TRANSCRIPTOME-WIDE INVESTIGATION OF NUCLEAR RNA-BINDING PROTEINS by ERIC NGUYEN B.A., B.S., University of Washington, 2009 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2017 This thesis for the Doctor of Philosophy degree by Eric Nguyen has been approved for the Molecular Biology Program by Arthur Gutierrez-Hartmann, Chair Anthony Gerber Thomas Evans Patricia Ernst Matthew Taylor Aaron Johnson, Advisor Date: December 15th, 2017 ii Nguyen, Eric (PhD, Molecular Biology Program) Transcriptome-Wide Investigation of Nuclear RNA-Binding Proteins Thesis directed by Assistant Professor Aaron M. Johnson ABSTRACT RNA-binding proteins play a number of important roles throughout the cell. In order to more closely investigate their activity, we have adapted high-throughput techniques to characterize their activity across the transcriptome. We have previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 as a potential adaptor protein for interactions between the chromatin silencing complex PRC2 and the RNA HOTAIR. We used enhanced cross- linking immunoprecipitation (eCLIP) to map the complete set of direct interactions between hnRNP A2/B1 and RNA in breast cancer cells. Surprisingly, a strong A2/B1 binding site occurs in the third intron of HOTAIR, which interrupts a known RNA- RNA interaction hotspot and is retained at a higher frequency than other HOTAIR introns. In vitro eCLIP experiments suggest that A2/B1 may redistribute to exonic binding sites once this intron is spliced. A2/B1 associates with multiple lncRNAs at regions that may contribute to regulation. Finally, we performed cellular fractionation to characterize the pattern of RNA association of A2/B1 in chromatin, nucleoplasm, and cytoplasm. We also examined the potential relevance of hnRNP A2/B1 in myogenesis. As A2/B1 has been associated with a number of muscle diseases, we performed eCLIP on A2/B1 in both undifferentiated mouse myoblasts and differentiated myotubes. We found that A2/B1 binds the 3′ UTR of transcripts in differentiated cells, and that these transcripts tended to be protein-coding. We also performed eCLIP on another iii protein TDP-43, that has been shown to directly interact with A2/B1 and be dysregulated in many of the same diseases. This experiment identified a number of exonic binding sites in myogenesis-associated transcripts, indicative of a role for TDP-43 in the nuclear export of long RNAs during myogenesis. Comparison of A2/B1 and TDP-43-bound transcripts shows some overlap, suggesting that they may act cooperatively in RNA regulation during myogenesis. Finally, we developed a novel method to investigate heterochromatin- associated RNA called hmRIP-seq, which was designed to differentiate between RNA-enzyme interactions leading to heterochromatin formation, and those that do not. This method identified a potential heterochromatin-interacting noncoding RNA, MTRNR2L12, that may direct silencing towards repetitive elements with similar sequence. The form and content of this abstract are approved. I recommend its publication. Approved: Aaron M. Johnson iv ACKNOWLEDGEMENTS This work would not have been possible without the assistance of many people. First and foremost, I would like to thanks my advisor, Aaron M. Johnson, for his invaluable advice and persistence in keeping the following projects on track as each one progressed. His copious comments on every paper, poster, and abstract were invariably helpful. I would also like to thank the graduate students with whom I have worked for the past four years, Alexis Zukowski and Maggie Balas, for their conversations and commiserations about life in the lab, and former lab members Emily Meredith and Karly Sindy for their invaluable mentorship and advice. Successful scientific endeavors rarely happen without outside help. For assistance with the eCLIP protocol I would like to thanks Gabriel Pratt and Eric van Nostrand, a chance conference encounter that became a lifeline for establishing their protocol in our lab. For assistance in imagining new frontiers for eCLIP I would like to thank Josh Wheeler and Tom Vogler for having the foresight to see how to include my work in their story. I would like to thank my thesis committee for their advice: Arthur Gutierrez- Hartmann, Tom Evans, Anthony Gerber, Patricia Ernst, and Matt Taylor. I would also like to acknowledge to contributions of former committee members Tobias Neff and David Bentley. I would like to thank the members of the Biochemistry and Molecular Genetics Department and Molecular Biology Program who have generously provided assistance: Nova Fong, Ryan Sheridan, Kerri York, and Monica Ransom for research v assistance; Sue Brozowski and Annie Vazquez for help navigating departmental