Depletion of the RNA Helicase SKIV2L2 Impedes Mitotic Progression and Histone Mrna Turnover in Murine Cell Lines Alexis Marie Onderak Marquette University
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Marquette University e-Publications@Marquette Dissertations (2009 -) Dissertations, Theses, and Professional Projects Depletion of the RNA Helicase SKIV2L2 Impedes Mitotic Progression and Histone mRNA Turnover in Murine Cell Lines Alexis Marie Onderak Marquette University Recommended Citation Onderak, Alexis Marie, "Depletion of the RNA Helicase SKIV2L2 Impedes Mitotic Progression and Histone mRNA Turnover in Murine Cell Lines" (2016). Dissertations (2009 -). 679. https://epublications.marquette.edu/dissertations_mu/679 DEPLETION OF THE RNA HELICASE SKIV2L2 IMPEDES MITOTIC PROGRESSION AND HISTONE MRNA TURNOVER IN MURINE CELL LINES By Alexis M. Onderak B.S. 2012, Marquette University A Dissertation submitted to the Faculty of the Graduate School, Marquette University, in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Milwaukee, Wisconsin December 2016 ABSTRACT DEPLETION OF THE RNA HELICASE SKIV2L2 IMPEDES MITOTIC PROGRESSION AND HISTONE MRNA TURNOVER IN MURINE CELL LINES Alexis M. Onderak Marquette University, 2016 RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This dissertation aimed to characterize the phenotype associated with RNAi knockdown of SKIV2L2 in two murine cancer cell lines: Neuro2A and P19. Skiv2l2 knockdown in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation. Analysis of the cell cycle revealed defective progression through mitosis following SKIV2L2 depletion. These results indicate that SKIV2L2 enhances mitotic progression, thereby maintaining cancer cell proliferation and preventing differentiation. Indeed, SKIV2L2 levels were found to be downregulated during chemically induced differentiation, further implicating SKIV2L2 in maintaining proliferation and multipotency in cancer cell lines. Because SKIV2L2 targets RNAs for processing and degradation via the nuclear exosome, it was hypothesized that with SKIV2L2 depletion, the accumulation of some RNA target triggers mitotic arrest. In search of such a target, RNA-seq was utilized to identify RNAs that were elevated in Skiv2l2 knockdown cells. SKIV2L2 depletion resulted in the accumulation of non-coding RNAs, intergenic RNAs, ribosomal protein mRNAs, and replication-dependent histone mRNAs. Given that the regulation of histone mRNAs is tightly linked to the cell cycle, further experiments were conducted to confirm SKIV2L2 targets histone mRNAs for degradation. RNA immunoprecipitation demonstrated direct binding between SKIV2L2 and histone mRNAs, and RNA degradation assays showed that the half-life of histone mRNAs doubles with SKIV2L2 depletion. The resulting histone imbalance following loss of SKIV2L2-directed RNA surveillance could impede mitotic progression, resulting in mitotic defects and indirectly triggering differentiation i ACKNOWLEDGMENTS Alexis M. Onderak I would like to first and foremost thank my advisor, Dr. Anderson, for his guidance and support throughout my dissertation research. He played such an important role in helping me develop as a scientist. I appreciated all his help and patience along the way. During my time in the Anderson laboratory, I lost both my grandfather and godfather, and I am grateful for Dr. Anderson’s compassion during those tough times. I would also like to thank my committee members: Dr. Abbott, Dr. Maki, Dr. Petrella, and Dr. Schlappi. I thoroughly enjoyed learning from each one in the classroom, and I appreciated their criticisms and suggestions for my research. Each committee member pushed me to see things in a different light and more importantly instilled in me a great enthusiasm for biology, for which I am very grateful. My research would not have been possible without the support of my fellow members of the Anderson laboratory: Dr. Jane Dorweiler, Yan Li, and Fengchao Wang. They helped me navigate and learn about both benchwork and genomic data analysis. Also, much gratitude is extended to the undergraduates Ryan O’Toole and Oleg Nagorny. I would also like to thank the Hristova laboratory members for technical support and use of their qPCR machine, the Schlappi laboratory for use of the UVP imaging system, the Yang laboratory for instruction on use of their GFP microscope, and the Petrella laboratory for help with the inverted microscope. I am also appreciative of all the faculty, students, and staff that not only help the Marquette Biological Sciences Department run smoothly but also make it an enjoyable workplace. Finally, I would like to thank my family and friends for their never-ending love and support. My family served as my rock throughout graduate school, always willing to listen and offer reassurance, and I am thankful to be surrounded by such kind and loving individuals. ii TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………….....vii LIST OF FIGURES………………..