is critical for maintaining cellular homeostasis. Despite efforts to characterize translation initiation in real time, we still lack a detailed understanding of how and why cap recognition varies between transcripts, and of how individual initiation factors contribute to these differences, due to an absence of methods that dissect the detailed molecular mechanisms on the initiation timescale. Initiation is a multi-step, multi-factor process, substeps of which are difficult to dissect in vivo due to its extreme complexity. We have developed single- molecule fluorescence assays to address this problem. Using single-molecule FRET, we reconstituted cap recognition by the eIF4F subunit eIF4E, and characterized it in real-time on full-length mRNA transcripts. With yeast as a model system, we applied this method to individual transcripts as well as mRNA populations, characterizing the extent to which eIF4E–cap recognition varies across populations in the transcriptome. Additionally, we parsed out the contributions of eIF4F subunits eIF4G1 and eIF4A to the dynamics and stability of the eIF4F-mRNA complex, establishing a kinetic scheme for mRNA cap recognition by eIF4F. Our results suggest that mRNAs are inherently recognized at different rates by eIF4E with contributions from each eIF4F subunit, the mRNA, and RNA helicase activities, that vary between transcripts. Specifically, our findings highlight how factor composition and variations in mRNA size, sequence, and structure could fine tune the dynamics of protein synthesis by controlling the rates of formation and stability of intermediates during early initiation

Presenting author email [email protected]

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Translation Mechanism 814 Particularities of dirithromycin interaction with the ribosome.

Alena Paleskava1,2, Evgeny Pichkur1,3, Andrey Tereshchenkov4, Ilya Osterman4,5, Yury Polikanov6, Alexander Myasnikov1,7, Andrey Konevega1,3 1Petersburg Nuclear Institute named by B.P. Konstantinov of NRC “Kurchatov Institute”, Gatchina, Russian Federation. 2Peter the Great St.Petersburg Polytechnic University, St. Petersburg, Russian Federation. 3NRC “Kurchatov Institute”, Moscow, Russian Federation. 4Lomonosov Moscow State University, Moscow, Russian Federation. 5Skolkovo Institute of Science and Technology, Moscow, Russian Federation. 6University of Illinois at Chicago, Chicago, IL, USA. 7St. Jude Children’s Research Hospital, Memphis, TN, USA

Abstract

High therapeutic efficacy and safety ensured that erythromycin, a macrolide antibiotic, and its semisynthetic analogs had joined aspirin and penicillin as the most widely used drugs in the history of medicine. Dirithromycin is one of the second-generation macrolides that differs from the prototype macrolide erythromycin by additional hydrophobic side chain that has significantly increased the delivery of this antibiotic to tissues due to its higher lipophilicity. Previously, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in uL4 of the Thermus thermophilus 70S ribosome [1]. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome [2] with unprecedentedly high resolution 2.24Å. Our study suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain. This work was supported by the Russian Science Foundation grant 17-14-01416 to A.L.K. (cryo-EM experiments). 1. Khabibullina, N.F., et al. (2019). Structure of dirithromycin bound to the bacterial ribosome suggests new ways for rational improvement of macrolides. Antimicrob. Agents Chemother. 63(6). pii:e02266-18. 2. Pichkur, E.B., et al. (2020) Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome. RNA. doi: 10.1261/.073817.119.

Presenting author email [email protected]

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Translation Mechanism 884 Labeling of Initiation Factors for exploring their role in IRES-mediated translation

Sangeetha Selvam1, Jyotsana Pandey2, Tianhan Huang2, Alexey Petrov2, Gabriele Fuchs1 1University at Albany, SUNY, Albany, NY, USA. 2Auburn University, Auburn, AL, USA

Abstract

Protein biosynthesis requires precise temporal and spatial interactions of ribosomes, mRNAs, tRNAs, and translation factors. Due to complexity, the molecular mechanism of initiation remains obscure. In recent years, single-molecule methods have been used to reveal the timing and role of interactions between translation components. Single-molecule methods provide the advantages of revealing reaction pathways, which are usually hidden by averaging in ensemble experiments. The main challenge in the application of single- molecule methods is the lack of fluorescently labeled and biotinylated reagents. In this work, we focus on tagging the eukaryotic translation initiation factors with a short peptide, the ybbR tag, derived from the genomic library of Bacillus subtilis. SFP synthase is used to transfer the dye-phosphopantetheinyl moiety from CoA substrate to ybbR tag resulting in covalently and site-specifically labeled protein. Being only 11 amino acids long, the ybbR tag is expected to have minimal interference with tagged protein activity and thus is a suitable candidate to label small translation factors. eIF2 plays a key regulator role in translation initiation. It functions as the carrier of initiator tRNA and is composed of three distinct subunits - alpha, beta, and gamma. To follow eIF2 using single-molecule FRET we introduced site-specifically labeled and biotinylated eIF2 subunits. We have established the human cell lines expressing eIF2 subunits carrying ybbR and avi tags. We have successfully labeled it with a fluorescent substrate of CoA and was biotinylated with recombinant BirA ligase. eIF2a was recombinantly expressed and site-specifically labeled with maleimide chemistry. The labeled antibody biotinylated subunits were used to reconstitute eIF2 trimer. Initial single-molecule studies demonstrated the FRET between eIF2g and eIF2a. The FRET signal is dynamic, indicating the conformational plasticity of eIF2. Currently, we are working on improving the robustness of FRET and single-molecule experiments. In the future, we will follow the conformation of eIF2 during translation initiation to reveal its role in the translation initiation process. This labeling approach may be later employed for other initiation factors to explore their transition dynamics during .

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Translation Mechanism 906 Molecular determinants of resistance to the termination inhibitor Apidaecin

Chetana Baliga, Teresa Szal, Tanja Florin, Nora Vazquez-Laslop, Alexander Mankin Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

Abstract

Many antibiotics act by targeting various steps of bacterial protein synthesis. Api137, a derivative of naturally produced Proline Rich antimicrobial peptide Apidaecin (Api), acts at the termination stage of translation. Following the release of the newly synthesized peptide, Api137 binds in the nascent peptide exit tunnel (NPET) where its C-terminal residues interact with the (RF1 or RF2) and arrest the ribosome at the . Binding of Api137 is mediated by its interactions with rRNA of the NPET. We have found that expression of wild type Api in the bacterial cell is toxic. In order to test whether endogenously-expressed Api inhibits cell growth through the same mechanism as exogenously added chemically synthesized Api137, we expressed it in the mutant cells we had previously selected for their resistance to Api137. Several of the previously selected Api137 resistance mutations, including 23S rRNA mutations A2059C, A2503C and A2503G; and a mutation R81C in the ribosomal protein L16, improved survival of Api-expressing cells. These results show that toxicity of the endogenously-expressed Api is mediated by its action upon the ribosome likely through a mechanism identical to that of the exogenously-added peptide. We used the single rrn-allele E. coli SQ110 strain to identify new Api-resistance mutations. Besides identifying new mutations in the release factors, we found that the rRNA mutation C2666U, located in the ribosomal GTPase-associated center also confers resistance to exogenously-added or endogenously-expressed Api. The C2666U mutation may confer resistance by increasing the rate of recycling of ribosome-associated RF1/RF2 prior to Api binding or by stimulating the dissociation of the ribosome-RF-Api complex by RF3 action. Our approach can aid in revealing interactions between nascent peptides, translation factors and the ribosome involved in modulation of translation termination.

Presenting author email [email protected]

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Translation Mechanism 918 Targeting the EIF4A dependent translation of KRAs signaling molecules as therapeutics in pancreatic adenocarcinoma

Kamini Singh1, Jianan Lin2, Nicolas Lecomte3, Steven D. Leach4, Zhengqing Ouyang5, Hans-Guido Wendel1 1Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA, New York, NY, USA. 2The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 and Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, Storrs, CT, USA. 3David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA, New York, NY, USA. 4Molecular Systems Biology and Surgery, Geisel School of Medicine, Dartmouth, Norris Cotton Cancer Center at Dartmouth-Hitchcock, Lebanon, NH 03756, USA, New York, NY, USA. 5Department of Genetics and Genome Sciences and Institute for System Genomics, University of Connecticut Health Center, Farmington, CT 06030, USA, Farmington, CT, USA

Abstract

Silvestrol analogues like CR-1-31B are highly effective inhibitors of the eIF4A/DDX2 RNA helicase. EIF4A is required for the translation of highly structured mRNAs such as those containing multiple G-quadruplex (GQ) elements.Computational structure analyses identify multiple GQ elements in the 5’UTRs of mRNAs encoding KRAS and several downstream signalling molecules. Accordingly, transcriptome-scale ribosome profiling reveals that eIF4A inhibition blocks the translation of important KRAS signalling molecules including PI3K, RALA, RAC2, MYC, MET, and YAP1. Notably, CR-1-31B and the natural compound silvestrol kill PDAC cells and organoids at nanomolar concentrations and show in vivo efficacy in mouse, xenograft, and primary PDX models of human PDAC at safe dose levels. Together, we report the targetable translation of important PDAC oncogenes that implicates pharmacological eIF4A inhibition as a new and feasible strategy against KRAS- driven cancers.

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Translation Mechanism 964 Ribosome Profiling in Archaea Reveals Leaderless Translation, Novel Translational Initiation Sites, and Ribosome Pausing at Single Codon Resolution

Diego Gelsinger1, Emma Dallon1, Rahul Reddy1, Fuad Mohammad2, Allen Buskirk2, Jocelyne DiRuggiero1 1Johns Hopkins University, Baltimore, MD, USA. 2Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

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Translation Mechanism 31 Multifaceted regulation of cellular homeostasis by microRNA-mediated translational control, effected by the cap-binding protein 4EHP

Xu Zhang1, Clément Chapat1, Peng Wang1, Sung-Hoon Kim1, Tommy Alain2, Long Yang1, Nahum Sonenberg1, Seyed Mehdi Jafarnejad3 1McGill University, Montreal, Canada. 2University of Ottawa, Ottawa, Canada. 3Queen’s University Belfast, Belfast, United Kingdom

Abstract

MicroRNAs (miRNAs) exert a broad influence over gene expression by regulating the translation efficiency and stability of their target mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-induced gene silencing. However, little is known about the mRNA repertoire that is controlled by this mechanism or its biological importance. By using a combination of genomics, biochemistry, and molecular biology approaches and knockout mouse model we demonstrate that this mechanism has a critical role in regulation of multiple vital cellular processes. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP- mediate silencing induced by miRNAs. This promotes ERK1/2 , resulting in augmented cell growth and reduced apoptosis. Thus, we empirically define the integral role of this process in the control of the ERK signaling cascade in mammalian cells. In addition, we reveal that 4EHP plays a key role in controlling the innate immune response to viral infection through suppression of IFN-β production by effecting the miRNA- induced translational silencing of Ifnb1 mRNA. Our findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP which impedes sustained IFN production. Our study highlights an intrinsic regulatory function for the miRNA and translation machinery in maintaining cellular homeostasis.

Presenting author email [email protected]

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Translation Regulation 44 Role of the ORB2 RNA-binding protein in

Timothy Low1, Sichun Lin2, Stephane Angers2, Howard Lipshitz1 1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 2Departments of Pharmaceutical Sciences and Biochemistry, University of Toronto, Toronto, Ontario, Canada

Abstract

RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory framework and can determine the translational fate of their RNA targets. In Drosophila, ORB2 is an RBP known to repress the translation of target transcripts, but relatively little is known about what it targets and the mechanisms by which it confers this repression. We have initiated a global study of the RNA targets and protein-binding partners of ORB2 in the context of the early Drosophila embryo. RNA co-immunoprecipitation followed by next generation sequencing identified >350 ORB2-associated transcripts. These RNAs have, on average, a lower translation index than co-expressed unbound transcripts. We have used a luciferase reporter assay in S2 cell culture to confirm that recruitment of ORB2 to a target mRNA represses its translation and have shown that a structured domain within the protein is both necessary and sufficient for repression. Protein co- immunoprecipitation followed by mass spectrometry has identified protein binding partners of ORB2. Several known translational are among these protein partners, of which several are known components of the ME31B-TRAL repressive complex.

Presenting author email [email protected]

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Translation Regulation 48 Human OAS1 activation by dsRNA is dependent on the context of RNA sequence and other activating RNA motifs

Samantha Schwartz, Esther Park, Virginia Vachon, Shamika Danzy, Anice Lowen, Graeme Conn Emory University, Atlanta, Georgia, USA

Abstract

2’-5’-oligoadenylate synthetase (OAS) enzymes are key players in innate immunity, acting as sensors of cytosolic double-stranded RNA (dsRNA) to limit viral infection. dsRNA binding allosterically induces structural changes in OAS1 that reorganize its catalytic center to drive synthesis of 2’-5’-oligoadenylates (2-5A) from ATP. This 2-5A second messenger activates a single known target, the latent ribonuclease (RNase L), which then degrades viral and cellular RNA to halt viral replication. Attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous strategies to evade OAS activation. Although several studies have provided insight into the molecular mechanisms of OAS activation, many details of its regulation by viral or cellular RNAs are not fully understood. Recent studies have shown, for example, that specific RNA sequences and structural motifs can strongly enhance activation of at least one form of OAS (OAS1) through a mechanism that remains undefined. We designed a series of dsRNAs to test the impact of specific sequence changes within a strongly activating 18 bp dsRNA. Remarkably, while a 3’-end single-stranded pyrimidine (3’- ssPy) motif on one strand caused increased OAS1 activation, the equivalent 3’-ssPy motif on the opposite strand led to no measurable changes either in vitro or human A549 cells. Our findings also showed that the OAS1 activation consensus sequence (WWN9WG) and its context within dsRNA are important for potentiating OAS1 activity: altering the distance between the 3’-ssPy motif and the consensus sequence impacts its ability to enhance activation. Together, a picture emerges in which sequence, structural motifs, and the context in which they are presented all dictate the ability of a given dsRNA to activate OAS1. Defining the full complement of RNA molecular signatures that activate OAS is essential to our understanding of how these proteins maximize their protective role against pathogens while avoiding inadvertent activation by cellular RNAs. A more complete knowledge of OAS regulation may serve as a foundation for developing novel antiviral therapeutic strategies and lead to a deeper understanding of currently unappreciated functions of the OAS/RNase L pathway in the absence of infection.

