עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By אורה חיימוב Ora Haimov חקר מנגנון התרגום הייחודי המתווך על-ידי TISU Elucidating the unique translation initiation mechanism directed by TISU element מנחה: :Advisor פרופ' רבקה דיקשטיין Prof.Rivka Dikstein כסלו תשע"ז December, 2016 Acknowledgments First and foremost, I would like to express my deep gratitude and appreciation to my advisor, Prof. Rivka Dikstein, for her excellent guidance, passion for science, critical and creative thinking, dedication, encouragement, support and mostly for being humane. Rivka, the things I learned from you will remain with me throughout my entire career. I would also like to thank my former and current colleagues for their scientific assistance, good advice and mostly for their friendship. Special thanks to Dr. Anat Bahat for being our ultimate staff scientist and a good friend, to Shaked Ashkenazi for her good spirit and contribution to the lab atmosphere, to Ana Tamarkin Ben-harush for many wonderful moments that I will always cherish, to Dr. Hadar Sinvani for collaboration and to Dr. Rafi Emmanuel for the initiation of the “TISU Project”. I owe many thanks to Anna Uzonyi and Adi Jacob, two young and talented scientists who contributed to this study. Last but not least, I wish to thank my family, especially to my parents, Mordechai and Geula, from whom I learned the value of knowledge and education, to my husband, Reuven, for his love, and to my four precious children, Ariel, Eitan, Naama and Yehonatan, who made this whole period challenging, meaningful and much more satisfying. Table of Contents Abbreviations ................................................................................................ 4 Abstract .......................................................................................................... 5 Introduction ................................................................................................... 6 The translation initiation process .................................................................................... 6 Regulation of translation initiation by eIF1 and eIF1A .............................................. 10 TISU- The Translation Initiator of Short 5’UTR .......................................................... 11 Objectives ..................................................................................................... 13 Materials and Methods ............................................................................... 14 Results .......................................................................................................... 25 Analysis of TISU element sequence requirements ....................................................... 25 The roles of eIFs in the regulation of translation initiation fidelity and ribosomal scanning ........................................................................................................................... 27 The effect of eIF1A and eIF1 overexpression on translation initiation fidelity and ribosomal scanning ......................................................................................................... 28 The effect of eIF1 and eIF1A overexpression on H2B mRNA translation ................. 33 The effect of eIF1A and eIF1 knockdown on translation initiation fidelity and ribosomal scanning ......................................................................................................... 35 eIF1 and eIF1A protein levels are cell cycle regulated ................................................ 40 eIF1 and eIF1A protein levels in different mouse cell types and Human tissue culture cell types .............................................................................................................. 41 The effect of eIF3c knockdown on translation initiation fidelity and ribosomal scanning ........................................................................................................................... 42 The role of eIF4A in translation initiation fidelity and ribosomal scanning ............. 44 2 The effect of eIF5 knockdown on translation initiation fidelity and ribosomal scanning ........................................................................................................................... 48 eIF4GI has differential effects on translation initiation fidelity and ribosomal scanning ………………………………………………………………………………...50 Analysis of eIF1-eIF4GI interaction.............................................................................. 54 The role of eIF4E in translation initiation fidelity and ribosomal scanning ............. 58 Interaction of eIF4E and eIF1 with eIF4GI is mutually exclusive…………………..61 Discussion ..................................................................................................... 64 Bibliography ................................................................................................ 74 Declaration ................................................................................................... 81 List of publications ...................................................................................... 81 3 Abbreviations TISU- Translation Initiator of Short 5’ UTR eIF- Eukaryotic translation initiation factor Met-tRNAi- Initiator methionyl tRNA m7G cap- 7 methylguanosine cap UTR- Untranslated region PIC- Preinitiation complex TC- Ternary complex PABP-Poly A binding protein ORF- Open reading frame US-AUG- Upstream AUG DS-AUG-Downstream AUG RL-Renilla Luciferase FL- Firefly Luciferase 4E-BP- eIF4E- Binding Protein 4 Abstract Translation Initiator of Short 5’UTR (TISU) is a unique regulatory element of both transcription and translation initiation. The core of the element has an invariable ATG sequence, and it directs efficient translation initiation from extremely short 5’UTR in a cap-dependent manner, but without scanning. The major goal of this study is to investigate the sequence and the factor requirements of the non-canonical mechanism of TISU. Comprehensive mutagenesis established TISU as a robust translation initiator of short 5’UTR mRNAs and revealed that all TISU-AUG flanking nucleotides contribute to its translational strength; however, position -3, +4, +5 and +6 are critical for its high- fidelity. We tested the role of several eIFs on translation of mRNAs with various AUG contexts and 5’UTR lengths using overexpression and knockdown experiments. Our findings show that the effects of eIF1A, eIF1, eIF4GI and eIF4E are not general but dependent on mRNA characteristics. For example, elevated levels of eIF1A promoted translation from cap-proximal AUG within canonical AUG context, while eIF1 overexpression exhibited an antagonistic effect. Regarding TISU translation, overexpression of either eIF1A or eIF1 had no significant effect. Interestingly, knockdown of eIF1, eIF1A, eIF4GI or eIF4E caused a dramatic decrease in TISU mediated translation, whereas translation from cap-proximal canonical AUG was almost unaffected, suggesting that TISU activity is highly dependent on those eIFs. The knockdown experiments also uncovered the regulatory roles that eIF4GI and eIF4E play in scanning. We show that eIF1 specifically interacts with the middle conserved domain of eIF4GI. Furthermore, our results suggest the existence of a novel eIF4E binding site which is also located in the middle conserved domain of eIF4GI. Interestingly, the interaction of eIF4GI with eIF4E and eIF1 is mutually exclusive. An eIF1 mutant impaired in eIF4GI binding fails to promote scanning but facilitates TISU activity, suggesting that eIF1-eIF4GI binding is required for scanning but not for TISU. Strikingly, eIF4E-eIF4GI antagonizes the scanning promoting activity of eIF1-eIF4GI. Therefore, we propose that the entrance into the scanning phase necessitates an exchange of eIF4GI from eIF4E to eIF1. These findings expand our understanding of scanning-dependent and independent translation initiation process and uncover important mechanistic aspects of the unique translation initiation directed by TISU. 5 Introduction The translation initiation process Translation of mRNA to protein in eukaryotes requires a complex apparatus consisting of mRNA, ribosomes, tRNAs and protein factors. mRNA translation is a cyclic process which can be divided into initiation, elongation, termination and recycling. Within this framework the initiation stage is considered as a major regulatory target. This stage involves different initiation factors (eIFs) and different mRNA features, all of which play important roles in translational control. The predominant form of eukaryotic translation initiation is the canonical cap-dependent scanning mechanism (Aitken & Lorsch, 2012; Hinnebusch, 2014; Marintchev & Wagner, 2004; Sonenberg & Hinnebusch, 2009) which depends on the m7G cap structure, present at the 5ʹ-end of the mRNA, and on ribosomal scanning. Translation initiation begins with formation of the 43S preinitiation complex (PIC) that is assembled from the ternary complex (eIF2–GTP–Met–tRNAi), several initiation factors (eIFs) that include eIF1, 1A, 3 and 5 and the 40S small ribosomal subunit (Fig. 1). This 43S PIC, then attaches the cap-proximal region of activated mRNAs. mRNA activation is driven by eIF4F which is composed of eIF4E- the cap-binding protein, eIF4GI -a scaffold protein, and eIF4A- a DEAD-box ATPase and RNA dependent helicase.