The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2016 MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION Niza Nemkul Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Nemkul, Niza, "MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION" (2016). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 646. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/646 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION by Niza Nemkul, B.S. APPROVED: ______________________________ Dr. Leslie Krushel, Ph.D. Advisory Professor ____________________________ Dr. Gilbert Cote, Ph.D. Thesis Chair ______________________________ Dr. William Mattox, Ph.D. ______________________________ Dr. Richard Lloyd, Ph.D. ______________________________ Dr. Sadhan Majumder, Ph.D. ______________________________ Dr. Pierre McCrea, Ph.D. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston i MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Niza Nemkul, B.S. Houston, Texas May,2016 ii This dissertation is dedicated to my family Mom, Dad, Nisha di, and Ojas. iii ACKNOWLEDGEMENTS I am forever grateful to my family, especially my parents and my sister Nisha, for being the most loving family anyone could ask for. I would not have made it here without their love and support. I am also grateful to my dear friend Subi, for being my support system in the US and for keeping me sane through the years. In the graduate school, I would first like to thank Dr. Leslie Krushel for providing me a home for the past five years. I also want to thank Krushel lab members- Bethany Veo for starting the project and helping me through my first few months in the lab, for helping me through the years, and being as excited as me about my data, other lab members– Tara Dobson, Kristopher Veo, Shihuang Su, Roy Voice III, and Juan Chen for making my lab experience a memorable one. Next I would like to thank my advisory committee– Dr. Gilbert Cote, Dr. William Mattox, Dr. Richard Lloyd, Dr. Sadhan Majumder, and Dr. Pierre McCrea for their guidance, critiques, and encouragement. I would especially like to thank Dr. Cote for undertaking the role of my thesis chair, and helping me through the last stretch of my graduate career and getting me through my thesis writing and defense. I really appreciate the amount of effort he put in to understand my work, and I enjoyed the many hours of discussion in his office. I also need to acknowledge Dr. Mattox for his guidance and counseling through some of my difficult times in the graduate school. iv Of course, this journey would be incomplete without my friends here in the graduate school. I would like to thank all my friends in the Genes and Development program, the G&D faculty, and our program manager Elisabeth Lindheim for providing such a supportive environment. A special thanks to my friends -Amanda Haltom, Sangita Pal, Pingping Wang- for celebrating with me in my good days and for getting me through my bad days. I really could not have survived graduate school without their help. I also must thank the Tyler lab and all Tyler lab members- especially Richard, Candice, Hillary, and Reva for letting me be a part of their lab family and for sharing their resources, and their wisdom. I will always appreciate their support. v MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION Niza Nemkul, B.S. Advisory Professor: Dr. Leslie Krushel, Ph.D. The translation of most eukaryotic mRNAs occurs in a cap-dependent manner. However, a subset of mRNAs are capable of initiating translation in a cap-independent manner by utilizing sequences in their 5’ UTR called IRES. It was previously shown that the 5’ UTR of the tau mRNA contains an IRES. In this study I show that IRES dependent translation of tau IRES is regulated at multiple levels in order to regulate the expression of the tau protein. Tau protein is ubiquitously expressed but is concentrated in the brain. In this study, I utilized neural and non-neural cell lines to show that tau IRES is utilized differently (in some cases, up to 50% of total tau translation) depending on the cell type. For many IRES containing mRNAs, IRES activity is enhanced in conditions when cap-dependent translation is shut down, such as during cellular stress and mitosis. In this study, I show that tau IRES activity is upregulated during increased iron, poly (I:C), and extracellular Aβ exposure, which are stress conditions commonly observed in neurodegenerative diseases. Further, I show that tau IRES is differentially regulated by various upstream stimuli through their downstream signaling kinases. However, a comparison of the effect of various signaling pathways on tau and APP IRES suggested that the specific regulation of these IRESes occur downstream of mTOR signaling. vi Most IRESes require binding by certain non-canonical factors called IRES trans acting factors (ITAFs) for internal initiation. ITAFs can be positive or negative, thus enhancing or inhibiting IRES function. Examination of sequences in the tau 5’ UTR led us to analyze four different RNA binding proteins as putative ITAFs for tau. Out of these, I identified two proteins – polypyrimidine tract binding protein (PTB) and neural PTB (nPTB) as inhibitory ITAFs of tau IRES. Altering the expression of PTB and nPTB in vitro and in cells negatively influenced tau IRES activity and protein expression. Along with sequences in the 5’ UTR, the sequences in the 3’ UTR of an mRNA may also affect its translation, either through direct interaction between the RNA sequences, or through interaction by RNA binding proteins. In this study, I show that the tau 3’ UTR enhances IRES-dependent translation of tau, and this interaction requires the entire tau 3’UTR. Overall, I show that the tau IRES is a unique tool utilized by the mRNA to regulate tau protein expression. vii TABLE OF CONTENTS Acknowledgements………………………………………………..………………………….iv Abstract……………………..…………………………………………………………….......vi Table of contents…………………………………………………………..………….…… viii List of illustrations………..………………………………...……………………...………..xiii List of tables…………………………………………..………………………………….….xvi Abbreviations……………………………………………...…………………………......…xvii Chapter I. Introduction………………………………………..……………………………….1 1.1 Microtubule associated protein tau (MAPT)……………………..……………….2 Transcriptional regulation and alternative splicing of tau protein…………..……..3 Regulation of tau by post-translational modifications…………………..…………4 Tau mutations in tauopathies…………………………………………………..…..6 Regulation of tau protein expression…………………………………..…………..7 1.2. Protein synthesis………………………………………………………..………...8 Regulation of cap-dependent initiation by mTOR………………………..……...11 MTOR independent regulation of cap-dependent translation…………..………..14 IRES-dependent translation initiation………………………………..…………..15 Cellular IRES………………………………………………………..…………...18 Defining the eukaryotic IRES…………………………………………..………..20 Regulation of IRES-dependent translation by signaling pathways……..………..22 ITAFs……………………………………………………...………………...…...22 1.3 Polypyrimidine tract binding protein (PTB)………………………...…………...24 Role of PTB in splicing…………………………………………..……………...25 viii Role of PTB in RNA transport and stability………………………..……………26 Role of PTB as an ITAF………………………………………………..………..27 1.4 Scope of the study………………………………………………………………..29 Chapter II. Materials and Methods………………………………………………..…………30 2.1 Constructs and cloning……………………………….…………………………..31 2.2 Cell Culture………………………………………………………………………35 2.3 In vitro transcription…………………………………………………………..…36 2.4 Cell treatments…………………………………………………………………...36 2.5 In vitro Translation………………………………………………………………39 2.6 Dual luciferase assay..……………………………………………………………40 2.7 Lentiviral Transduction…………………………………………………………..40 2.8 siRNA treatment………..………………………………………………………..41 2.9 Western Blot……………………………………………………………………..41 2.10 Polysome Analysis………………………………………………..…………….43 2.11 RNA extraction and Quantitative Reverse Transcription-PCR…………..…….44 2.12 RNA immunoprecipitation assay………………………………..……………...45 2.13 siRNA Human Kinase library screen………………………………..………….45 2.14 Statistical analysis………………………………………………..……………..46 Chapter III. Regulation of tau IRES: stress, kinases, and pathways………..……..…………47 3.1 Development of assay…………………………………………………..………..48 3.1A Tau IRES is differentially