The role of the RNA-associated proteins NOT4 and Bam in mRNA decay Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Csilla Keskeny aus Budapest, Ungarn Tübingen 2019 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 01.10.2019 Dekan: Professor Dr. Wolfgang Rosenstiel 1. Berichterstatter: Professor Dr. Ralf-Peter Jansen 2. Berichterstatter: Professor Dr. Thorsten Stafforst The work described in this thesis was conducted in the laboratory of Professor Dr. Elisa Izaurralde in the Department of Biochemistry at the Max Planck Institute for Developmental Biology, Tübingen, Germany, from September 2014 to January 2019. It was further supervised by Professor Dr. Ralf-Peter Jansen at the Eberhard Karls University Tübingen, Germany, and was supported by a fellowship of the Max Planck Society within the framework of the International Max Planck Research School “From Molecules to Organisms”. I declare that this thesis is the product of my own work. The parts that have been published or where other sources have been used were cited accordingly. Work that was carried out by my colleagues was also indicated accordingly. Table of Contents 1. ABBREVIATIONS ............................................................................................................... 1 2.1 SUMMARY ....................................................................................................................... 4 2.2 ZUSAMMENFASSUNG .................................................................................................. 5 3. LIST OF PUBLICATIONS ................................................................................................. 7 4. INTRODUCTION................................................................................................................. 8 4.1 Gene expression in eukaryotes............................................................................................ 8 4.2 mRNA turnover ................................................................................................................ 12 4.3 Protein degradation ........................................................................................................... 15 4.4 The CCR4-NOT complex ................................................................................................. 15 4.4.1 Functions of CCR4-NOT .......................................................................................... 15 4.4.2 Recruitment of CCR4-NOT to mRNA targets.......................................................... 17 4.4.3 Architecture of the CCR4-NOT complex ................................................................. 18 4.4.3.1 CAF40 ............................................................................................................... 19 4.4.3.2 NOT4 as a constitutive or facultative component of CCR4-NOT .................... 20 4.5 NOT4 ................................................................................................................................ 22 4.5.1 The role of NOT4 in mRNA deadenylation ............................................................. 23 4.5.2 NOT4 as an ubiquitin ligase ..................................................................................... 24 4.5.2.1 The RING domain of NOT4 ............................................................................. 24 4.5.2.2 Substrates of the NOT4 ubiquitin ligase ........................................................... 25 4.5.3 The role of NOT4 in co-translational quality control ............................................... 26 4.6 Bam (Bag-of-marbles) ...................................................................................................... 28 5. AIMS AND OBJECTIVES ................................................................................................ 30 6.1 SYNOPSIS OF PUBLICATION 1 ................................................................................ 32 6.2 SYNOPSIS OF PUBLICATION 2 ................................................................................ 33 7. DISCUSSION ...................................................................................................................... 35 7.1 The transient nature of the interaction between NOT4 and the CCR4-NOT complex in metazoans .................................................................................................................................. 35 7.2 The RNA-binding function of NOT4 ............................................................................... 36 7.3 NOT4 as an RNA-binding ubiquitin ligase ...................................................................... 