UC San Diego UC San Diego Electronic Theses and Dissertations Title Mechanisms of U6 small nuclear RNA gene expression in Drosophila melanogaster Permalink https://escholarship.org/uc/item/89r1v4c0 Author Verma, Neha Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO SAN DIEGO STATE UNIVERSITY Mechanisms of U6 small nuclear RNA gene expression in Drosophila melanogaster A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Neha Verma Committee in Charge: University of California, San Diego Professor Randolph Y. Hampton Professor Katherine A. Jones San Diego State University Professor William E. Stumph, Chair Professor Sanford I. Bernstein Professor Terrence Frey 2017 The Dissertation of Neha Verma is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ _________________________________________________________________ Chair University of California, San Diego San Diego State University 2017 iii DEDICATION This dissertation is dedicated to my parents, my husband, my in-laws, the rest of my family and my loving friends for their incredible support, continuous encouragement, and endless love. iv TABLE OF CONTENTS Signature Page ........................................................................................................ iii Dedication Page ...................................................................................................... iv Table of Contents ................................................................................................... v List of Figures ......................................................................................................... vii Acknowledgements ................................................................................................ ix Vita ......................................................................................................................... xi Abstract of the Dissertation .................................................................................... xiv General Introduction ............................................................................................... 1 A: References ............................................................................................ 13 Chapter 1 Differential utilization of TATA box-binding protein (TBP) and TBP- related factor 1 (TRF1) at different classes of RNA polymerase III promoters ... 18 A: Abstract .......................................................................................... 19 B: Introduction .................................................................................... 19 C: Materials and Methods ................................................................... 20 D: Results ............................................................................................ 21 E: Discussion ....................................................................................... 24 F: References ....................................................................................... 25 Chapter 2 SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter ........................................................ 27 A: Introduction .................................................................................... 28 B: Materials and Methods ................................................................... 31 C: Results ............................................................................................ 34 D: Discussion ...................................................................................... 45 E: References ....................................................................................... 48 v Chapter 3 Mapping a region of SNAPc responsible for recruitment of Bdp1 to the . Drosophila melanogaster U6 snRNA gene promoter ............................... 53 A: Introduction .................................................................................... 54 B: Materials and Methods ................................................................... 57 C: Results and Discussion ................................................................... 59 D: Refernces ........................................................................................ 61 Concluding Remarks .............................................................................................. 63 A: References ........................................................................................... 70 Appendix ................................................................................................................ 72 A: Detailed protocol for chromatin immunoprecipitation assay (ChIP) ... 73 B: Detailed protocol for in vitro transcription assay ................................. 85 C: Detailed protocol for immune-depletion assay .................................... 100 D: Detailed protocol for nickel-chelate chromatography ......................... 106 E: Detailed protocol for electrophoretic mobility shift assay (EMSA) .... 115 vi LIST OF FIGURES General Introduction Figure 1: Schematic representation of cis-acting elements in the 5’- flanking DNA of a variety of snRNA genes ............................................................. 2 Figure 2: Comparison of different classes of D. melanogaster snRNA gene promoters .................................................................................................... 4 Figure 3: Working model for RNA polymerase specificity at U1 (A) and U6 (B) snRNA promoters ....................................................................................... 7 Chapter 1 Figure 1: ChIPs indicating that D. melanogaster U6 snRNA gene promoters are occupied by TBP in vivo .............................................................................. 21 Figure 2: U6 snRNA transcription in vitro utilizes TBP ............................ 23 Figure 3: Manipulation of TBP and TRF1 levels in cells by overexpression (A) and by RNAi knockdown (B) ...................................................................... 24 Chapter 2 Figure 1: Assembly of DmSNAPc with individual subunits of TFIIIB on the U6 snRNA gene promoter ................................................................................. 35 Figure 2: A region of Bdp1 C-terminal of the SANT domain is required for its recruitment by DmSNAPc ........................................................................... 37 Figure 3: A Pol III-specific PSEA is necessary and sufficient for Bdp1 recruitment by DmSNAPc ........................................................................... 39 Figure 4: The DmSNAPc-Bdp1 complex efficiently recruits TBP to the U6 promoter .................................................................................................... 42 Figure 5: A TATA box is required for recruitment of TBP to the U6 promoter by DmSNAPc-Bdp1 ......................................................................................... 43 vii Figure 6: Mapping a region of Bdp1 that participates in the recruitment of TBP by the DmSNAPc-Bdp1 complex on U6 promoter DNA ........................... 44 Figure 7: Bdp1 is required, together with DmSNAPc bound to a Pol III-specific PSEA and TBP bound to a TATA box, to form a stable protein-DNA complex on the bipartite Drosophila U6 snRNA gene promoter ............................... 46 Chapter 3 Figure 1: Schematic representation of various DmSNAP43 constructs ...... 56 Figure 2: Mapping a region of DmSNAPc involved in interaction with Bdp1 on U6 promoter DNA ....................................................................................... 58 Concluding Remarks Figure 1: Schematic representation of transcription requirements at different classes of RNA polymerase III promoters ................................................... 66 viii ACKNOWLEDGEMENTS First of all, I would like to thank my thesis advisor Dr. Stumph for his mentorship, guidance, motivation and immense support for the years I have been in his lab. I learnt a lot and grew as a scientist under his leadership and could not have imagined a better advisor for my PhD. Besides my advisor, I would also like to thank Dr. Bernstein, Dr. Frey, Dr. Hampton and Dr. Jones for serving on my dissertation committee and giving me invaluable suggestions on my projects to make this thesis possible. I am grateful to all my past and current lab mates at San Diego State University for their help and encouragement that facilitated the accomplishment of my projects and made this experience so much fun. I thank Ko-Hsuan Hung and Jin Joo Kang for their contributions to the publication that constitutes Chapter 1 of this thesis. I am also grateful to Ann Marie Hurlburt, Yoon Soon Kang, Phuc Phan, and Angela Wolfe for their assistance in preparation of Bdp1 truncation constructs and performing nickel- chelate chromatography and EMSAs for Chapter 2 of this thesis. I would like to thank my parents for giving birth to me in the first place and also for supporting me throughout this journey. I am also deeply grateful to my husband,