Structural and functional characterization of the RING-like protein RTF2 by Andrew Zhai A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto Copyright © 2020 by Andrew Zhai Abstract Structural and functional characterization of the RING-like protein RTF2 Andrew Zhai Doctor of Philosophy Graduate Department of Biochemistry University of Toronto 2020 RING and RING-like domains represent the functional component on the vast majority of E3 ligases. With over 600 known members, the family of RING E3 ligases promote the direct transfer of ubiquitin from an E2 onto a substrate, acting as the key mediators of specificity during the reaction. However, beyond acting as a simple scaffold for E2-binding, many RING and RING-like domains have been found to play a more active role by inducing conformational selectivity in the otherwise flexible E2~ubiquitin conjugate. With their central role in the ubiquitination reaction, it is no surprise that RING and RING-like domains exhibit a wide array of regulatory mechanisms that govern their activity, including the ability to function in different oligomeric states. Specifically, while some function as monomers, many RING and RING-like E3 ligases are dependent on dimerization for their activity. This thesis explores the structure and function of a poorly characterized, evolutionarily conserved RING-like protein RTF2. In the first chapter, I examine the crystal structure of human RTF2 revealing two conserved RING-like domains: an N-terminal U-box-like domain associated with a SP-RING domain in an arrangement I describe as an intra-chain heterodimer. I then move on to describe a role for RTF2 in pre-mRNA splicing and begin to explore its interactome using proximity-dependent biotin identification (BioID). In the third chapter, I identify conserved functional elements on both RING-like domains and ii demonstrate the importance of these residues for RTF2-dependent splicing, possibly by facilitating E2 recruitment. Together, these results reveal a novel RING-like structural mode and provide a foundation for further work examining the mechanism underlying RTF2 function and its role in pre-mRNA splicing. iii Acknowledgments Of course, I extend my most immediate gratitude to my supervisor Gil Privé for his years of mentorship and allowing me the privilege of conducting research in his lab. Graduate school has been equal parts rewarding and challenging, but his guidance allowed me to emerge mostly intact. The lessons I have learned while under his tutelage are ones that I will remember throughout my life. I have also been fortunate enough to count two incredible scientists, Frank Sicheri and Brian Raught, as my committee members. They deserve thanks for their continuous support and advice during the completion of this thesis. I have always left our meetings with a renewed sense of enthusiasm and clarity due to their invaluable input. On top of this I also wish to thank Brian as well as Étienne Coyaud for their role as collaborators. Their patience in working with structural biologists on proteomics projects knows no bounds. Likewise, I would like to extend my gratitude towards Ben Blencowe and Sandy Pan for their collaboration on the RNA-seq and alternative splicing analysis portion of this thesis. I would also like to express my gratitude for Nana Lee and her unwavering dedication to my professional development. I wish to thank all past and present members of the Privé lab. While writing has mostly been an isolated experience, they ensured that my time at the bench was filled with lively discussion. In particular I owe much of the work presented here to Liyan Qiu who initiated the RTF2 project and conducted the crystallography and structure determination. I also thank Neil Pomroy for keeping the lab in check and making sure I didn’t break thousands of dollars’ worth of equipment. To Amie, Jess, Kyle, and the extended Anglo-Canadian Zinc Consortium: thank you for giving me life outside of the lab. Your friendship is something that I lean on. The endless jokes about crystallography didn’t make sense to me at first but I’ve learned to accept them. There are not enough words to express the appreciation I have for my parents as well as my grandparents. It is due to the sacrifices they’ve made that I have been able to pursue a fulfilling education in science. My mom, whose hard work and dedication I try to emulate in my own life, iv and my dad, who is to this day the most intelligent person I know. Whatever accomplishments I have are because of them. Finally, to my best friend and partner Rebecca. Every day I thank my lucky stars that I am able to share my life with you. Life after graduate school will bring even more adventures and obstacles, but with you I welcome the challenge with open arms. Also, thanks to my two unlicensed emotional support animals: Levi (left) and Trudy (right) v Table of Contents Chapter 1: Introduction ................................................................................................................. 1 1.1 The discovery of ubiquitin biology ........................................................................................... 2 1.2 Introduction to the ubiquitin system ....................................................................................... 3 1.3 Core components of the ubiquitination cascade ..................................................................... 5 1.3.1 Ubiquitin and ubiquitin-like modifiers .................................................................................................. 6 1.3.2 E1: ubiquitin activating enzymes ......................................................................................................... 10 1.3.3 E2-ubiquitin conjugating enzyme ........................................................................................................ 11 1.3.4 E3 – ubiquitin protein ligases .............................................................................................................. 13 1.4 The RING family of E3 ligases ............................................................................................... 16 1.4.1 Multi-subunit RING E3 ligases ........................................................................................................... 16 1.4.2 RBR E3 ligases .................................................................................................................................... 19 1.4.3 Simple RING E3 ligases ...................................................................................................................... 20 1.5 The RING domain ................................................................................................................... 23 1.5.1 History of the RING domain: From DNA binding to ubiquitin ligase ................................................ 23 1.5.2 Structure of the RING domain ............................................................................................................. 25 1.5.3 U-box and SP-RING domains ............................................................................................................. 27 1.5.4 E2 activation by RING-like E3 ligases ................................................................................................ 30 1.5.5 Oligomerization of RING and RING-like domains ............................................................................. 35 1.6 Introduction to pre-mRNA splicing ....................................................................................... 38 1.6.1 Mechanisms of alternative splicing ..................................................................................................... 42 1.6.2 Emerging roles for RING and RING-like E3 ligases in splicing ......................................................... 44 1.7 RTF2: An uncharacterized RING-like protein ..................................................................... 47 1.7.1 Cellular functions of RTF2 .................................................................................................................. 47 1.7.2 RTF2 is a core essential gene............................................................................................................... 48 1.8 Thesis rationale and overview ................................................................................................ 49 Chapter 2: Crystal structure of RTF2 ......................................................................................... 52 2.1 Abstract .................................................................................................................................... 53 2.2 Introduction ............................................................................................................................. 53 vi 2.3 Materials and methods ............................................................................................................ 56 2.3.1 Cloning, protein expression, and purification ...................................................................................... 56 2.3.2 Crystallization and structure determination ......................................................................................... 56 2.4 Results ....................................................................................................................................... 57 2.4.1 Structure of the RTF2 RING-like domains .........................................................................................