……………………………………………………viii LIST OF ABBREVIATIONS………………………………………………………...…xii CHAPTER I: INTRODUCTION 1.1 RNA surveillance…………………………..………………………………….1 1.2 The nuclear TRAMP complex………………………………………………...3 1.3 SKIV2L2 functions as a helicase in RNA decay pathways…………………...7 1.4 Stem cells are maintained through a variety of mechanisms …………………9 1.5 The potential role of SKIV2L2 in development……………………………..12 1.6 Aims and Hypotheses………………………………………………………..14 1.7 Summary of Findings…………………………..…………………………….15 1.8 Significance of Findings……………………………………………………..16 CHAPTER II: DIFFERENTIATION OF MURINE CELL LINES IS ASSOCIATED WITH DOWNREGULATION OF SKIV2L2 EXPRESSION 2.1 Introduction………………………………………………………………......18 2.2 Approach and Results…...…………………………………………………...21 2.2.1 N2a and P19 cells differentiate into cholinergic neurons and cardiomyocytes following chemical treatment…………………………..25 2.2.2 Skiv2l2 is downregulated during chemically induced differentiation of N2a and P19 cells……………………………………………………..30 2.2.3 Serum starvation is sufficient to downregulate Skiv2l2 in N2a cells.…………………………………..………………………………….34 2.2.4 Skiv2l2 downregulation during N2a cell differentiation does not occur due to post-transcriptional regulation via Skiv2l2’s 3'UTR……….36 iii 2.2.5 Multiple signaling cascades could indirectly or directly regulate Skiv2l2 expression.…………………………………………………….…40 2.3 Discussion…………………………………………………………...……….45 CHAPTER III: KNOCKDOWN OF SKIV2L2 VIA RNAI ENHANCES P19 CELL AND N2A CELL DIFFERENTIATION 3.1 Introduction…………………………………………………………………..48 3.2 Approach and Results………………………………………………………..50 3.2.1 RNAi knockdown reduces SKIV2L2 levels to negligible amounts in N2a and P19 cells………………………………………………………..50 3.2.2 SKIV2L2 depletion enhances morphological changes consistent with differentiation ……………………………………………………………53 3.2.3 Gene expression changes in Skiv2l2 knockdown cells reflect increased differentiation………………………………………………….56 3.2.4 SKIV2L2 depletion does not restrict differentiation to one cell type………………………………………………………………………61 3.2.5 Differentiation cannot be directly attributed to the loss of the TRAMP or NEXT complexes……………………………………………65 3.3 Discussion……………………………………………………………………72 CHAPTER IV: SKIV2L2 DEPLETION INDUCES A SLIGHT BUT SIGNIFICANT MITOTIC ARREST IN MURINE CELL LINES 4.1 Introduction…………………………………………………………………..76 4.2 Approach and Results………………………………………………………..79 4.2.1 SKIV2L2 depletion reduces the total number of proliferating cells by approximately 30%………………………………………………………79 4.2.2 Skiv2l2 knockdown does not induce cell death in cancer cell lines………………………………………………………………………83 4.2.3 SKIV2L2 depletion triggers a modest but statistically significant increase in G2/M phase cells…………………………………………….87 4.2.4 Skiv2l2 knockdown results in mitotic arrest as demonstrated through increased H3 phospho-S10 levels………………………………………..90 iv 4.2.5 Binuclear cells were detected following SKIV2L2 depletion……..94 4.2.6 Cell cycle genes were dysregulated following Skiv2l2 knockdown……………………………………………………………….97 4.3 Discussion……………………………………………………………………98 CHAPTER V: SKIV2L2 TARGETS SPECIFIC NON-CODING RNAS FOR PROCESSING AND DEGRADATION 5.1 Introduction…………………………………………………………………103 5.2 Approach and Results………………………………………………………109 5.2.1 TRAMP complex RNA targets possess short 3' tails consisting of three to six adenosines………...…………………………………..........109 5.2.2 The 5' leader of mir-322 is a definitive TRAMP target…………..114 5.2.3 SKIV2L2 is necessary for processing snoRNA Z18 from the long non-coding RNA Gas5………………………………………………….115 5.2.4 Forty seven non-coding RNAs accumulate in P19 cells following SKIV2L2 depletion……………………………………………………..119 5.2.5 SKIV2L2 depletion results in the accumulation of transcripts expressed from intergenic regions……………………………………...131 5.2.6 SKIV2L2 depletion impacts the steady state levels of certain mRNAs…………………………………………………………………137 5.3 Discussion…………………………………………………………………..141 CHAPTER VI: SKIV2L2 TARGETS HISTONE MRNAS FOR DEGRADATION IN THE NUCLEUS 6.1 Introduction…………………………………………………………………146 6.2 Approach and Results………………………………………………………150 6.2.1 Histone mRNAs H1, H2A, H3, and H4 accumulate in P19 and N2a cells following SKIV2L2 depletion…………………………………….150 6.2.2 Histone mRNAs accumulate slightly in Papd5 knockdown cells but not in Zcchc8 knockdown cells……………………………………..…..158 v 6.2.3 Histone mRNA accumulation with SKIV2L2 depletion correlates to an increase in at least one histone protein………………………………160 6.2.4 Histone accumulation with SKIV2L2 depletion may impact chromatin compaction…………………………………………………..162 6.2.5 Histone mRNAs are detected among RNAs bound to the