bureaucracy; and Sabrena Heilman, Michele Hwozdyk-Parsons, and Bob Sclafani for running the Molecular Biology Program. I would like to thank the Medical Scientist Training Program for their continued support: Arthur Gutierrez-Hartmann and Angie Ribera for heading the program; Jodi Cropper, Emily Thomas, and Katie Bidus for administrative assistance; Sally Peach, Laura Hancock, Greg Kirkpatrick, Tamara Garcia, Ariel Hernandez, and Leon Zheng, the classmates with whom I entered this program six years ago; and Jingjing Zhang, Tom Vogler, Josh Wheeler, Dan Youmans, Matt Becker, Kelly Higa, Taylor Soderborg, Sarah Haeger, Jason Silver, and Mindy Szeto for their continued willingness to adventure around Denver. I would like to thank my previous mentors for helping to guide me to this career path: Bertil Hille, Willie Swanson, Pat Navas, and Jay Hesselberth. I would like to thank my parents, Ann and Toan Nguyen, for their continued advice and support, as well as my brother, Grant Nguyen. Last but not least, I would like to thank Charlotte Siska for being there for me at any time of day or night. vi TABLE OF CONTENTS CHAPTER I. INTRODUCTION ...................................................................................................... 1 Chromatin Biology .................................................................................................... 1 Epigenetic Modifications Have an Effect on Gene Regulation .......................... 1 Polycomb Proteins Form Gene Silencing Complexes ........................................ 3 PRC2 Activity is Modulated by Protein and RNA Cofactors .............................. 5 PRC2 Can Bind to Many RNAs .......................................................................... 6 PRC2 and Disease ............................................................................................... 8 Long Noncoding RNAs and Their Effects on Chromatin ........................................ 8 Long Noncoding RNAs: A New Class of RNA .................................................... 8 lncRNAs Can Bind to Specific Sites in the Genome ......................................... 10 Many lncRNAs Can Bind to PRC2 .................................................................... 12 PRC2-lncRNA Interactions are Implicated in Disease ..................................... 12 Many Proteins Bind lncRNAs and Affect Chromatin State .............................. 14 hnRNP A2/B1: A Multi-Faceted Protein ................................................................ 15 hnRNPs Comprise a Diverse Family of Nuclear Proteins ................................ 15 The Structure of hnRNP A2/B1 ......................................................................... 16 The Potential Roles of hnRNP A2/B1 ............................................................... 17 hnRNP B1 Binds HOTAIR and Its Targets with Specificity ............................. 19 hnRNP B1 May Act as an RNA Matchmaker on Chromatin ............................ 19 hnRNP A2/B1 in Disease ................................................................................. 20 Functional Interactions between RNA-Binding Proteins ...................................... 21 vii TDP-43 is a Splicing Regulator that Interacts with hnRNP A2/B1 .................. 21 Cytoplasmic TDP-43 and hnRNP A2/B1 can be Pathologic ............................ 22 Scope of Thesis ................................................................................................. 23 II. THE RNA INTERACTOME OF HNRNP A2/B1 ................................................... 36 Introduction ........................................................................................................... 36 Materials and Methods ........................................................................................... 37 Lessons Learned from HITS-CLIP and iCLIP Methods ................................... 37 The Enhanced CLIP (eCLIP) Method .............................................................. 38 Computational Analysis of eCLIP-seq Samples ................................................ 41 In vitro eCLIP ................................................................................................... 42 Cellular Fractionation of MCF7 Cells ............................................................... 43 RNA Isolation and PCR .................................................................................... 43 Results .................................................................................................................... 43 The hnRNP B1 Exon is Well-Conserved and Expressed in Mouse and Human .........................................................................................................................