37 7.4 Recruitment of the CCR4-NOT complex ......................................................................... 38 7.5 CAF40 is a binding platform for RNA-associated proteins.............................................. 40 7.6 Conclusion ........................................................................................................................ 42 8. REFERENCES .................................................................................................................... 43 9. ACKNOWLEDGEMENTS ............................................................................................... 53 10. CURRICULUM VITAE ................................................................................................. 55 11. APPENDIX: ORIGINAL MANUSCRIPTS OF DISCUSSED PUBLICATIONS ... 57 Abbreviations 1 1. Abbreviations ABCE1 ABC-family ATPase 1 AGO Argonaute protein AREs AU (adenylate-uridylate)-rich elements Bam Bag-of-marbles protein Bgcn Benign gonial cell neoplasm BTG2 B-cell translocation gene 2 CAF1/40/130 CCR4-associated factor 1/40/130 CBM CAF40-binding motif of Roquin, Bam and NOT4 CC Coiled coil CCCH Zinc finger motif containing Cys-Cys-Cys-His, which coordinate a zinc ion CCR4 Carbon catabolite repressor 4 CCR4-NOT Carbon catabolite repressor 4; Negative on TATA-less CN9BD CAF40/CNOT9-binding domain of NOT1 DCP2 Decapping enzyme subunit 2 DDX6 DEAD-box helicase 6 DEAD-box Helicase domain characterized by a Asp-Glu-Ala-Asp tetrad DEDD Exonuclease domain characterized by a catalytic Asp-Glu-Asp-Asp tetrad Dm, D. melanogaster Drosophila melanogaster DNA Deoxyribonucleic acid dsRNA Double-stranded RNA EEP Exonuclease-endonuclease-phosphatase domain eIF Eukaryotic translation initiation factor EJC Exon junction complex eRFs Eukaryotic release factors GSC Germline stem cell GW182 182kDa protein containing glycine-tryptophan repeats HEAT Helical hairpin repeats HEK293T Human embryonic kidney 293T cells HeLa Henrietta Lacks; immortal cell line derived from cervical cancer cells Hs Homo sapiens; human kDa kilodalton Abbreviations 2 LRR Leucin-rich repeat domain LTN1 E3 ubiquitin-protein ligase listerin 1 Mei-P26 Meiotic-P26 met Met-tRNAi Initiator methionine transfer RNA MIF4G Middle domain of eukaryotic initiation factor 4G miRISC MicroRNA-induced silencing complex miRNA MicroRNA mRNA Messenger RNA mRNP Messenger-ribonucleoprotein particle NAC Nascent polypeptide-associated complex NGD No-go decay NMD Nonsense-mediated decay NOT1 Negative on TATA-less protein 1 NSD Nonstop decay ORF Open reading frame PABP Poly(A)-binding protein PAN2/3 Poly(A) nuclease 2/3 PAP Poly(A) polymerase PIC Pre-initiation complex Poly(A) Poly-adenosine stretch pre-mRNA Precursor messenger RNA PTM Post-translational modification RBP RNA-binding protein RING Really interesting new gene; E3 ubiquitin ligase domain type RNA Ribonucleic acid RNA pol II RNA polymerase II RNP Ribonucleoprotein RRM RNA-recognition motif rRNA Ribosomal RNA Sc, S. cerevisiae Saccharomyces cerevisiae, baker’s yeast / budding yeast SHD NOT1 superfamily homology domain shRNA Short hairpin RNA SLiM Short linear motif Sp, S. pombe Schizosaccharomyces pombe, fission yeast Abbreviations 3 Sxl Sex-lethal TNRC6 Trinucleotide repeat-containing gene 6 protein, human homolog of GW182 Tob Transducer of erbB-2 protein TRAMP Trf4/Air2/Mtr4p polyadenylation tRNA Transfer RNA TTP Tristetraprolin Tut Tumorous testis protein UBC Ubiquitin conjugating protein UTR Untranslated region XRN1 5’-to-3’ exoribonuclease 1 ZnF Zinc finger domain ZNF598 Zinc finger protein 598 Summary 4 2.1 Summary The multi-subunit CCR4-NOT complex is an important regulator of gene expression in eukaryotes. It affects most steps of the messenger RNA (mRNA) lifecycle, but as the major deadenylase in cells, its most studied function is the enzymatic removal of the 3’-poly(A) tail of mRNAs. In the past years, several studies were carried out to shed light on how the CCR4-NOT complex is directed to specific mRNA targets, and to identify proteins that regulate this process. The main aim of my doctoral work was to study metazoan-specific aspects in the assembly of the CCR4-NOT complex and to understand molecular details of its recruitment to mRNA by mRNA-associated proteins. The NOT4 E3 ubiquitin ligase is highly conserved in eukaryotes and has been shown to associate with the CCR4-NOT complex in budding yeast. Biochemical and structural studies also demonstrated that the C-terminal region of yeast NOT4 interacts with the C-terminal part of NOT1. In contrast, different studies indicated that NOT4 is not a stable component of the CCR4- NOT complex in human (Hs) and Drosophila melanogaster (Dm) cells.