Graduate Theses, Dissertations, and Problem Reports 2015 Enhancement of RNA Splicing by the Nutrient-Regulated Splicing Factor SRSF3 Amanda L. Suchanek Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Suchanek, Amanda L., "Enhancement of RNA Splicing by the Nutrient-Regulated Splicing Factor SRSF3" (2015). Graduate Theses, Dissertations, and Problem Reports. 6740. https://researchrepository.wvu.edu/etd/6740 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Enhancement of RNA Splicing by the Nutrient-Regulated Splicing Factor SRSF3. Amanda L. Suchanek Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry & Molecular Biology Committee Members Lisa Salati, Ph.D., Chair John Hollander, Ph.D. J. Michael Ruppert, M.D., Ph.D. Maxim Sokolov, Ph.D. Yehenew Agazie, Ph.D. Graduate Program in Biochemistry West Virginia University School of Medicine Morgantown, West Virginia 2015 Keywords: SRSF3, G6PD, RNA splicing, hepatocytes, adenovirus, intron retention © 2015 Amanda Suchanek ABSTRACT Enhancement of RNA Splicing by the Nutrient Regulated Splicing Factor, SRSF3 Amanda L. Suchanek Nutritional status is a powerful regulator of intracellular function. Dietary status can increase or decrease the rate of RNA splicing, thereby affecting gene expression; however, few molecular mechanisms have been identified that are responsible for this type of regulation. The glucose-6-phosphate dehydrogenase (G6PD) gene has provided a useful tool for the study of nutrient-regulated splicing because accumulation of G6PD mRNA is dependent solely on changes in the rate of mRNA splicing in response to nutritional stimuli, consistent with its enzymatic role of converting dietary energy to fatty acids. Treatment of primary hepatocytes in culture with insulin or feeding a high-carbohydrate, low fat diet to rodents increases splicing by 7- and 15-fold, respectively, and increases the cellular content of G6PD. In contrast, starvation or treatment of primary hepatocytes in culture with polyunsaturated fatty acids reduces G6PD mRNA splicing by 70% or more. The unspliced RNA is degraded in the nucleus, thus reducing expression of the G6PD enzyme. Our laboratory identified a splicing regulatory element within exon 12 of the G6PD transcript is required for splicing- related changes in G6PD expression in response to nutrient stimuli. This element is bound by two splicing regulatory proteins, SRSF3 and hnRNP K. To understand the mechanism of action of these proteins, we used a loss-of-function approach. SiRNA-mediated depletion of SRSF3 significantly decreased G6PD splicing and expression; in contrast, depletion of hnRNP K enhanced both splicing and mRNA accumulation. Consistent with their apparent roles as enhancers or silencers of splicing, respectively, the results of RNA immunoprecipitation (RIP) indicated binding of SRSF3 to exon 12 was enhanced 6-fold in the livers of refed mice and nearly undetectable in starved mice. Conversely, binding of hnRNP K to the regulatory element was increased 12.5-fold in the livers of starved mice and barely detectable in the livers of refed mice. Furthermore, RNA EMSA using purified SRSF3 and hnRNP K recombinant proteins, we demonstrated that SRSF3 and hnRNP K compete for binding to the same sequences within the regulatory element, which we hypothesize is a bifunctional ESE/ESS element. Thus, mutually exclusive binding of SRSF3 and hnRNP K to this regulatory element mediates the nutrient regulation of G6PD mRNA splicing and establishes a new intracellular mechanism for nutrient regulation of gene expression. We hypothesize that nutrient regulation of splicing is not unique to the G6PD mRNA, and thus the experiments described herein are focused identifying additional genes that are regulated by changes in alternative splicing in response to nutrient availability. ACKNOWLEDGMENTS I would like to thank all of the people who have stood by and supported me through this incredible journey. I’d like to thank my dissertation committee, and especially my advisor, Lisa Salati, for giving me a chance and for giving me the tools to be a successful research scientist. I’d also like to thank Brad Hillgartner, Roberta Leonardi, and Peter Stoilov for their advice and criticism during lab meetings and other presentations that have helped me become a stronger public speaker. I can say without hesitation