Presenting author email [email protected]

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Translation Regulation 67 Tissue-specific separates the catalytic and cell signaling functions of an essential translation protein

Maxwell Baymiller, Benjamin Nordick, Susan Martinis University of Illinois Urbana-Champaign, Urbana, Illinois, USA

Abstract

The aminoacyl-tRNA synthetases are an ancient and ubiquitous component of all life. Many eukaryotic synthetases balance their essential function, preparing aminoacyl-tRNA for use in translation, with diverse roles in cell signaling. Herein we describe a leukocyte-specific exon skipping event in human leucyl-tRNA synthetase (LRS). This splice variant, which we term LSV3, is highly expressed, and lacks any tRNA leucylation activity in part due to a 71 amino acid deletion in the canonical LRS catalytic domain. Surprisingly, LSV3 retains its role as a leucine sensor and signal transducer for the mTOR pathway despite this deletion. Expression of LSV3 is regulated by serine-arginine rich splicing factor 1 (SRSF1), and increases in SRSF1 during differentiation of some leukocyte cell lines results in decreased LSV3 exon skipping. In this case, alternative splicing has separated the ancient catalytic activity of a housekeeping enzyme from its evolutionarily recent cell signaling role, providing functional specificity in human immune cells.

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Translation Regulation 82 Bicaudal-C translational recognizes RNA substrates via a bipartite binding site

Megan Dowdle, Michael Sheets University of Wisconsin, Madison, Wisconsin, USA

Abstract

Bicaudal-C (Bicc1) is a conserved RNA binding protein and developmental regulator that functions in metazoans to selectively repress the translation of specific mRNAs. This repression is essential for controlling the synthesis of proteins that in turn regulate key developmental decisions. Despite its biological relevance, how Bicc1 selectively recognizes and binds specific mRNA targets for repression is not understood. Studies in Xenopus indicate that the N-terminal region of Bicc1, which contains three canonical KH-domains and two KH-like (KHL) domains, is both necessary and sufficient for mRNA binding. In depth in vitro and in vivo analysis revealed that the second KH domain (KH2) is critical for Bicc1’s mRNA binding activity, while the KH1 and KH3 domains make minimal contributions to binding. With regards to the RNA features that define Bicc1 binding sites, the most extensive information has come from studies of the Xenopus Cripto1 mRNA and the 32- nucleotide Bicc1 binding site that resides within its 3’ UTR. This 32nt-binding site consists of a stem-loop structure preceded by an unstructured region referred to as the leader. Previous studies demonstrated that the loop and the leader are both required for efficient Bicc1 binding, suggesting that these elements comprise a bipartite binding site. To analyze the functions of the leader and loop elements we systematically changed all the nucleotides in both regions and monitored Bicc1 binding via electrophoretic mobility shift assays (EMSAs) and fluorescent polarization. We observe that the important sequence features of the leader and loop elements are distinct, while both elements need to be present to form a high efficiency Bicc1 binding site. The requirement of both elements for efficient binding could reflect that the individual elements together form a higher order RNA structure required for Bicc1 recognition or alternatively that the two elements simply provide multiple points of contact for the Bicc1 protein. Our results provide new insights for understanding how multi- domain RNA binding proteins recognize specific target sequences within large complex mRNAs as well for understanding the specific mechanisms by which Bicc1 influences embryonic development through its RNA binding functions.

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Translation Regulation 138 The key parameters that govern translation efficiency

Khanh Dao Duc University of British Columbia, Vancouver, British Columbia, Canada

Abstract

Translation of mRNA into protein is a fundamental yet complex biological process with multiple factors that can potentially affect its efficiency. Here, we study a stochastic model describing the traffic flow of ribosomes along the mRNA and identify the key parameters that govern the overall rate of protein synthesis, sensitivity to initiation rate changes, and efficiency of ribosome usage. By analyzing a continuum limit of the model, we obtain closed-form expressions for stationary currents and ribosomal densities, which agree well with Monte Carlo simulations. Furthermore, we completely characterize the phase transitions in the system, and by applying our theoretical results, we formulate design principles that detail how to tune the key parameters we identified to optimize translation efficiency. Using ribosome profiling data from S. cerevisiae, we show that its translation system is generally consistent with these principles. Our theoretical results have implications for evolutionary biology, as well as for synthetic biology.

Reference: Erdmann-Pham, Dao Duc and Song (2020), Cell Systems

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Translation Regulation 141 Catch me if you can – how the ribosome melts mRNA secondary structures for translation initiation

Vanessa de Jesus1, Nusrat S. Qureshi1, Sven Warhaut1, Marina Dietz2, Jasleen K. Bains2, Julian Langer3, Mike Heilemann2, Harald Schwalbe1, Boris Fürtig1 1Institute of Organic Chemistry and Chemical Biology - Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, Frankfurt, Germany. 2Institute of Physical and Theoretical Chemistry, Goethe- University Frankfurt, Frankfurt, Germany. 3Max Planck Institute of Biophysics, Frankfurt, Germany

Abstract

Translation initiation is the rate limiting step in protein synthesis. To initiate translation the ribosome binding site (RBS) of the mRNA has to be single stranded. But what happens, when the RBS is not accessible and the ribosome cannot bind to the translation initiation region? The “standby model” describes the paradox of high translation rates from highly structured mRNAs around the RBS, which should be theoretically inhibitory. According to this model, the ribosome binds unspecifically to single-stranded regions of the mRNA flanking the structured RBS. As soon as the RBS opens, the ribosome slides into position and specifically binds the RBS, locked by the SD-antiSD (Shine-Dalgarno) interactions. In this model, translation initiation is not dependent on the recruitment of free ribosomes from the cytoplasm, rather on the unfolding kinetics of the structured mRNA around the RBS. Here, we investigate a highly structured 5’-untranslated region (UTR) containing a riboswitch to study the structural dynamics of an mRNA in the context of the standby model. Previous studies have focused on the understanding and characterization of the riboswitch alone. In this adenine-sensing riboswitch from Vibrio vulnificus, the apo conformation sequesters the RBS in stable base pair interactions. Adenine binding to the aptamer domain releases the expression platform, providing a single-stranded RBS. We demonstrate that adenine binding is not sufficient to accommodate the mRNA within the ribosomal mRNA-tunnel, as a hairpin flanking the RBS blocks full mRNA accommodation due to steric clashes. By employing an integrated NMR, MST and FCS approach, we find that the chaperone function of the ribosomal S1 protein bound to 30S is essential for a complete melting of the RBS and rS1’s presence enables the formation of an initiation complex. In fact, the investigated riboswitch does not function as a stand-alone RNA translation-regulatory system but relies on components of the translation machinery. Our findings suggest re-evaluating both the translational-regulation mechanism of riboswitches stemming from gram-negative bacteria, as their function appears to be intimately linked to the ribosomal S1 protein as well as the standby model, as parts of the ribosome actively interfere with the unfolding of structured 5'-UTRs.

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Translation Regulation 148 CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD transcripts

Laure SCHAEFFER1,2, Xin JIANG3, Aurélie DURAND1,2, Clotilde LAGIER-TOURENNE3, Franck MARTIN1,2 1Université de Strasbourg, STRASBOURG, France. 2Institut de Biologie Moléculaire et Cellulaire - CNRS, STRASBOURG, France. 3MassGeneral Institute for Neurodegenerative Disease - Massachusetts General Hospital and Harvard Medical School, CHARLESTOWN, MA, USA

Abstract

Expansion of repeats in the C9ORF72gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Bidirectional of the C9ORF72locus results in the production of either G4C2or C4G2repeats that undergo so-called Repeat-Associated Non-AUG (RAN) translation. Translation of G4C2repeats produces toxic dipeptide repeat (DPR) proteins from three reading frames, namely poly Gly-Ala (GA), poly Gly-Pro (GP) and poly Gly-Arg (GR). Using cell-free translation extracts and in vivoRAN translation in cells (HEK, neuronal progenitors and motor neuron-like cells NSC-34), we determined cisand trans-factors influencing RAN translation of the human C9ORF72expansion transcripts. G4C2RAN translation operates by a canonical cap-dependent mechanism that involves 5’-3’ scanning mechanism, and requires a Met CUG codon located 24 nucleotides upstream of the repeats and an initiator Met-tRNA i. Production of poly- GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG indicating that DPR production requires frameshifting events. RAN translation is also regulated by an upstream open (uORF) present in mis-spliced C9ORF72transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5’-flanking G4C2sequence block ribosomal scanning and prevent RAN translation in all frames. Finally, we identify an unexpected affinity of expanded transcripts for the ribosomal subunits that are sequestered independently from their translation. Reference: Tabet et al., 2018, Nature Communications 9, 152.

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[email protected]

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Translation Regulation 183 3’UTR-mediated Post-transcriptional regulation of the RNA-binding protein MEX-3 in Caenorhabditis elegans germline

Mennatallah Albarqi, Sean Ryder University of Massachusetts Medical School, Worcester, MA, USA

Abstract

In early embryonic development prior to the onset of zygotic transcription, post-transcriptional regulation of mRNA contributes to cell fate specification and patterning. In Caenorhabditis elegans, there are several germline RNA-binding proteins (RBPs) that coordinate oogenesis, spermatogenesis, and embryogenesis through post-transcriptional mechanisms. MEX-3 is a conserved KH-domain RBP essential for early embryogenesis. Loss of function mex-3 mutants are maternal-effect embryonic lethal with anterior cell fate specification defects. Previous studies in our lab demonstrate that the 3’UTR of mex-3 is sufficient to regulate its expression pattern. To test whether the 3’UTR of mex-3 is necessary to pattern MEX-3 expression and activity, we used CRISPR/Cas9 to make an allelic series of mex-3 3’UTR deletions in a strain where the endogenous MEX-3 is tagged with GFP (GFP::MEX-3). Among the 3’UTR deletion mutants, two mutants showed overexpression of MEX-3 and one of these showed strong germline defects leading to sterility. Our observations reveal that the expression pattern of MEX-3 is spatially regulated through multiple elements present throughout the 3’UTR. To determine which RBPs regulate localization of MEX-3, we used RNAi to knock down several candidate RBPs in the GFP::MEX-3 strain. Reduction of GLD-1, OMA-1/2, LIN-41, and DAZ-1 significantly changes the expression pattern of MEX-3. To determine whether effects are mediated by the 3’UTR, we repeated the experiments in a mex-3 3’UTR transgenic reporter strain and observed similar effects to those observed in the endogenously tagged strain (GFP::MEX-3). Previous studies showed that the poly(A) tail length of mex-3 mRNA is reduced in null mutants of gld-2, a cytoplasmic poly(A) polymerase. When we knocked down components of the cytoplasmic adenylation and de-adenylation complexes, we observed distinct changes in the MEX-3 expression pattern, suggesting that each mechanism acts with different priority in different regions of the germline. Our results demonstrate that the mex-3 3’UTR is critical to reproductive health and begin to reveal the complex network of pathways that coordinate its precise temporal and spatial pattern of expression.

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Topic category

Translation Regulation 187 Transcription Start Site selection within a single cluster: consequences on translation

Arif Anwer Surani1, Keith Spriggs2, Christoph Ufer3, Cristina Montiel-Duarte1 1Nottingham Trent University, Nottingham, United Kingdom. 2University of Nottingham, Nottingham, United Kingdom. 3Charité Universitätsmedizin, Berlin, Germany

Abstract

Transcription Start Site (TSS) selection greatly influences the translational ability of a transcript. It determines the 5’ untranslated region (UTR) boundary and the inclusion of the regulatory elements that affect gene translation. Although some studies have confirmed the effect of differential TSSs usage on translational efficiency, the evidence is restricted to TSSs in different clusters many nucleotides apart. The relevance of TSS selection within a single cluster, however, is currently unknown. Here, we present our findings on AGAP2 as an example of a single cluster TSS-mediated gene regulation mechanism. Using 5’ RLM-RACE, we have identified different TSSs usage for AGAP2 mRNA in chronic myeloid leukaemia (CML) and prostate cancer (PC) cell-lines, giving rise to populations of transcripts with variable lengths of 5’UTR. The population of longer 5’UTR were significantly higher in CML cell-lines (p<0.05) and those extra nucleotides contained the consensus sequence for a G-quadruplex (G4). The G4 formation was verified by CD spectroscopy. Additionally, we have developed an immunoprecipitation method termed ‘GRIP’ [G4-RNA- Immunoprecipitation] and demonstrated in vivo existence of these RNA secondary structures. To study the impact on translation efficiency, we cloned 5’UTR isoforms into a bicistronic plasmid and reported a significant decrease in luciferase activity due to the G4 in the longer 5’UTR (p<0.001). This result coincides with the protein levels found in these cell-lines, where AGAP2 protein levels were higher when the 5’UTR is shorter (PC cell-lines). Furthermore, polysome fractionation studies also confirmed that mRNA with longer 5’UTR associated less prominently with polyribosomes (p<0.001). Using in-house R and phyton scripts, we identified around 5000 transcripts in the FANTOM database that contained G4 consensus sequences between the major TSS and another TSS up to 100 nucleotides apart. This suggests that the regulatory mechanism we found for AGAP2 might also be relevant for regulating the expression of other genes. We have also employed RNA sequencing and SEASTAR bioinformatics pipeline to acquire CAGE-quality TSSs for validation of other gene targets. Overall, our study emphasises the relevance of single cluster TSS-G4-mediate regulation of gene regulation and provides direct evidence for RNA G4 in living cells.

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Translation Regulation 273 RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1

Yifan E. Wang1, Qingtang Shen2, Mathew Truong1, Kohila Mahadevan1, Jing Ze Wu1, Harrison W. Smith1, Craig A. Smibert1, Alexander F. Palazzo1 1Department of Biochemistry, University of Toronto, Toronto, ON, Canada. 2Laboratory of Scientific Research, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China

Abstract

Throughout their lifecycle, messenger RNAs are packaged into messenger ribonucleoprotein (mRNP) complexes and undergo a number of maturation events, where the associated protein factors are exchanged or post-translationally modified. Since all mRNPs need to cross the nuclear pore prior to translation, this provides an opportunity for nucleoporin proteins, especially those on the cytoplasmic face of the pore, to promote mRNP maturation. Ran-binding protein 2 (RanBP2), also known as Nucleoporin 358 (Nup358), is a component of the cytoplasmic filaments of the nuclear pore complex. It is a SUMO E3-ligase that post-translationally adds small ubiquitin-like modifiers (SUMO) onto its substrates. Four mutations in RanBP2 are associated with a disease called acute necrotizing encephalopathy 1 (ANE1), where patients experience excessive cytokine secretion upon influenza infection, which may cause long-term neurological damage and a high mortality rate. However, how RanBP2 contributes to ANE1 remains unclear. Here, we found that RanBP2-dependent sumoylation represses the translation of Interleukin 6 (IL6), one of the most up-regulated cytokines in ANE1 patients. We showed that Argonaute 1 (AGO1), a core component of the RNA-induced silencing complex, associates with IL6 mRNA in the nucleus. As this mRNP complex enters the cytoplasm, AGO1 becomes a substrate for RanBP2 and this sumoylation stabilizes AGO1 and enforces its mRNA silencing activity. These results provide an example of how a nuclear pore filament, RanBP2, contributes to mRNP maturation events and how such processes can affect the translational output of transcripts.

