Promoter directionality is controlled by U1 snRNP and polyadenylation signals in mouse embryonic stem cells By Albert E. Almada B.S. Biological Sciences University of California at Irvine, 2007 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY IN BIOLOGY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY © 2013 Massachusetts Institute of Technology All rights reserved September 2013 Signature of Author……………………………………………………………................................ Albert E. Almada Department of Biology August 29, 2013 Certified by………………………………………………………………………………………… Phillip A. Sharp Institute Professor of Biology Accepted by……………………………………………………………………………………....... Stephen P. Bell Professor of Biology Chairman, Biology Graduate Committee 1 Promoter directionality is controlled by U1 snRNP and polyadenylation signals in mouse embryonic stem cells By Albert E. Almada Submitted to the Department of Biology on August 29, 2013 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology Abstract RNA polymerase II (RNAPII) transcription is a tightly regulated process controlling cell type and state. Advancements in our understanding of how transcription is regulated will provide insight into the mechanisms controlling cell identity, cellular differentiation, and its misregulation in disease. It was generally presumed that RNAPII transcribed in a unidirectional manner to produce a coding mRNA. However, RNAPII has recently been found to initiate transcription upstream and antisense from active gene promoters in mammals and yeast. Although RNAPII initiates divergently from these promoters, efficient RNAPII elongation leading to the production of a full-length, stable, abundant RNA molecule is confined to the coding sense direction. These data suggest an unknown mechanism to suppress transcription from the upstream antisense region of divergent promoters. In Chapter 2, we describe an analysis of uaRNA at a candidate set of divergent promoters in mouse embryonic stem cells (mESCs). We reveal that upstream antisense RNAs (uaRNAs) are less than 1 kb in size, 5’-capped, heterogeneous at their 3’-ends, and accumulate to 1-4 copies per cell at the steady state. In addition, uaRNA are transcribed with comparable kinetics as their linked mRNA and undergo RNAPII pausing and pause release via the recruitment and activity of P-TEFb. Furthermore, uaRNA have short half-lives (15-20 minutes), likely due to them being targeted for rapid degradation by the RNA exosome. Altogether, these data indicate that the mechanism regulating promoter directionality at divergent promoters occurs after P- TEFb recruitment. In Chapter 3, we describe a genome-wide analysis to map the 3’-ends of polyadenylated RNAs in mESCs and reveal that uaRNAs terminate through a poly (A) site (PAS)-dependent mechanism shortly after being initiated. Interestingly, we find that an asymmetric distribution of encoded U1 snRNP binding sites (U1 sites or 5’ splice sites) and PASs surrounding gene transcription start sites (TSSs) enforce promoter directionality by ensuring uaRNAs are prematurely terminated and likely subsequently degraded. Together, these studies highlight the importance of early splicing signals in producing a full-length coding mRNA, but more importantly, our data reveals that the genomic DNA contains the necessary instructions to read the gene in the correct orientation. Thesis Supervisor: Phillip A. Sharp, Institute Professor of Biology 2 Acknowledgments First and foremost, I would like to thank my advisor, Phil, for his mentorship over the past 5 years and his active involvement in my development as an independent scientist. Thank you for giving me the opportunity to review manuscripts, write grants, give frequent talks in lab meeting, travel to scientific conferences, and for providing a great environment to do “good” science. As I move forward in my scientific career, I will always cherish the time I spent in the Sharp Lab. You pushed me harder than I could have ever done on my own, thank you. I would also like to thank my labmates, past and present, who have supported me over the years. I have learned a great deal from you and feel honored to have studied in the presence of such intelligent individuals. Amy Seila, for taking me under her wing the first few months in the lab and passing on to me a very fruitful project. Ryan Flynn, for our collaborations and providing a great example of a diligent, technically sound scientist. Jesse Zamudio, for your guidance on the work presented in Chapter 2. Xuebing Wu, you are an incredible scientist with unmatched computational prowess. I have learned a lot from you, about how to think about problems computationally as well as seeing the big picture. Mohini, from the beginning of graduate school you have always supported and encouraged me. Thank you for your friendship. I will miss you greatly as I move on. Allan, thank you for being a great example of a diligent, rigorous, and generous scientist. I have enjoyed our conversations about science and life over the years. Jeremy, thank you for your help on manuscripts, proposals, and presentations. Your feedback has been valuable and I have learned a lot from you. Lastly, I would like to thank my family. Amalia, thank you for all the love and support you have given me these past few years. To my second family, the Arudas, thank you for your encouragement and support. To my Mom, Dad, April, and Chris for being great examples of hard work, perseverance, and love. 3 Table of Contents Abstract.......................................................................................................................................2 Acknowledgements.................................................................................................................3 Table of Contents.....................................................................................................................4 Chapter 1: Introduction.........................................................................................................5 Chromatin and gene activation ....................................................................................................7 Transcription......................................................................................................................................8 RNAPII structure and the CTD...................................................................................................................9 RNAPII CTD couples RNA processing to transcription ...................................................................9 Transcription initiation .............................................................................................................................10 Transcription elongation ..........................................................................................................................11 Transcription termination........................................................................................................................13 Co-transcriptional processes ..................................................................................................... 15 5’ capping.........................................................................................................................................................15 RNA splicing....................................................................................................................................................16 Cleavage and polyadenylation ................................................................................................................20 Discovery of divergent transcription ...................................................................................... 26 References ........................................................................................................................................ 32 Chapter 2: Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome ............................................................................. 48 Introduction..................................................................................................................................... 50 Results ............................................................................................................................................... 52 Discussion......................................................................................................................................... 59 Methods............................................................................................................................................. 62 References........................................................................................................................................ 88 Chapter 3: Promoter directionality is controlled by U1 snRNP and polyadenylation signals....................................................................................................................................... 91 Introduction..................................................................................................................................... 93 Results ............................................................................................................................................... 93 Discussion........................................................................................................................................