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William Martin Gregor Neuert, Ph.D RNA Recognition by the Histone Demethylase LSD1 and Proteinaceous RNase P: Characterization of the Binding of Highly Structured RNAs by Enzyme Complexes By William Martin Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry February 28, 2018 Nashville, Tennessee Approved: Neil Osheroff, Ph.D. Nicholas Reiter, Ph.D. Martin Egli, Ph.D. David Cortez, Ph.D. Gregor Neuert, Ph.D. ACKNOWLEDGEMENTS I must first acknowledge my mentor Nick Reiter; he has encouraged me at every turn, especially when science was disheartening, and provided constant support while allowing me the flexibility to direct my own research while encouraging the pursuit of a full life outside of research as well. I must also thank the other members of our lab especially Alex Hirschi, who began the LSD1 story and taught me many lessons in the lab. I would also like to acknowledge the members of my thesis committee for providing regular feedback and advice. Vanderbilt has a reputation as an exceptionally collaborative environment for scientists. That reputation is well-deserved; I cannot mention all of the people who selflessly took the time to learn about my research and provide advice, training, materials, the use of equipment, or simply have discussions about my research. However, I would especially like to thank Manny Ascano and his lab for their critical assistance in setting up and performing PAR-CLIP; the Guengerich and York lab for generously allowing the use of their lab space and equipment; Hayes McDonald for his contributions to the mass spectrometry project; the Vickers lab for their assistance with cDNA preparation; Quanhu "Tiger" Sheng for his assistance with bioinformatics; and the Vanderbilt Center for Structural Biology for providing state-of-the art resources and training on those instruments. Numerous organizations made the following work financially possible. They are the National Institute of General Medical Sciences of the NIH through award number R01GM120572 and the Vanderbilt Molecular Biophysics Training Program T32GM008320; the American Heart Association through grant number GRNT20380334; and the Vanderbilt Biochemistry Department. Finally, I am grateful to my parents, Jay and Lezlie, and my wife Rebecca; without their constant support and patience this body of work would not have been possible. ii TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS .............................................................................. ii LIST OF TABLES .................................................................................... iv LIST OF FIGURES .................................................................................... v LIST OF ACRONYMS ............................................................................... vi ABSTRACT ........................................................................................... viii Chapter 1. Introduction ...................................................................................... 1 1.1: Role of Structure in RNA Biology .............................................. 1 1.2: RNase P: An Ancient Ribozyme and Modern Protein Enzyme ........ 4 1.3: TERRA: A Structured RNA at Telomeres .................................... 9 1.4: Epigenetic Regulation of Gene Expression by LSD1 .................. 16 2. Substrate Recognition by Proteinaceous RNase P ................................. 27 2.1 Introduction ......................................................................... 27 2.2: PRORP Activity and pre-tRNA Binding ..................................... 33 2.3: Crystallization Screens ......................................................... 37 2.4 Discussion ............................................................................ 41 2.5 Materials and Methods ........................................................... 43 3. Structure-Specific Recognition of G-Quadruplex RNA by LSD1 ............... 46 3.1 Introduction ......................................................................... 46 3.2: TERRA Forms a Stacked G-Quadruplex in K+ .......................... 49 3.3: LSD1 Recognizes a Stacked G-Quadruplex ............................. 53 3.4: An RNA—Binding Domain in the SWIRM Domain of LSD1 .......... 56 3.5: Discussion........................................................................... 63 3.6: Materials and Methods .......................................................... 67 4. Identification of RNAs Bound by LSD1 and CoREST in Cells ................... 76 4.1: Introduction ........................................................................ 76 4.2: PAR-CLIP Results ................................................................. 