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Translation Regulation 283 Inverted translational control of eukaryotic gene expression by ribosome collisions

Heungwon Park, Arvind Rasi Subramaniam Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Abstract

The canonical model of eukaryotic translation posits that efficient translation initiation increases protein expression and mRNA stability. Contrary to this model, we find that increasing initiation rate can decrease both protein expression and stability of certain mRNAs in the budding yeast Saccharomyces cerevisiae. These mRNAs encode a stretch of polybasic residues that cause ribosome stalling. Our computational modeling predicts that the observed decrease in gene expression at high initiation rates occurs when ribosome collisions at stalls stimulate abortive termination of the leading ribosome or cause endonucleolytic mRNA cleavage. Consistent with this prediction, the collision-associated quality-control factors Asc1 and Hel2 (orthologs of human RACK1 and ZNF598, respectively) decrease gene expression from stall-containing mRNAs only at high initiation rates. Remarkably, hundreds of S. cerevisiae mRNAs that contain ribosome stall sequences also exhibit lower translation efficiency. We propose that inefficient translation initiation allows these stall- containing endogenous mRNAs to escape collision-stimulated reduction in gene expression.

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Translation Regulation 287 mTORC1 enhances ribosome processivity

Erin An, Kiera Borthwick, Sherry Gu, Kyle Friend Washington and Lee University, Lexington, VA, USA

Abstract

During translation elongation, the ribosome serially adds amino acids to a growing polypeptide over many rounds of catalysis. The ribosome remains bound to mRNAs over these multiple catalytic cycles, requiring high processivity. Despite its importance to translation, relatively little is known about how mRNA sequences or signaling pathways might enhance or reduce ribosome processivity. Here, we describe a metric for ribosome processivity, the ribosome processivity index (RPI), which is readily calculated from ribosome profiling data. We show that ribosome processivity is not strongly influenced by open-reading frame (ORF) length or codon optimality. However, we do observe that ribosome processivity exists in two phases and that the early phase of ribosome processivity is enhanced by mTORC1, a key translational regulator. Importantly, we also observe protein accumulation when ribosomes prematurely abort translation. By showing that ribosome processivity is regulated and that abortive translation can give rise to stable polypeptides, our findings suggest an additional layer of control that the cell can exert to govern gene expression.

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Translation Regulation 324 Pyruvate Kinase Regulates mRNA Translation

Nevraj Kejiou1, Lena Ilan1, Stefan Aigner2, Enching Luo2, Ines Rabano2, Nishani Rajakulendran1, Hamed Najafabadi3, Stephane Angers1, Gene Yeo2, Alexander Palazzo1 1University of Toronto, Toronto, ON, Canada. 2University of California San Diego, San Diego, CA, USA. 3McGill University, Montreal, QC, Canada

Abstract

Many metabolic enzymes associate with translating mRNAs - however, the biological relevance of their interactions are unknown. We predict that these interactions represent a putative link between metabolic pathways and translation regulation. Here, we report that the glycolytic enzyme, Pyruvate Kinase M (PKM), binds specific polysomes and inhibits translation upon elevated levels of glucose. Furthermore, we find that adenosine 5'-diphosphate (ADP) inhibits PKM binding to polyribosomes. We also find that PKM binds polysomes translating poly-glutamic acid or poly-lysine stretches. Additionally, PKM-regulated mRNAs tend to encode cell cycle regulators - suggesting a link between cell division, glycolysis and protein synthesis. This work unearths an intimate relationship between glycolysis and mRNA translation that likely coordinates the flux of metabolites with the regulation of protein synthesis.

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Translation Regulation 326 Mechanisms of stress-induced translational reprogramming in budding yeast

Stefan Bresson, Vadim Shchepachev, Tomasz Turowski, Christos Spanos, David Tollervey University of Edinburgh, Edinburgh, United Kingdom

Abstract

Cells respond to environmental stress by reprogramming all aspects of gene expression. This process is mediated by RNA-binding proteins (RBPs), which collectively regulate RNA transcription, translation, and turnover. Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock using TRAPP (total RNA-associated protein purification). In the minutes following each stress, we observed robust changes in RNA binding for dozens of RBPs, while protein abundance was largely unaltered. Consistent with a general shutdown of translation in response to stress, ribosomal proteins which normally contact the translating mRNA showed decreased binding to RNA. Notably, mapping of the precise amino acids at sites of protein-RNA contact, using iTRAPP, indicated the pathway of the mRNA across the surface of the ribosome.

Throughout eukaryotes, phosphorylation of the highly conserved eIF2α leads to translation shutdown in response to various stresses. However, both glucose starvation and heat shock trigger translational repression via a different, but still unknown, pathway. For both stresses, we find that a specific subset of translation initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1) rapidly lose RNA binding. Crosslinking and sequencing analysis (CRAC) revealed that all three factors primarily target the 5′ ends of mRNAs in unstressed cells, consistent with their role in translation initiation. Following glucose withdrawal, mRNAs remain stable but eIF4A, eIF4B and Ded1 binding to the 5′ ends of most transcripts is abolished within 30 seconds, explaining the rapid translation shutdown. Only small numbers of stress-induced mRNAs retain binding. This abrupt loss of translation factor binding requires the glycolytic enzyme and intracellular glucose sensor Hxk2. Under heat shock conditions, loss of RNA binding by these translation factors happens gradually over 16 minutes. During this period, we see a global decrease in mRNA levels, particularly strongly (~90%) for translation-related factors. These mRNAs are stabilized by co-treatment with cycloheximide, suggesting that translational arrest and subsequent ribosome runoff precede mRNA decay. We further show that the 5′ to 3′ exonuclease Xrn1 is required for degradation. Taken together, our results shed light on the mechanisms underlying translational control of gene expression during stress.

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Topic category

Translation Regulation 334 Unraveling the influence of sequence features and position on uORF activity using massively parallel reporter systems and machine learning

Gemma May, Christina Akirtava, Matthew Agar-Johnson, Jelena Micic, John Woolford, Joel McManus Carnegie Mellon University, Pittsburgh, PA, USA

Abstract

Upstream open reading frames (uORFs) are potent cis-acting regulators of translation and mRNA turnover. Ribosome profiling studies suggest that uORFs are quite common, with thousands of uORFs initiating at non- AUG start codons. However, few predicted non-AUG uORF have been tested for regulatory functions. Furthermore, the relative influences of sequence and structural features on uORF functions are currently unknown. To address this, we developed a massively parallel reporter assay (FACS-uORF) to test thousands of AUG- and non-AUG uORFs from three Saccharomyces yeast species. While nearly all AUG uORFs were repressors, only 30% of non-AUG uORFs were functional, with roughly equal numbers of modest enhancers and repressors. These results were validated in a complimentary reporter system that assayed the effects of uORFs on polysome association. Testing our reporter library in a Dupf1 yeast strain (lacking NMD) revealed NMD accounts for roughly one third of uORF repressive effects, and showed that stop codon sequence, uORF position, and uORF length influence the strength of NMD induction. In wildtype yeast, strong Kozak contexts were associated with the most repressive uORFs but were not required for robust repression. Furthermore, repression was stronger from uORFs located proximal to the 5’-cap, and alternative transcription initiation sites led to corresponding changes in uORF activity. Using machine learning, we developed a model that explains 39% of the variance in uORF function using Kozak sequence, structural accessibility, uORF position, length, codon usage, and peptide charge. Kozak context and uORF position have similar predictive power. Together, our results define the scope of uORF functions in gene regulation, identify features associated with uORF repression and NMD, and suggest that uORF functions are impacted by the frequency and speed of their translation.

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Topic category

Translation Regulation 355 Control of PABP1 multifunctionality by lysine acetylation or methylation: are cytoplasmic RNA-binding proteins regulated by ‘histone code-like’ switches?

Etienne Dubiez1,2, Taj Blee1, Atlanta Cook3, Satpal Virdee4, Niki Gray1, Matthew Brook5 1University of Edinburgh, Edinburgh, United Kingdom. 2MRC CRH, Edinburgh, United Kingdom. 3Wellcome Trust Centre for Cell Biology, Edinburgh, United Kingdom. 4MRC Protein Phosphorylation and Ubiquitylation Unit, Dundee, United Kingdom. 5Edinburgh Centre for Cardiovascular Science, Edinburgh, United Kingdom

Abstract

Poly(A)-binding proteins are multifunctional central regulators of gene expression. The best characterised family member, PABP1, acts as a primary determinant of translation efficiency and stability, regulates specific mRNA fate, and participates in miRNA-mediated regulation and nonsense-mediated mRNA decay (NMD). These disparate, and sometimes antagonistic, functions require PABP1 to interact with numerous, functionally diverse, protein partners. However, many PABP1 partners bind overlapping sites, e.g. up to 16 different PABP- interacting motif 2 (PAM2)- containing proteins compete to bind with similar affinity to the same C-terminal PABC domain surface. Thus, it is unclear how these interactions, and therefore different PABP1-mediated functions, are co- ordinated. Crucially, we identified that PABP1 is subject to extensive, dynamically-regulated, post-translational modification, including mutually-exclusive acetylation or methylation of lysine residues, and molecular modelling indicated that modification of Lysine-606 may act as a switch to differentially affect PABC- PAM2 interactions and confer regulated function, in a manner reminiscent of histone regulation. Using an orthogonal synthetic biology approach [BM1] with evolved tRNA-synthetase/tRNACUA pairs, in combination with chemoselective reactions, we have optimised methods for the quantitative, site-specific installation of acetyl-lysine or dimethyl-lysine in recombinant PABP1 PABC domain, allowing comparative kinetic and X-ray crystallographic structural studies. Here we show first pivotal insights into the molecular and structural consequences of PABP1 modification on PABP1-PAM2 interactions that will inform on PABP1 multifunctionality in post-transcriptional control of gene expression. Given the mass spectrometric documentation of similarly acetylated/methylated lysine residues in a rapidly growing number of multifunctional RNA-binding proteins (RBPs), we previously proposed the existence of an ‘RBP-code’, akin to the histone-code, and here use PABP1 as a prototype RBP to support this new paradigm.

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Topic category

Translation Regulation 360 Mitochondrial volume fraction and translation speed impact mRNA localization and production of nuclear-encoded mitochondrial proteins

Tatsuhisa Tsuboi1, Matheus Viana2,3, Fan Xu1, Ximena Arceo1, Wanfu Hou1, Susanne Rafelski2,3, Brian Zid1 1UC San Diego, La Jolla, CA, USA. 2UC Irvine, Irvine, CA, USA. 3Allen Institute for Cell Science, Seattle, WA, USA

Abstract

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial outer membrane, the importance of this localization in altering mitochondrial protein composition is unclear. We have found that, as yeast switch from fermentative to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. This drives the localization of certain nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through tethering experiments, we show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to the mitochondria. We have also found that global changes in translation elongation are sufficient to modulate mRNA localization and mitochondrial composition. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle- specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome.

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Topic category

Translation Regulation 424 Translational switch during integrated stress response: the examples of p53 and UPF1

Rafaela Lacerda1,2, Bruna Pereira1,2, Juliane Menezes1,2, Ana Ramos1,2, Ana Rita Neves1,2, Marco M. Candeias1,3, Luísa Romão1,2 1Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal. 2University of Lisboa, Faculty of Sciences, BioISI – Biosystems and Integrative Science Institute, Lisboa, Portugal. 3Molecular and RNA Cancer Unit, Department of Anatomy and Developmental Biology, Graduate School of Medicine Kyoto University, Kyoto, Japan

Abstract

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds to the cap structure at the 5’ end of the mRNA and scans the 5’ untranslated region (5’UTR) until an initiation codon is found. However, when cells are exposed to stress stimuli, cap- dependent translation is inhibited, while the synthesis of some proteins is maintained by alternative mechanisms of translation initiation, which are vital for cell survival and stress recovery. Here we show two examples in which a translational switch occurs during integrated stress response (ISR). In the first case, tumor suppressor p53, we show that the ISR leads to the specific induction of a shorter p53 isoform (Δ160p53 isoform). This induction is dependent on translation elongation but does not require the eIF4E-eIF4G interaction. Studies using bicistronic constructs with wild-type Δ160p53 or reporter genes confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA, being eIF2α phosphorylation a key event leading to cap-independent expression of Δ160p53 during ISR. Interestingly, cancer-specific mutations in p53 also enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Our data support a model in which an IRES structure in the coding region of p53, and the cancer-specific mutations that affect this structure, control p53 oncogenic functions by regulating Δ160p53 protein expression. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. Indeed, our results show that UPF1 5’UTR is able to mediate cap-independent translation in a bicistronic luciferase vector expressed in cervical and colorectal cancer cell lines. Such activity is maintained under endoplasmic reticulum stress. Interestingly, we found that the UPF1 5’UTR IRES function is inhibited when the first 100 nucleotides, or the last 125, are absent or altered. Understanding these IRESs mechanism of function and their biological relevance might provide tools for developing new therapies for human diseases such as cancer.

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Topic category

Translation Regulation 432 Structural basis of transcription-translation coupling and collision in bacteria.

Michael Webster, Maria Takacs, Chengjin Zhu, Vita Vidmar, Ayesha Eduljee, Mo'men Abdelkareem, Albert Weixlbaumer IGBMC, Strasbourg, France

Abstract

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit- channel. In this collided complex, NusG-linkage is no longer possible. These structures show at high resolution a molecular assembly line, which executes the entire process of gene expression. The structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.

