IDENTIFYING ENDOGENOUS BINDING PARTNERS FOR BTF AND TRAP150 A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By JAYLEN BRAXTON HUDSON B.S. UNIVERSITY OF DAYTON, 2018 2020 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL April 28, 2020 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Jaylen Braxton Hudson ENTITLED Identifying Endogenous Binding Partners for Btf and TRAP150 BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ________________________________ Paula A. Bubulya, Ph.D. Thesis Director ________________________________ David L. Goldstein, Ph.D. Chair, Department of Biological Sciences Committee on Final Examination: ________________________________ Quan Zhong, Ph.D. ________________________________ Labib Rouhana, Ph.D. ________________________________ Barry Milligan, Ph.D. Interim Dean of the Graduate School ABSTRACT Hudson, Jaylen Braxton. M.S. Department of Biological Sciences, Wright State University, 2020. Identifying Endogenous Binding Partners of Btf and TRAP150. Since being discovered in the early 1990s, the primary functions of classical serine- arginine rich (SR) proteins and non-classical SR-related proteins have been demonstrated in gene regulatory processes such as pre-mRNA processing, mRNA metabolism, and nuclear export of mRNAs. Bcl-2-associated transcription factor 1 (BCLAF1, also called Btf) and Thyroid hormone receptor associated protein 3 (THRAP3, also called TRAP150) are homologous non-classical SR-like splicing factors. In this thesis, I have identified endogenous binding partners of Btf and TRAP150. I used statistical analysis to select common versus distinct protein partners and compared previously documented subcellular localization of these identified proteins with studies for Btf/TRAP150. Previous studies indicate that Btf and TRAP150 have overlapping roles in maintaining cell cycle progression and DNA damage repair, but different roles in regulating subcellular mRNA distribution. Therefore, I hypothesized that Btf and TRAP150 interact with a common set of protein partners for pathways in which they share functions yet have distinct protein partners that impart unique functions. MS-based proteomic analysis of immunoprecipitated Btf and TRAP150 complexes revealed novel interactions with FXR1P and FXR2P, autosomal paralogs of Fragile X mental retardation protein (FMRP). FXR1P is a common partner for both Btf and TRAP150 whereas FXR2P is distinct to TRAP150. Future studies will confirm interaction between these binding partners as well as others from the analysis. Also, siRNA depletion studies will investigate the mechanisms for how Btf/TRAP150 impact subcellular distribution and function of Fragile X proteins. iii Table of Contents CHAPTER 1: SIGNIFICANCE AND BACKGROUND………………………………….…..1 1.2 Nuclear speckles…………………………………………………….…..14 1.3 SR and SR-related proteins…………………………………...……….19 1.4 Btf and TRAP150…………………………………………………………19 1.5 Subcellular mRNA distribution………………………………..………23 1.6 Regulation of DNA Damage Repair transcripts………..……..……24 CHAPTER 2: HYPOTHESIS AND EXPERIMENTAL AIMS……………………………..27 2.1 Specific Aims…………………………………………………….……….27 CHAPTER 3: MATERIALS AND METHODS…………………………………..………….31 3.1 Thawing/Freezing Cells………………………………………...……....31 3.2 Cell Culture…………………………………………………….………….31 3.3 Plating Cells………………………………………………………………32 3.4 Whole Cell Nuclear Extraction (WCNE) preparation ……...………32 3.5 Protein Quantification (Bradford Assay)………………….…………33 3.6 SDS-PAGE and Immunoblotting………………………………………33 3.7 Immunoprecipitation……………………………….……………………35 3.8 Silver Staining of SDS-PAGE gels……………………………………36 3.9 Mass Spectrometry and Proteomics…………….…………………...37 3.10 Immunofluorescence…………………………….…………………….37 iv CHAPTER 4: RESULTS OF IMMUNOPRECIPITATION AND MASS SPECTROMETRY…………………………………………………………………………….39 4.1 Isolation of Btf and TRAP150 complexes by Immunoprecipitation (IP)…………..............................................................................................39 4.2 Using silver staining to detect endogenous binding partners…..47 4.3 Mass spectrometry results and data analysis…………………...…54 CHAPTER 5: DISCUSSION………………………………………………………………….80 REFERENCES………………………………………………………………………….……..87 ABBREVIATIONS……………………………………………………………………..………95 v List of Figures Figure 1. A schematic diagram of co/post-transcriptional regulation of mRNAs….4 Figure 2: Pre-mRNA splicing in the absence of an SR protein RS domain…………8 Figure 3: Regulation of post-transcriptional gene expression and protein-protein interaction by the EJC……………………………………………………………………….12 Figure 4: A schematic diagram of nuclear domains in the mammalian cell nucleus………………………………………………………………………………………...15 Figure 5: Diversity of gene expression from the regulation of nuclear speckle proteins………………………………………………………………………………………...17 Figure 6: Alignment data for Btf and TRAP150 showing sequence similarity……21 Figure 7: Immunoblot images for Btf and TRAP150 IPs (Replicate #1)………….