79 4.3: Validation of LSD1/CoREST Bound RNAs ................................. 83 4.4: Implications of PAR-CLIP Study ............................................. 84 4.5: Materials and Methods .......................................................... 86 5. Discussion and Conclusions ............................................................... 92 WORKS CITED ..................................................................................... 98 iii LIST OF TABLES Table Page 1. Lysine Demethylase Enzymes............................................................ 22 2. Affinity of LSD1/CoREST for GQ RNA Panel. ......................................... 54 3. Enrichment of Different RNAs in Gene Clusters vs. Reads. ..................... 82 4. G-Quadruplex Enrichment.....................................................................82 iv LIST OF FIGURES Figure Page 1. Structured RNA scaffolds are at the Core of RNP Complexes. ................... 3 2. Structure and Mechanisms of RNase P Enzymes. .................................... 8 3. Telomere T-Loop Blocks the DNA Damage Response. ............................ 11 4. RNA G-Quadruplex Structure. ............................................................ 13 5. Mechanism of LSD1 and JmjC-class Demethylases. ............................... 22 6. Overview of Approach. ..................................................................... 26 7. PRORP3 pre-tRNA Cleavage Assays. ................................................... 34 8. Calcium Inhibits PRORP3 Activity. ....................................................... 36 9. tRNA Design for Crystallographic Studies. ............................................ 39 10.PRORP3 Crystal. ............................................................................... 40 11.Representative PRORP3 Purification. ................................................... 44 12.TERRA RNA recruits LSD1 to deprotected telomeres.............................. 50 13.Monovalent ions dramatically alter the structure of GQ-forming RNAs. .... 52 14.Affinity and specificity of LSD1/CoREST for GQ RNA. ............................. 54 15.Nucleic acid binding specificity of LSD1. .............................................. 57 16.Figure 16: UV Light specifically cross-links LSD1/CoREST to a GQ RNA. 58 17.Identification of the LSD1 GQ RNA binding domain via mass spec. .......... 59 18.Identification of candidate GQ binding regions on LSD1 via XL-MS. ......... 60 19.Purity of LSD1/CoREST constructs prepared for this study. .................... 70 20.PAR-CLIP Theory and Workflow. ......................................................... 78 21.Overlap between genes containing PAR-CLIP clusters. ........................... 81 22.LSD1-CoREST Binds PAR-CLIP Identified GQ RNAs. ............................... 85 23.Induced expression of FLAG-HA LSD1 and Co-IP of CoREST. .................. 89 24.RNA binding by LSD1 and CoREST after Cross-link and IP. .................... 90 v LIST OF ACRONYMS 4SU 4-thiouridine AOD amine oxidase domain ATM ataxia telangiectasia mutated ATR ataxia telangiectasia and Rad3-related CD circular dichroism ChIP chromatin immunoprecipitation CK2 casein kinase 2 COREST corepressor of RE1 silencing transcription factor CRB CREB-binding protein CtBP1 C-terminal binding protein 1 CytB cytochrome B DMS dimethyl sulfate DNMT1 DNA methyltransferase 1 dsDNA double-stranded DNA E2F1 E2F transcription factor 1E2F1 EMSA electromobility shift assay EPHB1 EPH receptor B1 FAD flavin adenine dinucleotide FAM57B family with sequence similarity 57 member B FMRP fragile X mental retardation protein fRIP formaldehyde RNA immunoprecipitation GQ G-quadruplex HDAC histone deacetylase HOTAIR HOX transcript antisense intergenic RNA HOX homeobox HP1 heterochromatin protein HR homologous recombination lncRNA long non-coding RNA LSD1 lysine-specific demethylase-1 LSD2 lysine-specific demethylase-2 MALAT1 metastasis associated lung adenocarcinoma transcript 1 MRE11 MRE11 homolog, double strand break repair nuclease MRN Mre11/Rad50/Nbs1 MRPP1 mitochondrial RNase P protein 1; also, tRNA methyltransferase 10C (TRMT10C) MRPP2 mitochondrial RNase P protein 2; also, hydroxysteroid 17-beta dehydrogenase 10 (HSD17B10) MYO1B myosin 1B MYPT1 myosin phosphatase target subunit 1 NAI 2-methylnicotinic acid imidazolide Nbs1 nibrin ncRNA non-coding RNA ND5 NADH dehydrogenase subunit 5 NEAT1 nuclear paraspeckle assembly transcript 1 vi NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NHEJ non-homologous end joining PAR-CLIP photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation PDB protein data bank pre-tRNA precursor tRNA PPR pentatricopeptide repeat PRC2 polycomb remodeling complex 2 PRORP proteinaceous RNase P protein Rad50
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