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[email protected]

Topic category

Translation Regulation 443 Characterization of small RNAs as ribosomal regulators in bacteria

Sydnee H. Calhoun, Cristina Carvalho Barbosa, Hans-Joachim Wieden Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada

Abstract

Gene expression is a highly regulated process that occurs in all living cells. Recently it has been shown that several types of small, non-coding RNA (sRNA) exist that do not encode proteins but are involved in the regulation of gene expression [1,2]. One of the later steps in gene expression is translation, the ribosome- dependent process of decoding messenger RNA transcripts resulting in the synthesis of the corresponding protein. Translation is the target of over 50% of current antibiotics and is therefore a promising target for the development of novel anti-microbial strategies. A recent addition to the classes of sRNAs have been shown to regulate translation via direct interactions with the ribosome [3-7]. These sRNA-dependent mechanisms of ribosomal regulation have been identified in eukaryotes and archaea, but not bacteria. Since the ribosome is highly conserved among the three domains of life, it is likely that sRNAs exist that also regulate translation in bacteria by directly interacting with the ribosome. Here we report work studying the ability of these sRNAs to act as regulators in Escherichia coli. To identify potential sRNAs, published bacterial databases were analyzed and candidates were identified based on sequence alignment to 23S and 16S ribosomal RNA [8,9]. Candidate and ribosome interactions were then biochemically validated by determining the affinity of the candidates to 70S, 50S, and 30S ribosomes and ribosomal subunits. Secondly, in vitro and in vivo translation assays were used to assess the potential of these candidate sRNAs to regulate translation. References: 1. Pircher, A., et al. (2014). RNA Biol 11. 2. Carvalho Barbosa, C., Calhoun, S.H., et al. (2019). BCB 98. 3. Gebetsberger, J., et al. (2012). Archaea 2012. 4. Pircher, A., et al. (2014). Mol Cell 54. 5. Bakowska-Zywicka, K., et al. (2016). FEMS Yeast Res 16. 6. Gebetsberger, J., et al. (2017). RNA Biol 14. 7. Fricker, R., et al. (2019). Nat Commun 118. 8. Waters, S. A., et al. (2017). EMBO J 36. 9. Smirnov, A., et al. (2016). Proc Nat Acad Sci 113.

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Topic category

Translation Regulation 483 Systematic analysis of gene expressions during recovery from severe heat shock in Escherichia coli.

Muyang Huang1,2, Masahiro C. Miura1,2, Rerina Inose1, Asako Sato1, Keita Kamezaki1, Masaru Tomita1,2, Masaru Mori1,3, Kazuharu Arakawa1,2, Akio Kanai1,2 1Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan. 2Faculty of Environment and Information Studies, Keio University, Tokyo, Japan. 3Systems Biology Program, Graduate School of Media and Governance, Keio University, Tokyo, Japan

Abstract

Heat shock response is one of the well-characterized examples of stress adaptation in Escherichia coli. However, the detailed molecular mechanism of the recovery from heat shock is poorly understood. To clarify the factors involved in the recovery phase, we adopted systematic approaches such as proteome and transcriptome analyses, and characterized the mutant (single gene-knockout) strains for candidate factors. After preculturing at 30°C, E. coli cells were exposed to a severe heat shock (50°C, 90 minutes), which is lethal for about 30~40% of the cells. Bacterial pellets were collected at 7-time points at approximately 30 minutes interval both for heat shock and control samples. We then first conducted a shotgun proteomic analysis by nanoLC-MS/MS and obtained time-course data for 1,425 proteins. As a result, we confirmed the stress induced expression of known heat shock proteins (HSP), RpoH and DnaK. Other protein groups induced during the recovery phase include, for example, approximately 30 ribosomal proteins induced just after the heat shock, and tRNA/rRNA modification enzymes induced just before restarting the exponential growth. Principal component analysis of proteome data suggested that many of tRNA/rRNA modification enzymes and proteins involved in ribosome biogenesis were key molecules to recover from the heat shock. To verify their significance to heat recoveries, using series of single-gene knockout mutants in E. coli (Keio collection), we showed that rRNA modification enzymes such as RluA, RluF, and RlmH, and tRNA modification enzymes such as MnmA and QueA were necessary to recover from the severe heat shock condition but not for the normal growth. We also performed RNA-Seq analysis using Illumina NextSeq 500 for the samples of the same time-points, obtaining time-series expression profiles for 4,424 mRNAs. We found that changes in protein level and mRNA level did not always correlate with each other. Therefore, we are currently focusing on small RNAs (sRNAs) as representative translation suppressors and are conducting a comprehensive analysis of each sRNA that may involve in post-transcriptional regulation of specific genes. Based on these results, we would like to discuss the framework of the regulatory mechanisms for recovery from severe heat shock.

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Topic category

Translation Regulation 507 Computational Prediction of Programmed Ribosomal Frameshifting

Mary Richardson, Sean Eddy Harvard University, Cambridge, MA, USA

Abstract

Programmed ribosomal frameshifting (PRF) is an important translational control mechanism that can allow an mRNA to encode multiple functional protein products. Sequence and structural elements of some mRNAs cause ribosomes to slip predictably during translation, resulting in a change in reading frame that alters the downstream protein sequence. A handful of PRF instances have been found in species ranging from viruses to humans, but the prevalence of PRF remains unknown. The regulatory elements that lead to PRF are also not well understood in some cases, making it difficult to develop a predictive model for novel PRF events based on mRNA sequence alone. Ribosome profiling data is a promising alternative, since it can provide nucleotide- resolution information about ribosome position during translation. We developed a hidden Markov model to infer reading frame from high-resolution ribosome profiling data. Our model is capable of predicting known frameshifts in E. coli and may reveal novel PRF events throughout the transcriptome of E. coli and other model organisms when applied more broadly. These predictions should contribute to an improved understanding of the role of frameshifting in translation.

Presenting author email [email protected]

Topic category

Translation Regulation 548 Ribosomal protein RACK1 is enhances both viral and cellular IRES-mediated translation

Clare Miller, Ethan LaFontaine, Natasha Permaul, Elliot Martin, Dr. Gabriele Fuchs University at Albany, SUNY, Albany, New York, USA

Abstract

The ribosomal protein RACK1 has been shown to be required for translation initiation of the hepatitis C virus and the 5’ IRES of cricket paralysis virus (CrPV) but not the intergenic CrPV IRES. Here we tested if RACK1 is also required for other viral IRESs. Indeed, we found that both encephalomyocarditis virus (EMCV) and poliovirus (PV) also require RACK1 for translation of the viral IRESs. The observed decrease in IRES-mediated translation in RACK1 knockout cells was rescued by expression of exogenous RACK1. Further, in cells lacking RACK1, we observed that PV plaques are smaller compared to wildtype cells suggesting that loss of RACK1 slows down the virus life cycle. Since RACK1 appears to be a required ribosomal protein for translation initiation of viral IRESs, we examined if RACK1 is also required for translation initiation of cellular IRESs. Using dual luciferase reporter constructs, we observed that the cellular IRESs myb, Bag-1, c-myc, L-myc, RUNX1T1 and Set7 also require RACK1 for translation. Our data suggest that RACK1 and ribosomal protein eS25, previously shown to be required for IRES-mediated translation, functionally overlap. While eS25 appears to directly interact with the HCV IRES RNA by cryoEM, RACK1 might indirectly regulate IRES-mediated translation. Using RNA-sequencing on wildtype, RACK1 knockout cells and eS25 knockout cells, we are currently identifying cellular targets of RACK1 and eS25 to determine the level of overlapping targets. Knowing RACK1 targets and understanding the mechanistic involvement of RACK1 in IRES-mediated translation may aid in developing therapeutics for disorders that are based in cap-independent IRES-mediated translation.

Presenting author email [email protected]

Topic category

Translation Regulation 584 Cis-elements and trans-factors involved in efficient translation reinitiation after premature termination

Laura Baquero Galvis, Sujatha Jagannathan University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA

Abstract

Nonsense-mediated decay (NMD) is a translation-dependent mRNA surveillance system that targets transcripts containing premature termination codons (PTC). However, certain nonsense-containing mRNAs evade NMD by implementing translation reinitiation. During translation reinitiation, the terminating ribosome fails to be recycled properly at the premature termination site, allowing for further scanning of the transcript and subsequent reinitiation at a downstream AUG. Furthermore, the continued scanning of the transcript by the ribosome leads to the removal of RNA binding proteins (RBPs) that would otherwise trigger NMD. Although reinitiation events have been cataloged in a number of mammalian genes, the elements that influence the efficiency at which this process occurs are not fully understood. Here, we have developed a reporter system that faithfully reports on reinitiation to characterize specific mRNA cis-elements and trans-factors that may influence incidence of translation reinitiation. Additionally, we are developing selective ribosome profiling strategies to identify possible initiation factors that remain in contact with the ribosome post-premature termination, and which may have a role in reinitiation events. These data will contribute to a better understanding of the possible mechanism used by the ribosome to resume scanning downstream of a PTC, and how the cis-elements in an mRNA and trans-factors together influence reinitiation

Presenting author email [email protected]

Topic category

Translation Regulation 601 Translation initiation site profiling reveals widespread synthesis of non- AUG-initiated protein isoforms in yeast

Amy Eisenberg1, Andrea Higdon1, Ina Hollerer1, Alexander Fields2, Irwin Jungreis3, Paige Diamond1, Manolis Kellis3, Marko Jovanovic4, Gloria Brar1 1UC Berkeley, Berkeley, CA, USA. 2GRAIL, Inc., Menlo Park, CA, USA. 3MIT, Cambridge, MA, USA. 4Columbia University, New York, NY, USA

Abstract

Genomic analyses in budding yeast have helped to define the foundational principles of eukaryotic gene expression, but have systematically excluded specific classes of potential coding regions, including those with non-AUG start codons. Without methods to define coding regions empirically, the prevalence of these non- canonical coding regions has been impossible to assess. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast during meiosis and identified a class of genes that encode N-terminally extended alternate protein isoforms that result from translation initiation at non-AUG codons upstream of the annotated AUG start codon. These alternate isoforms are produced in concert with canonical isoforms and are translated with a high degree of specificity, resulting from initiation at only a small subset of possible start codons in 5’ leader regions. These findings reveal widespread production of non-canonical protein isoforms and, more generally, show unexpected complexity to the rules by which the budding yeast genome is decoded.

Presenting author email [email protected]

Topic category

Translation Regulation 612 Visualizing dynamic tethering of Argonaute to single mRNA in live human cells

Charlotte Cialek, Tatsuya Morisaki, Taiowa Montgomery, Timothy Stasevich Colorado State University, Fort Collins, CO, USA

Abstract

Argonaute proteins are essential for microRNA (miRNA)-mediated silencing of messenger RNA (mRNA). Though well-studied, this silencing mechanism remains ambiguous due to limitations in the spatiotemporal resolution of techniques such as ribosome profiling and more traditional bulk assays. To overcome these limitations, we developed technology to directly visualize how Argonaute impacts the live-cell translation dynamics of single mRNA. Our methodology uses three complementary fluorescent probes to simultaneously track single mRNA, their translational status, and Argonaute tethering in real time. At the single-cell level, our system in steady- state confirms Argonaute-tetherable mRNA are fewer in number, less likely to be translated, and produce less mature protein than non-tetherable mRNA. At the single mRNA level, our system reveals heterogeneity in the type and spatial distribution of mRNA. Early on, a small, dynamic fraction (46%) of tethered mRNA were still translationally active. By tracking thousands of single mRNA, we were able to capture rare Argonaute-tethering events, which led to translational silencing in just 20 minutes on average. This timescale is on par with the time it takes ribosomes to run-off transcripts upon treatment with the translation initiation inhibitor Harringtonine. The similarity of these timescales suggests Argonaute binding mainly blocks translation initiation rather than elongation. Though Argonaute-tetherable cells had less total reporter mRNA, whether Argonaute induced decay remains unclear. Translationally silent, Argonaute-tethered mRNA had a high propensity to accumulate in P bodies and persist for hours, in stark contrast to non-tetherable mRNA and control mRNA tethered to an inert protein, both of which did not accumulate in P bodies. Beyond Argonaute, researchers can now use our tethering technology to visualize in real time the impact of other proteins of interest on translating mRNA in live cells.

Presenting author email ccialek@colostateledu

Topic category

Translation Regulation 618 The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

Anne Ramat, Martine Simonelig IGH, Montpellier, France

Abstract

The regulation of maternal mRNAs is determinant for the first steps of life as they govern body axis establishment and cell divisions before the zygotic genome starts to be expressed. Drosophila embryo is a well-established model to study maternal mRNA regulation. Antero-posterior axis is specified through the localisation of key mRNAs, one of them being nanos (nos), a key determinant of the posterior structures. Piwi-interacting RNAs (piRNAs) and PIWI proteins, at first known for their role in the repression of transposons in the germline, have later been shown to post-transcriptionally regulate cellular mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) regulate maternal mRNA stability. Recently, we described a new function of Aub in translational regulation: Aub is required for translational activation of nos mRNA at the posterior germ plasm. Aub acts at the translation initiation step through its interaction with translation initiation factors including the poly(A) binding protein PABP and several subunits of the eIF3 complex. Furthermore, in the germplasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, suggesting that translation might take place at the edge of germ granules (Ramat et al., Cell Research 2020). Here, we characterize germ granule architecture using super-resolution microscopy. We find that Aub forms doughnut-like structures and co-localizes with Oskar and Tudor, two key components of germ granules assembly. eIF3d is present at the periphery of these Aub structures. Strikingly, Nos protein is also detected at the edge of Aub doughnut-like structures, strengthening our hypothesis that the periphery of germ granules is the site of Aub-mediated translation. We have set up an assay to tether Aub to an mRNA encoding GFP. We find that Aub binding significantly increases GFP levels, consistent with Aub function in translational activation. This tool will help us deciphering in more details the molecular mechanisms underlying this new function of Aub.

Presenting author email [email protected]

Topic category

Translation Regulation 649 Translation control of seven mRNA communities connected with Spinal Muscular Atrophy by SMN-primed ribosomes.