that I would not have reached this point without the steadfast support of my family and friends. I’d first like to thank my father and mother, Joe & Cindy Suchanek, and my sister, Michelle Navarro, for always encouraging me to pursue my goals, for their emotional support and calming influences during the ups and downs of these last several years. I’d also like to thank my “Pennsylvania family,” especially my uncle Gary and aunt Cathy Robinson, cousins Cristen and Cris Lambert, and David, Tina, Jon, Jessi, and Courtney for their support (and Thanksgiving/Easter dinners!) while I have been pursuing this degree. I would like to thank my former lab mates Holly and Travis Cyphert for their friendship, advice, and our always exciting scientific discussions. I’d like to thank Jess Gibat and Kim Alonge for their friendship and support here in the biochemistry department. Our always- colorful conversations, scientific and otherwise, have gone a long way in keeping me sane. Thank you to my friends Gina Mazzetti, Rosemary and Jerry Munsey, without whom these final months spent finishing this degree would have been next to impossible. Finally, thank you to my other “sisters,” Alisa Elliott, Bahar Mihalcin, and Courtney Newhouse. Their unwavering support, encouragement, friendship, and laughter has kept me going through some of the toughest years of my life. iii TABLE OF CONTENTS LIST OF FIGURES vi. LIST OF ABBREVIATIONS viii. CHAPTER 1: LITERATURE REVIEW 1 I. RNA PROCESSING 1.1 INTRODUCTION 1 1.2 CAPPING 1 1.3 POLYADENYLATION 3 1.4 SPLICING 4 1.5 POST-SPLICING 8 1.6 ADDITIONAL SPLICING REGULATORY SEQUENCES 9 II. TYPES OF SPLICING 2.1 INTRODUCTION 10 2.2 ALTERNATIVE 5’ OR 3’ SPLICE SITE CHOICE 11 2.3 CASSETTE EXON SKIPPING/INCLUSION 15 2.4 INTRON RETENTION 16 III. REGULATION OF SPLICING 3.1 INTRODUCTION 17 3.2 hnRNPs 18 3.3 SR PROTEINS 20 3.4 REGULATION OF SR PROTEINS 22 3.5 SRSF3 AND REGULATION OF G6PD 24 IV. HYPOTHESIS 26 V. REFERENCES 27 CHAPTER 2: SERINE ARGININE SPLICING FACTOR 3 (SRSF3) IS INVOLVED IN ENHANCED SPLICING OF GLUCOSE-6- PHOSPHATE DEHYDROGENASE (G6PD) RNA IN RESPONSE TO NUTRIENTS AND HORMONES IN LIVER 42 CHAPTER 3: CONSTRUCTION AND EVALUATION OF AN ADENOVIRAL VECTOR FOR THE LIVER-SPECIFIC EXPRESSION OF THE SERINE/ARGININE-RICH SPLICING FACTOR, SRSF3 89 iv CHAPTER 4: INTRON RETENTION IS NOT A MAJOR REGULATOR OF HEPATIC GENE EXPRESSION 118 CHAPTER 5: SUMMARY 150 APPENDIX: I. CLIP-SEQ PROTOCOL 153 II. DUAL ISOLATION OF PROTEIN AND RNA FROM CELLS IN CULTURE 161 III. RNA IMMUNOPRECIPITATION PROTOCOL 164 v LIST OF FIGURES CHAPTER 1: Figure 1: mRNA and the spliceosome. 6 Figure 2: Splicing catalysis. 7 Figure 3: Types of splicing. 12-13 Figure 4: The SR protein family. 21 CHAPTER 2: Figure 1. Insulin and arachidonic acid regulate the amount of phosphorylated SR proteins in the nuclei of primary rat hepatocytes. 82 Figure 2. Refeeding increases the binding of phosphorylated SR proteins to the regulatory element of G6PD exon 12 in vivo. 83 Figure 3. SRSF3 and SRSF4 bind to the splicing regulatory element. 84 Figure 4. Purified SRSF3 binds to the regulatory element in exon 12. 85 Figure 5. SiRNA-mediated depletion of SRSF3 reduces the splicing of a G6PD reporter and the endogenous G6PD mRNA. 86 Figure 6. SiRNA-mediated depletion of SRSF3 reduces splicing of G6PD in HeLa cells. 87 Figure 7. SRSF3 specifically binds to the splicing regulatory element in vivo and refeeding enhances the binding of SRSF3 to the splicing regulatory element in mouse liver. 88 CHAPTER 3: Figure 1: Cloning schemes used to construct adenoviral shuttle vectors. 114 Figure 2: CMV-driven FLAG-SRSF3 is highly expressed in HEK293T packaging cells while albumin-driven FLAG-SRSF3 is not. 115 Figure 3: FLAG-SRSF3 is expressed in HepG2 cells infected with albumin FLAG-SRSF3 adenovirus. 115 vi Figure 4: FLAG-SRSF3 is detected in primary rat hepatocytes infected with albumin FLAG-SRSF3 adenovirus. 116 Figure 5: Overexpression of FLAG-SRSF3 regulates mRNA abundance of mRNA targets. 117 CHAPTER 4: Figure 1: Experimental approach. 129 a. Mouse experiment workflow. b. Agarose gel of total RNA collected from whole mouse liver. c. Representative BioAnalyzer traces of total RNA collected from whole mouse liver. Figure 2: qRT-PCR analysis of genes known to respond to starvation and refeeding. 129 Table 1a: Intron Retention Events (sorted