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Discovery and characterization of stable introns in yeast by Jeffrey T. Morgan B.S., Biochemistry (2011) University of Michigan SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY SEPTEMBER 2018 c 2018 Massachusetts Institute of Technology All rights reserved Signature redacted Signature of Author: Jeffrey T. Morgan Department of Biology August 2, 2018 Signature redacted Certified by: David P. Bartel Professor of Biology Thesis Supervisor Signature redacted Accepted by: Amy E. Keating MASSACHUSETTS INSTITUTE Professor of Biology OF TECHNOLOGY Co-Chair, Biology Graduate Committee AUG 6jj018 LIBRARIES I 2 Discovery and characterization of stable introns in yeast by Jeffrey T. Morgan Submitted to the Department of Biology on August 2, 2018 In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Abstract Spliceosomal introns are a defining feature of eukaryotes; they are present in all known eukaryotic genomes, absent from all known non-eukaryotic genomes, and their accurate removal is essential for mRNA maturation. Although smaller ncRNAs can be processed from introns, the introns themselves are considered biologically inert byproducts of splicing; their collective fate post-splicing is to be de-branched and rapidly degraded. This dissertation details the first described instance of a regulated fate and function for excised and de-branched introns in eukaryotes. We observed a set of introns in the budding yeast Saccharomyces cerevisiae's transcriptome that, although rapidly degraded during log-phase growth as expected, accumulate as linear RNAs under saturated-growth conditions and during inhibition of TORC 1, a key integrator of growth signaling. At least 34 introns-1 1% of the introns in S. cerevisiae-show this change in stability. We find no evidence that this stability can be attributed to intron retention in the mature transcript. Instead, introns that become stabilized remain associated with components of the spliceosome post-splicing, likely resulting in their protection from degradation. Compared to other yeast introns, these stable introns have no enriched sequence motifs but do share a short distance between their lariat branch point and the 3' splice site. Indeed, by manipulating this distance, we are able to show a causal relationship between branch-point position and stable-intron formation. To test for cellular functions of stable introns, we created strains with precise intron deletions. We created 20 strains with combinations of up to five introns deleted, with the quintuple mutant eliminating >60% of the stable-intron molecules in the transcriptome. When these strains are challenged with the TORC I inhibitor rapamycin, their growth exceeds that of the parental strain, with a striking relationship (R 2 = 0.9) between the fraction of SI molecules removed from the transcriptome and the rate of growth under TORC 1 inhibition. Overexpression of native or engineered stable introns suppresses this aberrant rapamycin response. These results indicate that stable introns function within the TOR-mediated growth-signaling pathway of S. cerevisiae, and more broadly, excised introns can be stabilized and coopted to perform biological functions in eukaryotic cells. Thesis Advisor: David P. Bartel Title: Professor 3 4 Acknowledgements This work was possible because of the mentorship and trust of my advisor Dave Bartel. Dave has an endless ability to think critically and rigorously about the diverse projects in the lab. He taught me how to ask the right questions, how to conclusively answer them, when to focus on one experiment until you crack it, and when to seek outside input on a problem. He also tried to teach me a great deal about very specific aspects of grammar; I have assuredly erred in these aspects in the writing to follow. I was fortunate to land with great mentors since I started cold e- mailing Pls before my sophomore year of college: Steve Ragsdale and Li Yi at the University of Michigan, and Richard Leapman and Alioscka Sousa at the NIH. I did not fully appreciate them at the time, but I would not have ended up at MIT without their support and example. I am grateful to many faculty members (and one fellow) of the MIT and Whitehead communities for their insight over the years: Chris Burge, Gerry Fink, Dennis Kim, David Pincus, David Sabatini, and Phil Sharp. Gerry and Phil have been instrumental in the success of this project since its inception and have been constant sources of guidance during thesis committee meetings. I additionally thank Gerry for his advice as I looked for postdocs, convincing me success lies foremost in looking where others aren't already looking. I thank members of the Fink lab for input and discussion on my work during Fink group meetings, and a special additional thanks to David Pincus for a great deal of input and suggestions over the past few years. I knew effectively nothing about what the Bartel lab studies when I joined. Because of the creative and generous members of the lab, that quickly changed. I have overlapped with many amazing scientists in the lab: Weinberg, Igor, Vincent (during his brief return), Alex, Olivia, DK, Vikram, Sue-Jean, Katrin, Stephen, Junjie, Grace, Asia, Ben, Wenwen, Coffee, Jamie, Xuebing, Namita, Dan, Jarrett, Matt, Sean, Tim, Charlie, Danny, Kathy, Elena, Justin, Glenn, Thy. and Emir. I am additionally grateful to Laura, the lab's administrative manager, for processing my 657 (and counting) orders, and for being an endless source of positivity in the lab. I must point to a few lab members in particular: Stephen for advice-both big- and small-picture; Alex for being an endless fount of timeworn impressions and timeless experimental designs; Olivia for guidance when I was starting out in the lab, and for the brief period when I was super into the Tour de France; Grace for always having the big soccer matches streaming on her computer for quick check-ins; Thy for many unsolicited pictures of her cat; and Sean for bringing his light-hearted presence, thoughtful opinions, and so much of the cafeteria's flatware to our bay. I still believe a basic tenet of MIT Biology's recruitment pitch: it is special because of the focus on one's cohort. Mine contained many amazing people. I especially want to thank Eric, Erik, Ian, Julie, Kevin, and Sahin for their friendship and support. Finally, I thank my parents for supporting me, my education, and for pushing my siblings and I to follow our interests-even if those interests led us far from home. Many aren't lucky enough to do something they enjoy for a living, and I don't take that for granted. I thank my partner, Zo , for her support, putting up with my inability to accurately predict the time needed to finish up in lab, her love of Jeopardy! and national parks, making sure I eat decent food, and generally for making all aspects of my life much richer. Last but not least, I would like to thank our cat, Ollie, for being a good boy. 5 6 Table of Contents A bstract.......................................................................................................................................... 3 A cknow ledgem nents ....................................................................................................................... 5 Ta ble of C ontents .......................................................................................................................... 7 C hapter 1. Introduction ............................................................................................................... 9 Part 1. Pre-m R N A processing.......................................................................................... 11 Intron recognition.............................................................................................................. 1 1 Chem istry of splicing ........................................................................................................ 13 Figure 1. Tw o-step m echanism of pre-m RN A splicing................................................. 14 The spliceosom e: a dynam ic ribonucleoprotein m achine............................................... 15 Figure 2. Schematic view of the spliceosome cycle in S. cerevisiae.............................. 18 Spliceosom e disassem bly and lariat-intron degradation............................................... 19 Part 2. Introns qua introns................................................................................................ 21 Evolution of introns .......................................................................................................... 21 Loss and gain of introns................................................................................................ 23 Figure 3. Intron density of eukaryotes .......................................................................... 24 Function of introns in alternative splicing .................................................................... 25 Other functions of introns in m odern eukaryotes ......................................................... 26 N otable post-splicing fates of intact introns ................................................................. 28 Part 3. S. cerevisiae outside of log-phase grow th............................................................ 29 G row th phases................................................................................................................... 30
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