…41 Figure 8: Immunoblot images for Btf and TRAP150 IPs (Replicate #2)…………….43 Figure 9: Immunoblot images for Btf and TRAP150 IPs (Replicate #3)………….…45 Figure 10: Silver stain image for Btf/TRAP150 (Replicate #1)………………....…….48 Figure 11: Silver stain images for Btf/TRAP150 (Replicate #2)………………………50 Figure 12: Silver stain images for Btf/TRAP150 (Replicate #3)………………….…..52 Figure 13: Linear regression analysis for Btf binding partners...…….……………..74 vi Figure 14: Linear regression analysis for TRAP150 binding partners……………..77 vii List of Tables Table 1: Category description for variables in MS-based proteomics analysis.…55 Table 2: Quantification of identified endogenous binding partners for Btf and TRAP150……………………………………………………………………………….………58 Table 3: Common binding partners for Btf and TRAP150 w/ unique biological function………………………………………………………………………………………...62 Table 4: Biological pathways associated with Btf binding partners..……….……..65 Table 5: Biological pathways associated with TRAP150 binding partners………..68 Table 6: Correlation between variables for Btf and TRAP150………………………..72 viii ACKNOWLEDGMENTS I would like to recognize and express sincere gratitude to my advisor, Dr. Paula Bubulya, for her supervision of my research. Her constant support, guidance, and encouragement in the past two years has helped me grow as a graduate student in many aspects. I would also like to thank my Thesis committee consisting of Dr. Quan Zhong and Dr. Labib Rouhana. Their insight as professors in Molecular Genetics have allowed me to develop important skills during my study. In addition, I am thankful for the students in the Bubulya lab, Melissa Ward, Jacob Ward, and Rawan H. Alqahtani for our productive discussions during weekly meetings. I appreciate the strong relationships that we were able to build during my time at Wright State University. I greatly appreciate The Ohio State University for accepting me into their Molecular, Cellular, and Developmental Biology Program. I look forward to doing future research along with their professors and Ph. D. students. Lastly, I would like to thank my parents, Beverly Hudson and Tyrone Stepter for showing support, as well as my brother James Hudson. I am grateful for all of my loved ones, especially Sinclaire Smith for her belief in my life goals. This study was supported by NIH Award grant 2R15GM084407-03 and -04. The Bubulya lab would like to thank the Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility at The Ohio State University. Dr. Liwen Zhang provided valuable contributions to our endogenous binding partner study. The Fusion Orbitrap Instrument at OSU was supported by NIH Award Grant S10 OD018056. The lab also thanks Dr. Volker Bahn at Wright State University for aiding with R statistical analysis during the study. Gene Ontology studies were supported by grant U41 HG002273 from the National Human Genome Research Institute. ix CHAPTER 1: SIGNIFICANCE AND BACKGROUND 1.1 INTRODUCTION Gene expression is a fundamental multistep process that regulates all aspects of the eukaryotic cell. The final gene products, proteins and RNA, are essential for maintaining cell structure, function, and homeostasis. The events of gene expression are represented in the mRNA production cycle which consists of transcription, pre-mRNA splicing, mRNA export, translation, mRNA stability, and mRNA decay (Martinez, 2018). While transcription is the most highly regulated step during RNA processing, co-transcriptional events such as pre-mRNA splicing have the potential to affect biological processes outside of gene expression (Li, 2006). Although early studies suggest that the stages of gene expression are independent, genomic analyses confirm a functional connection and coordination between events (Komili, 2008). Several transcription factors and RNA processing factors are coupled through similar binding patterns that help recruit RNA polymerase II (Komili, 2008; White, 2010). Before nuclear export, the mRNA transcript undergoes 5’ capping, splicing, and 3’polyadenylation modifications coordinated by the carboxy terminal domain (CTD) of RNA polymerase II (Figure 1; Ramirez-Clavijo, 2013). After mRNA processing, the mature molecule is exported from the nucleus to the cytoplasm where it is impacted by other regulatory pathways including mRNA decay/stabilization, mRNA localization, and mRNA translation (Figure 1; Le Hir et al., 2001; Woodward, 2017; Martinez, 2018). The CTD of RNA polymerase II is a hypophosphorylated complex