Fabio Lauria1, Paola Bernabò1, Toma Tebaldi2, Ewout Groen3,4, Elena Perenthaler1, Federica Maniscalco1, Annalisa Rossi5, Deborah Donzel1, Massimiliano Clamer6, Marta Marchioretto1, Neža Omersa7, Julia Orri1, Mauro Dalla Serra1, Gregor Anderluh7, Alessandro Quattrone5, Alberto Inga5, Tom Gillingwater3, Gabriella Viero1 1Institute of Biophysics, CNR Unit at Trento, Trento, Italy. 2Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA. 3Edinburgh Medical School: Biomedical Sciences & Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom. 4Department of Neurology and Neurosurgery, UMC Utrecht Brain Center, Utrecht, Netherlands. 5Department CIBIO, University of Trento, Trento, Italy. 6IMMAGINA Biotechnology s.r.l, Trento, Italy. 7National Institute of Chemistry, Ljubljana, Slovenia

Abstract

Translation is the most energy consuming process in cells and represents a core mechanism coordinating multiple post-transcriptional processes. Hence, it is not surprising that several mRNAs are largely controlled at the translational rather than transcriptional level. Indeed, dysregulation of translation has been implicated as a key driver pathogenesis across a wide range of conditions including cancer, autism and neurological disorders. However, the contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains enigmatic. Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder caused by genetic alterations of the Survival Motor Neuron (SMN1) gene and low levels of the SMN protein. We recently showed that SMN associates with polysomes in cell culture, as well as in mouse spinal cords and brains in vivo, where it directly influences translation. Moreover, genome-wide defects in mRNA recruitment on polysomes have previously been observed in SMA, but the mechanisms linking SMN to these defects have yet to be elucidated. Here, we investigated the hypothesis that SMN binding to ribosomes regulates ribosome recruitment on a specific set of mRNAs. By using multiple complementary approaches, we demonstrated that SMN protein binds to ribosomes and that SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs. These SMN-specific mRNAs are enriched in cap-independent/”IRES-like” sequences in the 5’UTR and in rare codons at the beginning of their coding sequence. We showed that SMN-specific mRNAs organized in seven functionally well-defined communities, associated with processes known to be defective in SMA, such as neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion from SMN-specific mRNAs, especially at the beginning of the coding sequence, leading to impairment of proteins involved in motor neuron function and stability, including acetylcholinesterase. Overall, these findings suggest a critical role of SMN in the regulation of ribosome fluxes along mRNAs which encode proteins relevant to SMA pathogenesis.

Presenting author email [email protected]

Topic category

Translation Regulation 683 Suppression of a MEHMO syndrome mutation in eIF2 by small molecule ISRIB

Sara Young-Baird1, Maíra Bertolessi2, Megan Elder3, Eric Klann3, Stefan Liebau2, Thomas Dever1 1National Institutes of Health, Bethesda, MD, USA. 2Eberhard Karls University Tübingen, Tübingen, Germany. 3New York University, New York, NY, USA

Abstract

Dysregulation of cellular protein synthesis is linked to a variety of diseases. Mutations in EIF2S3, encoding the gamma subunit of the heterotrimeric eukaryotic translation initiation factor eIF2 (eIF2gamma), cause MEHMO syndrome. MEHMO syndrome is an X-linked intellectual disability disorder in which patients exhibit mental (intellectual) deficiency, epilepsy, hypogenitalism, microcephaly, and obesity. Binding of GTP and initiator Met methionyl-tRNA (Met-tRNAi ) to the eIF2 heterotrimer forms the eIF2 ternary complex (TC) that delivers Met- Met tRNAi to the ribosome and participates in selection of the translation start site on a mRNA. Accordingly, MEHMO syndrome mutations in eIF2gamma are predicted to impact both global and gene-specific protein synthesis.

Using patient-derived induced pluripotent stem cells, we show that a mutation at the C-terminus of eIF2gamma impairs the activity of the eIF2 chaperone CDC123, reducing eIF2 complex formation and the level of eIF2 TC. This reduction in eIF2 abundance results in reduced global protein synthesis and enhanced gene- specific translation of mRNAs encoding ATF4 and CHOP, key regulators of the cellular stress response that are known to be translationally induced under limiting eIF2 TC conditions. Enhanced basal expression of these stress responsive factors primes the cell for an elevated response upon stress induction that culminates in cell death. Addition of the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor eIF2B, restores eIF2 TC levels and rescues cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation. Overall, our results demonstrate that MEHMO syndrome mutations in EIF2S3 impair eIF2 TC formation and result in dysregulated protein synthesis. Furthermore, bolstering eIF2 TC formation with ISRIB treatment rescues the translational and cellular effects of the eIF2gamma mutation, offering the possibility of therapeutic intervention for MEHMO syndrome.

Presenting author email [email protected]

Topic category

Translation Regulation 689 Understanding the dynamic interplay between SR45 and AtSAP18 in Arabidopsis thaliana

Anna Hu, Xiao-Ning Zhang St. Bonaventure University, St. Bonaventure, NY, USA

Abstract

RNA metabolism plays an important role in controlling mRNA abundance and diversity. In metazoans, the conserved apoptosis and splicing-associated protein (ASAP) complex is composed of RNPS1, apoptotic chromatin condensation inducer in the nucleus (Acinus), and Sin3-associated protein 18 (SAP18). It is involved in the regulation of transcription via histone deacetylation and pre-mRNA splicing. In Arabidopsis thaliana, an orthologous AtASAP complex exists. Previous studies suggested that SR45 (AtRNPS1) could function as a scaffold regulator for pre-mRNA splicing, and that SR45 may be required for maintaining a normal level of the AtSAP18 protein in the nucleus without changing the RNA expression and splicing pattern of the AtSAP18 transcript. In addition, all components of AtASAP have been found at the FLOWER LOCUS C (FLC) locus, to suppress gene expression. According to these prior findings, it is possible that the AtASAP complex participates in the regulation of multiple processes in RNA metabolism, as reported for their animal ortholog. We hypothesize that the reduced nuclear AtSAP18 protein levels in the sr45-1 null mutant could be due to translational repression, a lack of nuclear import or increased degradation in the nucleus. To test these hypotheses, two experiments were set up: (1) To address the differential accumulation of nuclear AtSAP18, sr45-1 null mutant seedlings overexpressing AtSAP18-GFP were treated with 50 μM of MG132. The total AtSAP18-GFP protein in seedlings was examined by western blot, and the subcellular localization of AtSAP18 was visualized via confocal imaging. 24 hours after MG132 treatment, a strong nuclear accumulation of AtSAP18 in root and guard cells was observed. A significant increase in the level of total AtSAP18 under MG132 treatment was also confirmed by western blot. These results suggest that SR45 may slow down AtSAP18 protein degradation in the nucleus in these seedlings. (2) To test whether the nuclear localization of AtASAP18 is required for the transcriptional silencing of FLC without SR45, AtSAP18-GFP was fused with the coding sequence of the -binding domain of rat glucocorticoid receptor and introduced into the sr45-1 mutant. The transgenic lines were examined for the subcellular localization of AtSAP18-GFP and the expression of FLC upon dexamethasone treatment.

Presenting author email [email protected]

Topic category

Translation Regulation 691 Mitochondrial Co-translational Protein Import and Network Structures Impact Mitochondrial Fragment Composition

Tatsuhisa Tsuboi1,2, Matheus Viana2,3, Susanne Rafelski2,3, Brian Zid1 1University of California San Diego, La Jolla, CA, USA. 2University of California Irvine, Irvine, CA, USA. 3Allen Institute for Cell Science, Seattle, WA, USA

Abstract

Mitochondria are hubs for metabolite and energy generation and have been shown to be very important for age-related processes, including cancer and neurodegeneration. Fragmented mitochondrial morphology is a hallmark of the dysfunction of mitochondrial activity and observed in those disease phenotypes. However, it remains unclear how eukaryotic cells coordinate protein production for those fragmented mitochondria. Mitochondrial proteins are mostly encoded in the nuclear genome and are imported from the cytoplasm to mitochondria. A fraction of mitochondrial protein-coding mRNA is localized to the mitochondrial outer membrane and co-translationally import proteins into the mitochondria, yet the impact of localized translation to these organelles on mitochondrial function is poorly understood. We made the novel observation that Tim50 protein, the translocase of the inner mitochondrial membrane and a co-translationally imported protein, is evenly distributed to mitochondria on average, however, the variation of the protein concentration was higher in mitochondrial fragments of the smaller size. We also observed the same phenotype in fzo1∆ mutant strains, in which the mitochondria showed fragmented morphology. To further test the involvement of translational regulation on this variability, we analyzed the mRNA movement on the mitochondrial outer membrane surface by utilizing the analysis of mitochondrial morphology through computational modeling and the analysis of single molecule mRNA visualization using the MS2-MCP system. We observed that TIM50 mRNAs were trapped at single fragmented mitochondria in fzo1∆ mutant strains. This suggests that the co-translational protein import mechanism restricts mRNA movements to single mitochondrial fragments. We are currently testing whether the variation of the protein concentration in each mitochondrial fragment is based on the translation by changing the copy number of the mRNA. Our work shows that mitochondrial morphology affects mRNA localization and this could potentially result in heterogeneity of protein composition in each mitochondrial fragment. We propose this is a way to regulate the quality of mitochondrial fragments and accelerate the degradation of nonfunctional mitochondrial fragments.

Presenting author email [email protected]

Topic category

Translation Regulation 706 Characterization of EBV Protein BGLF2 and its role in SUMO regulation through miRNAs

Ashley Campbell, Carlos De La Cruz-Herrera, Edyta Marcon, Jack Greenblatt, Lori Frappier Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

Abstract

Epstein-Barr virus (EBV) is a human herpesvirus that establishes life-long infections in ~90% of the global adult population, in which it alternates between latent and lytic modes of infection. Lytic infection involves the expression of ~80 viral proteins and multiple (miRNAs), many of which are poorly characterized but appear to function to modulate cellular responses to promote cell survival and infection. BGLF2 is a virion tegument component that is delivered to the cell upon infection, where it would be available to immediately affect the cellular environment. In a screen for EBV proteins that affect modification of cellular proteins with the Small Ubiquitin-like Modifier (SUMO), we showed that BGLF2 induced global SUMOylation. To gain insight into the mechanism of this effect, we performed affinity-purification coupled to mass spectroscopy (AP-MS) with BGLF2 to identify host proteins that physically interact with BGLF2. Multiple TNRC6 (GW182) and Argonaute proteins were identified, showing that BGLF2 interacts with the miRNA-Induced Silencing Complex (RISC), and implicating BGLF2 as a regulator of miRNA function. The BGLF2-RISC interaction was verified in multiple cell lines and shown to be a unique feature of EBV BGLF2 that does not occur in the homologues from other herpesviruses. To test the potential miRNA-regulating activity of BGLF2, we first assayed its effects on the well-studied let-7 family of miRNAs, which is known to target Dicer and SUMO mRNAs through the 3’UTRs. Assays with a luciferase reporter plasmid containing either let-7 target sites or the Dicer 3’UTR showed that expression of BGLF2 increased luciferase levels dependent on the 3’UTRs (containing let-7 sites), consistent with inhibiting let-7 activity. SUMO1, SUMO2 and SUMO3 transcripts are known to containing multiple let-7 binding sites in their 3’UTRs, prompting us to examine effects of BGLF2 on SUMO levels. We found that expression of BGLF2 increased the level of SUMO1 and SUMO2/3 proteins without affecting the levels of the transcripts, consistent with BGLF2 inhibition of let-7 miRNAs. The results as a whole suggest that BGLF2 is a miRNA regulator and that one outcome is the induction of SUMOylation by increasing the levels of free SUMO.

Presenting author email [email protected]

Topic category

Translation Regulation 719 RNA polymerase I and III common subunit influences its own expression

Neuton Gorjão, Monika Wisniewska, Damian Graczyk Institute of Biochemistry and Biophysics Polish Academy of Sciences, warsaw, Poland

Abstract

Colorectal cancer (CRC) is the second-most and third-most common cancer in women and men, respectively. Most patients with metastatic colorectal cancer fail to respond or develop resistance to the conventional treatments. Therefore, the identification of molecular mechanisms underlying colorectal cancer progress is critical to improve patient outcome and has enormous clinical value. POLR1D is a small subunit that is common to RNA polymerase I and III, which synthesize rRNA and tRNA, respectively. The products of these polymerases are crucial for protein synthesis. POLR1D is frequently upregulated in colorectal cancer and its high expression is positively correlated with tumour size and poor survival of CRC patients. In contrast, POLR1D knock-down inhibits CRC cells proliferation and tumour-growth in mouse xenograft model. Here, we provide evidence that that the ectopic overexpression of POLR1D in normal colon cells and several colorectal cancer cell lines decreases the expression of endogenous POLR1D. Furthermore, this phenomenon seems to be conserved in evolution, since we observe this effect also in budding yeast. Thus, there is a mechanism that tightly controls the levels of POLR1D protein. Interestingly, our recent work demonstrated that POLR1D is not regulated at transcription level or degraded at the proteasome or by autophagy. Another possible mechanism will be proposed and discussed.

This study is supported by the Foundation for Polish Science, FirstTEAM Programme (First TEAM/2016-3/19). D.G. was also supported by the stipend from the Polish Ministry of Science and Higher Education.

Presenting author email [email protected]

Topic category

Translation Regulation 721 Berberine analogues of chloramphenicol as novel translation inhibitors

Andrey Tereshchenkov1, Julia Pavlova2, Ilya Osterman2,3, Natalia Sumbatyan2, Alexey Bogdanov1,2 1A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation. 2Department of Chemistry, Lomonosov Moscow State University, Moscow, Russian Federation. 3Skolkovo Institute of Science and Technology, Skolkovo, Moscow region, Russian Federation

Abstract

Antibiotic resistance is one of the biggest threats to human health, gaining momentum over the past decades. Therefore, the search and development of new antibacterial drugs is a prerequisite in order to turn the tide. Chloramphenicol is a widely used antibiotic that inhibits . Recent studies have shown that its activity depends on the sequence of the nascent chain carried by the ribosome. Replacement of the dichloroacetyl residue by positively charged groups in the chloramphenicol molecule changes its context- specificity and leads to an increase in the affinity for the ribosome. Berberine is an isoquinoline alkaloid, which also shows antibacterial effect as well as anti-inflammatory and antioxidant activity. The presence of a condensed system in the structure of the berberine molecule, by which a positive charge of a quaternary nitrogen atom is delocalized, makes this compound an excellent candidate for modification of chloramphenicol in order to enhance its affinity and eliminate context-specificity. The aim of this research is the design, synthesis and study of the antibacterial activity of berberine derivatives of chloramphenicol. Molecular docking of the proposed conjugates into the structure of the E. coli ribosome revealed additional stacking interactions between the berberine residue and the heterocyclic bases of 23S rRNA leading to an increase in binding affinity compared to the initial antibiotic. Based on the results obtained, a number of conjugates of berberine or its reduced analogue with chloramphenicol amine connected via linkers of different lengths were synthesized. The study of the binding of the obtained compounds to bacterial ribosomes showed a more than twofold increase in the affinity of some of them for the ribosome in comparison with chloramphenicol. Evaluation of antibacterial activity using the pDualrep2 double reporter system [1] revealed that the action of the synthesized compounds is caused by both ribosome stalling due to interactions with rRNA nucleotides and DNA damage. This work was supported by the grant RFBR 20-04-00873А. [1] Osterman I.A., et al. 2016. Antimicrob Agents Chemother. 60 (12): 7481-7489.

Presenting author email [email protected]

Topic category

Translation Regulation 723 Dhx36 promotes satellite cell proliferation during skeletal muscle regeneration by binding 5' UTR G-quadruplex of mRNAs and facilitating translation

Xiaona Chen1, Jie Yuan1, Guang Xue1, Yu Zhao1, Liangqiang He1, Yuying Li1, Silvia Sanz2, Joan Marin2, Wen Wang3, Xi Mou4, Mubarak UMAR4, Zhongzhou Yang5, Wei Chen3, Yuanchao Xue6, Chun Kit Kwok4, Hao Sun1, Pura Muñoz-Cánoves2, Huating Wang1 1The Chinese University of Hong Kong, Hong Kong, China. 2University Pompeu Fabra, Barcelona, Spain. 3Southern University of Science and Technology, Shenzhen, China. 4The City University of Hong Kong, Hong Kong, China. 5Nanjing University, Nanjing, China. 6Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

Abstract

RNA G-quadruplexes and rG4 resolvase play important roles in regulating RNA functions during various biological processes. Skeletal muscle consists around 40% of human body mass and has remarkable regeneration capacity due to the muscle satellite cells, which are also known as muscle stem cells. We find that Dhx36, which is one of the major helicase for binding and unwounding rG4 structures is highly induced during SC activation. To understand rG4 formation and the function and targets of Dhx36 in satellite cells and muscle regeneration, in the present study we generate inducible satellite cell-specific Dhx36 knock-out mice and find that deletion of Dhx36 in adult mice SCs hampers SC cell cycle progression, resulting in impaired SC proliferation and regeneration. Transcriptomic mapping of Dhx36 binding sites on RNAs reveals that Dhx36 bind with thousands of RNAs at various regions. Together with polysome profiling of wildtype and Dhx36 KO myoblast and rG4 prediction in mouse myoblast cells we identify that Dhx36 bind with the rG4 formed at the 5’UTR of target gene Gnai2 and modulate gene expression at the translational level through regulating the formation of rG4 during SC activation. Altogether our findings identify Dhx36 as an important regulator for SC function and muscle regeneration through modulating translation by binding and unwinding the rG4 structure formed at the 5’UTR of target mRNAs.

Presenting author email [email protected]

Topic category

Translation Regulation 730 Development of axonal tag-free polysomal profiling to study translational regulation in ALS

Marta Marchioretto1, Federica Maniscalco1, Fabio Lauria1, Aurora Badaloni2, Renato Arnese1,2, Gian Giacomo Consalez2, Gabriella Viero1 1Institute of Biophysics, CNR Unit of Trento, Trento, Italy. 2Division of Neuroscience, San Raffaele Scientific Institute, Milano, Italy

Abstract

Amyotrophic Lateral Sclerosis is a disease characterized by motor neurons degeneration. The vast majority of involved genes encode for RNA-binding proteins (RBPs). RBPs play different roles (mRNA maturation, localization and translation) shaping cell-type and sub-cell-specific proteomes. Mutations in Tardbp gene are major contributors of ALS. Tardbp encodes for TDP-43, a ubiquitously expressed RBP. According to our preliminary data cytoplasmic TDP-43 co-sediments with polysomes in brain suggesting its involvement in mRNA translation. Our hypothesis is that TDP-43 mutations may impair transport and translational control of RNA processing in axonal polysomes of ALS affected neurons, dysregulating local translational activity. Existing approaches to isolate mRNAs associated to axonal ribosomes (translatome) rely on immunoprecipitation of tagged ribosomes (TRAP). However, with this method, the corresponding transcriptome cannot be determined, impeding the estimate of local translation efficiencies with respect to transcriptional changes. To overcome this problem, we developed an axonal tag-free polysomal profiling coupled to axonal transcriptional profiling using microfluidic chamber and primary neurons. Using miniaturized sucrose gradient fractionation on axonal lysate we purified and sequenced the axonal translatome and transcriptome. Next, we applied this technique to investigate axonal translational defects in ALS in neurons overexpressing either A315T or Q331K mutant TDP-43. We validated our sequencing results by qPCR and immunofluorescence.

Presenting author email [email protected] , [email protected]

Topic category

Translation Regulation 737 Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation

Rachel Niederer1, Maria Rojas-Duran1, Boris Zinshteyn2, Wendy Gilbert1 1Yale University, New Haven, CT, USA. 2Johns Hopkins Medical Institution, Baltimore, MD, USA

Abstract

Translational control of gene expression plays an essential role in a wide range of cellular processes, ranging from stress responses to immune regulation. The protein output per mRNA is ultimately governed by a combination of cis elements and trans factors. In particular, transcript leaders (TLs) of mRNAs have been shown to confer a thousand-fold range of translation efficiencies (TE) both in vivo and in vitro. However, the key mRNA features that distinguish efficiently translated from poorly translated mRNAs remain largely unknown. To elucidate the varied mechanisms by which translational control is achieved, we developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5′ untranslated regions that confer thousand-fold differences in ribosome recruitment in vitro. Using DART, we identified novel translational enhancers and silencers, determined a functional role for most alternative 5′ UTR isoforms expressed in yeast, and revealed a general mode of increased translation via direct binding to a core translation factor. Our analysis revealed both anticipated and novel trends in the data. For example, engineered stems are generally inhibitory to recruitment at levels proportional to their folding strength. However, the effects are highly context-dependent with some strong stems apparently promoting recruitment in specific positions. Strikingly, we observed a strong global anticorrelation between %C and ribosome recruitment (Spearman R = -0.544) and found C-rich motifs over-represented among poorly recruiting TLs. We have shown that the identified C-rich motifs are sufficient to repress translation in vitro. DART enables systematic assessment of the translational regulatory potential of 5′ UTR variants, whether native or disease- associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

Presenting author email [email protected]

Topic category

Translation Regulation 741 Investigating the translational control mechanism of glp-1 in the germline of Caenorhabditis elegans

Peren Coskun, Sean Ryder UMass Medical School, Worcester, MA, USA

Abstract

Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. In Caenorhabditis elegans, the maternal gene glp-1 encodes a homolog of the Notch transmembrane protein required for cell proliferation in the germline, and cell fate specification in the embryo. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3’ untranslated region (3’ UTR). When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3’ UTR transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of pos-1 increases the average polyA tail length of endogenous glp-1 transcripts in embryos. Here, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection, and has no effect on polyA tail length distribution, in young adults. These results rule out alternative polyA site usage as a mechanism of regulation. We also have shown that wild-type glp-1 transgenic reporter embryos have a shorter average polyA tail length compared to mutant transgenic reporters in GLD-1 or POS-1 binding motifs. We will expand our analysis by measuring reporter mRNA abundance and polysome association. When complete, our studies will provide insights of into the mechanism of glp-1 translational regulation in C. elegans germline.

Presenting author email [email protected]

Topic category

Translation Regulation 742 A versatile strategy to reduce UGA-selenocysteine recoding efficiency of the ribosome using CRISPR-Cas9-Viral-Like-Particles targeting selenocysteine- tRNA[Ser]Sec gene

Caroline Vindry, Olivia Guillin, Philippe Mangeot, Théophile Ohlmann, Laurent Chavatte CIRI, Centre International de Recherche en Infectiologie, INSERM U1111, CNRS/ENS/UCBL1 UMR5308, Lyon, France

Abstract

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: the selenocysteine insertion sequence (SECIS) located in the 3’UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting proteins partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR- Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells since these CRISPR-Cas9-VLPs have a wide tropism.

Presenting author email [email protected]

Topic category

Translation Regulation 756 A roadmap of ribosome heterogeneity and its impact on cellular differentiation

Naomi Genuth, Zhen Shi, Rachel Shulman, Irving Weissman, Kyle Loh, Maria Barna Stanford University, Stanford, California, USA

Abstract

The ribosome is a complex macromolecular machine that has recently been suggested to be a source of heterogeneity. Our lab has previously shown that ribosomes within a single cell type are heterogeneous in composition and that ribosome diversity alters the capacity of ribosomes to translate specific mRNAs. To determine the magnitude of ribosome composition changes and its functional contribution to cell fate specification, we developed a sophisticated model system to measure ribosome heterogeneity by quantitative mass spectrometry on a day-by-day basis as human embryonic stem cells differentiate in a step-wise fashion down two distinct endoderm and mesoderm lineages. In this first-ever roadmap of ribosome composition dynamics during cellular differentiation, we identified numerous core ribosomal proteins (RPs) as changing significantly in abundance in actively translating ribosomes. Interestingly, a comparison of the abundance of each RP in the polysomes versus the whole cell or cytoplasm frequently showed distinct mechanisms for changes in RP stoichiometry, suggesting multiple levels of control to ribosome composition. At the organismal level, we further generated a unique loss-of-function mouse model for a heterogeneous, large subunit RP and observed a remarkable series of phenotypes that include striking defects in mesodermal cell populations where the RP is most dynamically incorporated into the ribosome. These findings set the stage for studies of ribosome heterogeneity and specialization, creating a tool box for elucidating the functions of ribosomal heterogeneity from cells to organisms.

Presenting author email [email protected]

Topic category

Translation Regulation 766 Translational Regulation During Mitochondrial Biogenesis in Brown Adipogenesis

Jun Yu Ip1,2, Indrik Wijaya1, Li Ting Lee1, Cheryl Chan3, Peter C. Dedon3,4, Huili Guo1,5 1Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore. 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore. 3Singapore-MIT Alliance for Research and Technology, Singapore, Singapore. 4Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 5Department of Biological Sciences, National University of Singapore, Singapore, Singapore

Abstract

The vast majority (>99%) of proteins that constitute functional mitochondria are encoded by nuclear genes, synthesized in the cytoplasm and imported into the organelle. Whether these genes are co-ordinately regulated at the translation level, and whether they undergo localised translation at the vicinity of mitochondria (as in the situation for ~50% of such genes in yeast) during mitochondrial biogenesis is not well- studied. Using brown adipogenesis as a model system, we isolated bulk cytoplasm, cytosolic and crude mitochondrial fractions from various time points during brown adipogenesis. We aim to study local translation outside of mitochondria in a genome-wide manner using RNA-Seq and ribosome profiling .

At the whole-cell level, translational changes were not observed until the second half of brown fat differentiation; however, at the crude mitochondria level, translational changes were already observed early on in differentiation. At the same time, mRNAs with a higher frequency of codons ending in G or C tend to be more upregulated translationally. Intriguingly, this phenomenon occurs early on in differentiation in the crude mitochondria fraction but is not seen in the cytosolic fraction till the second half of differentiation. The preferential translational upregulation of mRNAs with more G- and C-ending codons implies there could be differences in the pool of tRNAs in the vicinity of mitochondria, versus the rest of the cytoplasm. This initial discrepancy dissipates as cells become fully differentiated, and could be linked to the increased production of mitochondrial reactive oxygen species (ROS) upon differentiation induction. We performed LC-MS/MS to determine the dynamic changes of tRNA modifications in crude mitochondria and cytosolic fractions and observed that tRNA modifications on uridine at position 34– catalysed by the Elongator complex– are reduced in the crude mitochondrial fraction during differentiation. ELP3, the catalytic subunit of the Elongator complex is an iron-sulfur protein, the activity of which is sensitive to ROS levels. The reduction in tRNA modifications may reduce the translational efficiency of A-ending codons and thus bias protein synthesis towards the translation of G-ending codons.

In conclusion, differential codon recognition and localised translation influence the translation of mRNAs, including those encoding mitochondrial proteins during mitochondrial biogenesis in brown adipogenesis.

Presenting author email [email protected]

Topic category

Translation Regulation 769 The uncharacterized S. pombe La-related protein 1 functions in translation and affects RNA abundance

Farnaz Mansouri-Noori1, Andreas Pircher1, Danielle Bilodeau2, Lidia Siniavskaia1, Jorg Grigull1, Olivia S. Rissland2, Mark A. Bayfield1 1York University, Toronto, ON, Canada. 2University of Colorado Denver, Denver, CO, USA

Abstract

La is an abundant RNA-binding protein that is best characterized to bind nascent polymerase III transcripts and promote their maturation. Genuine La and several La-related proteins (LARPs) have been identified in nearly all eukaryotes with conserved functions. The four known LARP families each have evolved with distinct roles in RNA metabolism. LARP1 family members bind 5’-terminal oligopyrimidine (5’TOP) mRNAs encoding several factors important in the process of translation. While LARP1 is thought to modulate both the translation and the stability of these mRNAs, the mechanisms of its function remain elusive. Schizosaccharomyces pombe contains an uncharacterized LARP phylogenetically grouped in the LARP1 family that was named the S. pombe La-related protein 1 (Slr1p). While yeasts are not known to have 5’TOP mRNAs with an oligopyrimidine track as they are characterized in other eukaryotes, we show Slr1p nevertheless plays a role related to LARP1 family members in the translation and stability of functionally similar mRNAs. Slr1p co- sediments with subpolysomes, the pre-initiation complex and with polysomes. Immunoprecipitation of endogenous complexes shows Slr1p engages various components of the cap-dependent translational machinery and specifically that of translation initiation. Whole transcriptome RNA-Seq and translational profiling further show slr1 null cells have an altered abundance of mRNAs encoding the proteins associated with the translational apparatus. Investigation of transcriptome-wide RNA decay rates using in vivo metabolic labeling in wildtype and slr1 null strains, confirm a role for Slr1p in the stability of such mRNAs. These transcripts contain two possible common motifs: a U-rich or an AC-rich motif. Renilla reporter assays show harbouring these specific motifs allows for higher reporter expressions in wildtype cells in a Slr1p-dependent manner. Slr1p engages poly(A) and poly(U) homopolymers as expected from La motif-containing proteins as well but does not affect reporter expressions through such regions in vivo. Thus, Slr1p appears to affect the translation and the stability of the yeast 5’TOP-equivalent mRNAs and our data are consistent with a role for Slr1p that places it as an early progenitor of the LARP1 family.

Presenting author email [email protected]

Topic category

Translation Regulation 773 Homeostatic Control of Cellular Polyamine Levels in S. cerevisiae through Translational Regulation of the High-affinity Polyamine Transporter Hol1

Arya Vindu, Byung-Sik Shin, Thomas Dever Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Bethesda, MD, USA

Abstract

Cellular polyamines (PAs) putrescine, spermidine, and spermine play critical roles in various processes including cell proliferation, development, signaling, transcription and translation. In eukaryotes, intracellular PA pools are homeostatically regulated by PA control of the synthesis of key biosynthetic and regulatory proteins. For example, PA-induced ribosomal frameshifting governs the synthesis of antizyme (OAZ1), an inhibitor of ornithine decarboxylase (ODC), which catalyzes the first step in polyamine synthesis. Moreover, PAs act via regulatory upstream open reading frames (uORFs) to control the synthesis of both S-adenosylmethionine decarboxylase (AdoMetDC), an enzyme required for spermine and spermidine synthesis, and antizyme inhibitor (AZIN1). The AZIN1 uORF, referred to as a uCC (for upstream conserved coding region), encodes a conserved peptide terminating with the sequence motif xEPPWxPS* (* indicates stop codon). Studies in our lab have shown that translation elongation and termination on this sequence motif is controlled by PAs via altered function of the eukaryotic translation factor eIF5A. In addition to de novo synthesis of PAs, cells also acquire PAs from extracellular sources via membrane transporters. Although the proteins Gap1, Agp2, Sam3 and Dur3 have been reported as PA transporters in S. cerevisiae, the identity and possible regulation of PA transporters is not well defined. In the present study, we have identified Hol1 as the major high-affinity polyamine transporter in S. cerevisiae. Confocal microscopy studies localized GFP-tagged Hol1 protein on the S. cerevisiae plasma membrane, and [14C]-spermidine uptake assays demonstrated that HOL1 is required for PA transport. We identified a conserved uORF encoding the peptide MLLLPS* in the 5’ leader of the HOL1 mRNA. Using a HOL1-FLuc reporter, we found that that the PS* motif in the uORF is critical to mediate PA control of HOL1 expression via altered function of the translation factor eIF5A. Thus, similar to the feedback mechanisms regulating the expression of PA metabolizing enzymes and regulators, we hypothesize that the PA transporter Hol1 is also under PA autoregulation. To conclude, our studies have identified Hol1 as the high-affinity PA transporter in yeast and characterized autoregulation of its expression by PAs.

Presenting author email [email protected]

Topic category

Translation Regulation 782 TGF-beta induces ribosome activity, alters ribosome composition and inhibits IRES-mediated translation in chondrocytes.

Guus van den Akker1, Donatus Surtel1, Alzbeta Chabronova1, Bas Housmans1, Marjolein Caron1, Mandy Peffers2, Tim Welting1 1Maastricht University, Maastricht, Netherlands. 2University of Liverpool, Liverpool, United Kingdom

Abstract

Ribosomes are required for the continuous translation of all proteins in cells. In addition to the Cap-mediated translation, Internal Ribosomal Entry Site (IRES) mediated translation is a distinct level of translational regulation. Thousands of human mRNAs contain a 5’ untranslated region that regulates protein translation through IRESes. We used a candidate approach in a chondrocytic cell line to identify growth factors or cytokines that differentially regulate IRES-mediated translation.

Our initial IRES activity screening revealed that Transforming Growth Factor beta 3 (TGFβ) consistently decreased IRES activity in favor of Cap-mediated translation. A concentration of ≥ 1 ng/ml TGFβ and at least two days of stimulation was required for this effect. Subsequently, we tested whether TGFβ increased total protein translation in SW1353. TGFβ significantly increased this by 1.1-1.5 fold without affecting proliferation.

To identify what proteins were differentially expressed, we utilized mass-spectrometry label-free quantification. In the cellular proteome we found that TGFβ induced protein expression of known TGFβ transcriptional target genes at day 3 compared to control, (JUNB, FN1, TGFB2). This was also reflected in the secretome. Interestingly, ALPP and ALPP2 were repressed by TGFβ in the cellular proteome and MMP3 in the secretome.

Finally, we isolated ribosomes with their associated proteins from control and TGFβ3-treated cells. Ribosomes isolated from TGFβ-treated cells contained significantly lower amounts of the Heterogeneous RiboNuclear Protein (HNRNP) family members A0, A1, A3, C, H1, L, M, R and U. The majority of these HNRNPs were not differentially expressed in the cellular proteome or only to a small degree (1.2x). Significantly increased association of eukaryotic initiation factor 2 (A, S1, S2, S3) and six tRNA ligases were found on ribosomes isolated from TGFβ3-treated cells, supporting the observed increased incorporation rate of 35S- methionine/cysteine. Finally, a small but significant increase of specific ribosomal proteins was found in ribosomes extracted from TGFβ-treated cells.

We demonstrate that a TGFβ can induce ribosome activity, alter ribosome composition and modulate the preferential mode of translation in eukaryotic cells. Future experiments will focus on functional validation of the link between ribosome-associated HNRNP abundance and alterations in IRES-mediated translation.

Presenting author email [email protected]

Topic category

Translation Regulation 784 Novel mechanisms of translational control enable cells to recover in response to severe environmental stress

Raul Jobava1, Yuanhui Mao2, Donny Licatalosi1, Shu-Bing Qian2, Maria Hatzoglou1 1Case Western Reserve University, Cleveland, Ohio, USA. 2Cornell University, Ithaca, New York, USA

Abstract

Cellular responses to environmental stresses are sophisticated and fine-tuned. The duration and magnitude of the response is commensurate with the severity of perturbation. Molecular mechanisms that enable cells to function as such rheostat are multi- layered and are associated with distinct gene expression signatures. We used ribosome footprinting and RNA-seq to characterize these gene expression landscapes in mammalian cells exposed to a gradient of hyperosmotic stress (from mild to moderate to severe), a physiologically relevant stress involved mostly in the normal function of kidney and immune cells and implicated in several inflammatory disorders. We identified widespread reprogramming of gene expression at the level of mRNA translation that promotes synthesis of proteins important for cell survival. Cells exposed to the mild and moderate stress shut down global protein synthesis and only translate the select pool of mRNAs, which is distinct from other stresses and includes genes coding for the components of cytoskeleton, magnesium and polyamine metabolism and inflammation. Unexpectedly, severe stress that inhibits protein synthesis by 95% preserved a single ribosome on the specific group of mRNAs that include ribosomal proteins and different proteins of oxidative phosphorylation. This ribosome was paused at the start codon of these mRNAs. Interestingly, the pausing increased even further during early recovery from this stress but was relieved later into the recovery. Overall, out data suggests hyperosmotic stress not only inhibits recruitment of ribosomes to the mRNAs but also prevents already assembled 80S ribosome to transition into the productive elongation. Our preliminary data suggests the involvement of eIF5B and eIF2A in these processes. Studying these novel, multi- layered mechanisms of gene expression regulation will shed light on how cell responds to various environmental cues that eventually lead to the disease development.

Presenting author email [email protected]

Topic category

Translation Regulation 785 Specialized ribosomes in erythroid differentiation

Jiemin Nah1, Yue Zhao2, Wei Ting Ng1, Fauziah Ally3, Claire Swa1, Harvey Lodish4, W.Y.K. Hwang5, J Gunaratne1, Huili Guo1 1A*STAR, Singapore, Singapore. 2NUS, Singapore, Singapore. 3-, Singapore, Singapore. 4Whitehead Institute for Biomedical Research, Cambridge, USA. 5SGH, Singapore, Singapore

Abstract

Eukaryotic ribosomes are made up of ~80 ribosomal proteins (RPs) and 4 ribosomal RNAs (rRNAs) and are traditionally thought to be invariant in all cells. Recent studies have found that ribosomes can be heterogeneous in different cell types and can play a role in regulating translation of certain subsets of mRNAs. Through the proteomics study of actively translating polysomes in differentiating erythroid cells, we show that although the expression of most RPs stay the same, certain RPs show changes in expression on ribosomes during erythropoiesis. Selective ribosome profiling experiments show that certain subsets of mRNAs, including those important in red blood cell development, are preferentially bound to ribosomes containing such RPs. The mRNAs that are preferentially associated with these ribosomes exhibit differences in translation initiation contexts, when compared to background. Such a phenomenon could form an additional layer of translation regulation to drive erythropoiesis.

Presenting author email [email protected]

Topic category

Translation Regulation 804 Deep mutational scan of the ErmD leader peptide reveals a dual mechanism for macrolide antibiotic sensing

Elodie Leroy1, Michael Graf2, Stefan Arenz2, Maha Abdelshahid2, Daniel N. Wilson2, C. Axel Innis1 1Inserm U1212 - CNRS UMR 5320, Bordeaux, France. 2Department of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany

Abstract

Infections caused by multidrug resistant pathogens have increased steadily over the last few decades. In contrast, the number of new antibiotics entering the market has decreased dramatically and antibiotic resistance is now a real threat to public health. More than half of all antibiotics used today target the bacterial ribosome and block bacterial protein synthesis. During this process, certain nascent polypeptides interact with the interior of the ribosome and block translation in a drug-dependent manner. This in turn can trigger the expression of some antimicrobial resistance genes. Understanding the sequence determinants underlying this phenomenon could therefore help to develop drugs that prevent such activation from taking place. Macrolide antibiotic-dependent arrest on the ermD leader (ermDL) sequence leads to the expression of the ermD gene (erythromycin resistance methyltransferase), conferring resistance to MLS antibiotics (macrolide, lincosamide, streptogramin B). The ErmDL peptide contains a +X+ arrest motif, where “+” stands for positively charged amino acids and “X” for any amino acid. This motif can stall the ribosome in the presence of different macrolides, including erythromycin and telithromycin. In the case of erythromycin-induced arrest, this “+X+” motif can be mutated without losing the stalling of the ribosome. In order to identify the primary sequence determinants that lead to translational arrest, we investigated the sequence dependence of the ErmDL peptide using inverse toeprinting. This high-throughput in vitro technique allowed us to perform a deep mutational scan of the ermDL sequence in the presence or absence of erythromycin or telithromycin. Here, we present our characterization of ErmDL variants in light of two cryo- electron microscopy structures of macrolide-bound E. coli ErmDL-70S ribosomes complexes. Our data indicate that ErmDL possesses two distinct arrest determinants: the previously identified +X+ motif and an N-terminal segment that is important for erythromycin-dependent arrest, but dispensable for macrolides that lack a cladinose moiety, such as telithromycin.

Presenting author email [email protected]

Topic category

Translation Regulation 811 Microbial weapon against antibiotics - translational activity of methicillin- resistance Staphylococcus aureus upon antibiotic stress.

Klementyna Marciniak1, Michał Chodań1, Kamilla Bąkowska-Żywicka1,2, Marek Żywicki3 1Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań, Poland. 22 Centre for Advanced Technologies, Poznań, Poland. 3Department of Computational Biology, Institute of Molecular Biology and Biotechnology Adam Mickiewicz University, Poznań, Poland

Abstract

Staphylococcus aureus gained methicillin resistance by acquiring MecA gene encoding novel penicillin binding protein PBP-2a, which allows this strain to grown in the presence of antibiotics, e.g. methicillin, nafcillin, dicloxacillin or flucloxacillin. Furthermore, by spontaneous mutations and horizontal gene transfer many strains become resistant to other commonly used groups of antibiotics. This ability to quickly adopt a novel resistance mechanisms became an urgent medical concern in bacteriosis treatment. In this work I present global changes in the protein biosynthesis in methicillin-resistant strain of S. aureus under various concentration of vancomycin. Translational activity was monitored by polysome profiling, where the amount of polysomes reflects the cellular levels of translation of mRNA encoding proteins essential for survival under antibiotic stress. The differences in polysome profiles depends on vancomycin concentration and exposing time.

Presenting author email [email protected]

Topic category

Translation Regulation 850 A G-quadruplex RNA-binding protein which regulates the translation of genes related to Parkinson's disease

Marc-Antoine Turcotte, Jean-Michel Garant, Hélène Roberge-Cossette, Jean-Pierre Perreault University of Sherbrooke, Sherbrooke, Quebec, Canada

Abstract

Only 1.5% of the genome is translated into proteins but more than 90% undergoes transcription into RNA. That phenomenon brings the importance of RNA regulation in cells. G-quadruplexes (G4) are noncanonical secondary structures present in eukaryotic RNA and DNA. These RNA motifs have been associated with many types of post-transcriptional regulations. Our laboratory is interested in the role of these structures in the 5’UTR of mRNA and their implication in translation regulation. Some proteins have already been identified to fold, stabilize or unfold G4. Moreover, studies showed that RNA are highly regulated in neurodegenerative diseases and few mutations can affect the fate of neuronal cells. The impact of G4 regulation in some neurodegenerative diseases, like Parkinson’s disease, has never been analyzed. The goal of this project is to identify proteins able to modulate translation through the binding of G4 structures found in 5’UTR of mRNAs that are involved in Parkinson's disease. To achieve our goal, RNA G4 candidates were characterized by in vitro fluorescence assays and circular dichroism. Moreover, in cellulo luciferase assays were done to determine the role of these G4s on translation in SH-SY5Y cells. Proteins able to bind these G4s have then been identified by an RNA pull-down with SH-SY5Y proteins lysates followed by label-free mass spectrometry. Finally, the interaction between RNA and proteins were further characterized by electromobility shift assay. Our results showed two new RNA G4 located in the 5’UTR of PRKN and VPS35. Both G4s were found to have an inhibitory effect on translation in cells. We also identified GNL1 as a protein partner of both G4. This interaction between GNL1 and the G4s could be the key to regulate the translation of PRKN and VPS35 in Parkinson’s disease.

Presenting author email

[email protected]

Topic category

Translation Regulation 856 Influence of the identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap on protein expression in mammalian cells.

Pawel Sikorski1, Marcin Warminski2, Dorota Kubacka2, Tomasz Ratajczak1, Dominika Nowis1,3, Joanna Kowalska2, Jacek Jemielity1 1Centre of New Technologies, University of Warsaw, Warsaw, Poland. 2Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland. 3Department of Immunology, Medical University of Warsaw, Warsaw, Poland

Abstract

Recently, much effort has been made to generate in vitro transcribed messenger RNA (IVT mRNA) with superior biological properties, increase protein biosynthesis yield or improve its cellular stability. Interestingly, all previously synthetized cap analogues are based on m7GpppG dinucleotide, where guanine is at position of the first transcribed nucleotide. However, there is not much known how presence of other nucleotides and especially presence of 2’-O methylated versions of nucleotides influence biological properties of mRNA. It is postulated that addition of methyl group to the 2’-O position of the ribose of the first transcribed nucleotide (cap 1) should lead to reduction of immunogenicity of exogenously delivered mRNA and results in higher protein biosynthesis yield. Therefore, to shed some light on how nucleotide composition of 5’ end of mRNA influence its biological properties, we developed new molecular tools – trinucleotide cap analogues which allow to introduce any nucleotide at the position of the first transcribed nucleotide.

Presenting author email [email protected]

Topic category

Translation Regulation 858 Translational repression of NMD targets by GIGYF2 and EIF4E2

Boris Zinshteyn, Usman Enam, Joodh Waleedh, Rachel Green Johns Hopkins University, Baltimore, MD, USA

Abstract

Messenger RNAs that encode premature translation termination codons can produce truncated proteins that misfold or produce dominant-negative effects. Production of these potentially toxic peptides is prevented through a process known as nonsense-mediated mRNA decay (NMD), which degrades the offending mRNA. NMD can be inhibited by stop codon readthrough, presumably by displacing NMD-promoting factors such as exon-junction complexes downstream of the premature termination codon. We developed a fluorescent reporter that simultaneously measures NMD (independent of downstream splicing) and stop codon readthrough allowing us to perform a genome-wide CRISPR flow cytometry screen for factors involved in both processes in K562 cells. Our screen recovered nearly all of the known core NMD factors, which increase fluorescence from our reporter when depleted. Among the novel NMD factors identified by the screen were the ribosomal protein RACK1 and translational repressors GIGYF2 and eIF4E2. These factors have been shown to repress translation in other regulatory pathways, but not in NMD. Ribosome profiling revealed a subset of NMD target transcripts that are translationally repressed by GIGYF2 and eIF4E2, as well as other transcripts that are translationally repressed by these factors as well as the NMD machinery. Preliminary data suggest that GIGYF2 and eIF4E2 interact with the core NMD factors both genetically and physically, raising the possibility that recognition of a stop codon as premature by the NMD machinery leads to its translational repression through GIGYF2 and eIF4E2.

Presenting author email [email protected]

Topic category

Translation Regulation 866 Elucidating the role of Zuo1 protein on the translational control of gene expression in yeast cells

Mehmet Tardu, Kristin Koutmou University of Michigan, Ann Arbor, Michigan, USA

Abstract

The ribosome associated complex (RAC) is a ribosome bound protein chaperone complex reported to surveil the translation of proteins with positively charged regions. It has been posited that RAC might be able to directly regulate translation by coupling co-translational folding with the peptide-elongation cycle. To identify the targets of RAC in cells and test the hypothesis that the complex modulates translation, we performed ribosome profiling on wild-type yeast cells and cells lacking a key component of the RAC that binds near the ribosome active site (zuo1Δ). Ribosome profiling is a sequencing-based technique that allows us to take a nucleotide resolution snapshot of where every ribosome sits on every mRNA in a cell at a given point in time. This powerful approach can provide information about the contributions of individual proteins to the translational landscape of a cell. We identified >300 targets for the RAC, and unexpectedly observed that the ribosome stalls on ~10% of the targets and frameshifts on ~1% in zuo1Δ cells. The maintenance of ribosome reading frame is essential for cell health because frameshifts can result in the production of non-functional truncated and extended protein products. These studies have the potential to uncover RAC as a critical determinant of translational fidelity in eukaryotic cells.

Presenting author email [email protected]

Topic category

Translation Regulation 871 The new mechanism of the dynamic regulation of the initiating ribosome during stringent response.

Daria Vinogradova1,2, Victor Zegarra3, Elena Maksimova1, Jose Alberto Nakamoto3, Pavel Kasatsky1, Alena Paleskava1,4, Pohl Milon3, Andrey Konevega1,4 1Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre "Kurchatov Institute", Gatchina, Russian Federation. 2NanoTemper Technologies Rus, St. Petersburg, Russian Federation. 3Universidad Peruana de Ciencias Aplicadas – UPC, Lima, Peru. 4Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russian Federation

Abstract

During stringent response in the bacterial cell the protein biosynthesis and the cell duplication slow down. The mechanism of the bacterial survival during stress conditions is mediated by the accumulation of the alarmone (p)ppGpp in the cell. Previously it was generally accepted that this regulation is carried out exclusively at the transcription level. Here we present the previously unknown mechanism of translation initiation regulation modulated by (p)ppGpp. In this work we studied (p)ppGpp-mediated regulation of the initial stage of protein synthesis using modern techniques of fluorescent spectroscopy stopped flow and microscale thermophoresis. Using a set of natural and model mRNAs allowed us to establish that the inhibitory effect of (p)ppGpp on translation initiation is highly dependent on the programmed mRNA [1]. mRNA mTufA (coding for EF-Tu) provided the least sensitivity of translation initiation to high concentrations of ppGpp. Structural modeling and biochemical analysis of the mTufA demonstrated two consecutive hairpins proximal to the translation initiation region that unveiled as structured enhancer of translation initiation (SETI). It determines an increase in both 30SIC production and IF2 affinity for GTP, as well as an increase in ppGpp tolerance under physiological concentrations of all guanosine nucleotides. We found this effect not only for mTufA but also for another SETI-containing mRnr (coding for RNAse R). In addition, we show that IF2 (with higher factor concentration) can use pppGpp to promote the formation of 30S initiation complexes. Our studies provide new details about the dynamic regulation of the initiating ribosome and describe new mechanism that bacteria can use to translate mRNA during a stringent response. The work is supported by the Russian Science Foundation (grant 17-14-01416) and RFBR (grant 17-00-00368) to A.L.K. 1. Vinogradova, D.S., et al. (2020) How the initiating ribosome copes with ppGpp to translate mRNAs. PLoS Biol. 18(1):e3000593. doi: 10.1371/ journal. pbio.3000593.

Presenting author email [email protected]

Topic category

Translation Regulation 895 Dissecting the Regulation of Translation Start Site Selection in Human CEBPA

Samantha Fernandez, Nicholas Ingolia UC Berkeley, Berkeley, CA, USA

Abstract

The discovery of thousands of alternative translation initiation sites in mammalian genomes by ribosome profiling has revealed a hidden diversity in the proteome and an extensive complexity in translation initiation. Initiation at these sites has the potential to yield functionally and structurally distinct protein isoforms that can play decisive roles during various cellular processes. Furthermore, alternative translation initiation can serve as a dynamic point of control in gene expression, enabling a rapid response to changes in the cellular environment. Re-initiation after the translation of upstream ORFs (uORFs) has been shown to regulate start site selection in cis, yet the molecular players and mechanism underlying this choice has been poorly understood. Here we have developed a dual-fluorescent reporter assay coupled with CRISPRi in a screen to identify the trans-acting factors that regulate start site usage in the human transcription factor, CEBPA, which plays a major role in driving diverse cellular differentiation programs.

Presenting author email [email protected]

Topic category

Translation Regulation 912 A Mechanism of tRNA Supply-Codon Demand Implicated in Coordinating the Synthesis of Functionally Related Proteins in Mycobacterium

Margaret Saks, John Oh, Austin Deets, George Mastorakos, Susan Martinis University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Abstract

Mycobacterium tuberculosis (Mtb) is one of the world's deadliest infectious agents. Consequently, a top priority is to identify its virulence genes and to elucidate mechanisms that regulate the synthesis of the corresponding proteins. The genome-wide nucleotide content of Mtb is biased towards GC. This bias chiefly affects the identity of the nucleotide at the third codon position which, in turn, imposes a demand for a commensurate bias in anticodon usage in the tRNA repertoire. Unexpectedly, rare AU-rich codons, particularly those in the leucine family, are not randomly distributed among the Mtb protein coding genes. Rather, they are overrepresented in Mtb virulence genes. Mtb is also unusual in that most of its tRNAs are single-copy and appear to be expressed from their own dedicated . Our work indicates that Mtb coordinates the synthesis of functionally related proteins by regulating the expression of the tRNAs that are needed to translate their rare codons.

Presenting author email [email protected]

Topic category

Translation Regulation 913 Frankenbodies, genetically encoded probes for imaging nascent and mature small linear epitopes tagged proteins in vivo

Ning Zhao1, Kouta Kamijo2, Philip Fox1, Haruka Oda2, Tatsuya Morisaki1, Yuko Sato2, Hiroshi Kimura2, Timothy Stasevich1 1Colorado State University, Fort Collins, CO, USA. 2Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

Abstract

To expand the toolbox of imaging in living cells, we have engineered genetically encodable single chain variable fragments binding small and linear epitope tags with high affinity and specificity in vivo. We call the resulting probes frankenbodies, among which we have demonstrated the anti-HA frankenbody in the following applications. First, the anti-HA frankenbody can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. It also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the anti-HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. Besides the anti-HA frankenbody, we are developing orthogonal frankenbodies to facilitate multicolor imaging. The versatility of the small and linear tags makes frankenbodies powerful tools for imaging protein dynamics in vivo.

Presenting author email [email protected]

Topic category

Translation Regulation 932 Identification of essential RNA regulatory elements in trypanosomatids by clustering co-regulated genes based on various data sources

Motahareh Sobat1, Pooria Mirzavand Boroujeni2, Reza Salavati1 1Institute of parasitology, Mcgill university, 21, 111, Lakeshore road, Sainte Anne de Bellevue,, Montreal, Quebec, Canada. 2Department of biotechnology, college of Science, University of Tehran, Tehran, Iran, Islamic Republic of

Abstract

The disease-causing trypanosomatid parasites must respond to transitions between mammalian and invertebrate hosts by extensively remodeling themselves to survive in these very different environments. They transcribe their genome mostly via constitutive transcription and lack the transcription factor network that regulates gene expression in their hosts. Trypanosomatid gene regulation instead mainly occurs post- transcriptionally (i.e., mRNA stability and translational control), through the interaction of RNA- binding proteins (RBPs) with their cis-acting RNA regulatory elements (RREs) of ~10 nucleotide within 3’ untranslated region (UTR) of mRNA. We hypothesize that this interaction is essential for gene regulation and parasite survival. The predicted RREs largely overlap with regulatory events occurring during the life cycle of trypanosomatids and/or lead to transcriptome remodeling in response to external and internal perturbations. We collected all predicted/experimentally validated RREs in a database and searched them against 3’UTR of all trypanosomatid genes to find the carrier genes. Next, various data such as mRNA co-expression in different life stages, riboprofiling, protein-protein interaction, GO term, and presence of each RRE in 3’UTR were used to cluster the genes that co-regulate with each other and share the same RRE. Mapping this result to the trypanosomatid essential genes (based on RNAi experiment) identified potentially crucial RRE candidates necessary for gene regulation and parasite survival.

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Topic category

Translation Regulation 945 Exogenous Leucyl-tRNA Synthetase Inhibits Mouse Myoblast C2C12 Differentiation

Dayu Zhang, Kiranmai Poruri, Mee-Sup Yoon, Robin L. Holland, Lin-Feng Chen, Steven R. Blanke, Auinash Kalsotra, Jie Chen, Susan A. Martinis Univ of Illinois, Urbana-Champaigne, Urbana, IL, USA

Abstract

Leucyl-tRNA synthetase (LeuRS) performs one of the first steps of translation by attaching leucine to its cognate tRNA. In addition to this “housekeeping” function, LeuRS also has non-canonical functions in cell signaling. We identified that exogenous LeuRS inhibits mouse myoblast C2C12 cell line differentiation, decreasing myotube formation and myosin heavy chain (MHC) expression. Confocal microscopy supported that both LeuRS and an N-terminal fragment of the protein bind to the cell surface and are endocytosed. We hypothesized that the N-terminus of LeuRS plays an important signaling role in regulating C2C12 cell differentiation.

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Topic category

Translation Regulation 950 Translational regulations for nuclear-encoded mitochondrial mRNAs identified in monosome

Sachiko Hayashi, Kazumi Iwamoto, Tohru Yoshihisa Graduate School of Life Science, University of Hyogo, Hyogo, Japan

Abstract

Mitochondria play crucial roles for numerous cellular processes, including the biosynthesis of heme and iron- sulfur clusters, the metabolism of amino acids and lipids, and synthesizing the bulk of ATP. In yeast, only 8 proteins are encoded by the mitochondrial genome, while 99% of all remaining mitochondrial proteins are encoded by the nuclear genome and translated by cytosolic ribosomes as their precursor form. Thus, correct sorting of mitochondrial proteins is the first step to ensure organellar functionality. A classical targeting pathway for mitochondrial proteins is used mitochondrial targeting sequences (MTS) mainly located on their N- terminus, whereas a part of nuclear-encoded mitochondrial mRNAs are also known to be transported to mitochondria and translated locally. To understand these two sorting mechanisms in terms of mRNAs, we focused on 204 monosome-enriced mRNAs identified by ribosome profiling in Heyer & Moore (Cell, 2016). These monosome-enrich mRNAs longer than 590 nt mainly included mRNAs for nuclear proteins, however, mRNAs for mitochondrial proteins are also included noticeably. We first analyzed their published Ribo-Seq data in silico, then performed polysome analysis followed by northern blotting to examine the mRNA distribution pattern in vivo. As we expected, categorized monosome-enriced mRNAs, such as AIM17, RSM10 and MRPL16, tended to be occupied with small number of ribosomes if it compared to polysome-enriched nuclear-encoded mRNA, ILV5 or ACT1 mRNA. Additionally, translational patterns of some of those monosome-enriched mRNAs were altered in the absence of PUF3 involving in the targeting of mRNAs to mitochondria, while polysome- enriched ILV5 mRNA was unaffected.

Presenting author email [email protected]

Topic category

Translation Regulation