How Cis Elements Direct Alternative Splicing by Modulating Spliceosome Assembly Patterns

1 To assemble or not to assemble: How cis elements direct alternative splicing by modulating spliceosome assembly patterns A Thesis Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Master of Science by Rachel Sandman December 2020 2 © 2020 Rachel Sandman 3 ABSTRACT Eukaryotic genes are generally composed of multiple exons with intervening introns that are spliced to form mature RNA molecules. Intron removal is catalyzed by the spliceosome, a large complex of proteins and 5 RNA-protein molecules known as snRNPs. In each splicing event, snRNPs assemble anew on the transcript, distinguishing exons from introns. Alternative splicing, the process by which portions of the pre-mRNA are alternatively included from the mRNA, involves differential spliceosome assembly upon essential cis elements in the pre- mRNA: the 5’ splice site, branch point and 3’ splice site. These are partially conserved motifs that are recognized by the U1 and U2 snRNPs. Currently, there are two models for how spliceosomes recognize the appropriate splice site -intron definition, where U1 and U2 interact across the intron, and exon definition, where the U1 snRNP initially pairs with the upstream U2 snRNP across the exon, followed by a rearrangement to form interactions with the downstream U2 snRNP across the intron. Subsequent steps of splicing are thought to proceed in a standard fashion regardless of the splice site recognition mode. Additional cis elements have been reported to regulate alternative splicing by modulating the stoichiometry and interactions of splicing activators and inhibitors as well as the steric conformation and accessibility of the splice sites and branch point to block or enhance splicing at specific locations. Studies have shown that even a base pair mutation of an additional cis acting element can cause a change in the splicing outcome of a transcript, yet, it is not well understood how prevalent these elements are in a transcript and how they affect spliceosome assembly outcomes. Recently, three cases of environmentally regulated alternative splicing and hundreds of alternative splicing events were reported in S. pombe , a fission yeast with splice sites that closely match the splice site degeneracy seen in higher eukaryotes but does not have many auxiliary splicing proteins. The environmentally regulated alternative splicing of the srrm1 pre- mRNA provides the opportunity to investigate the mechanistic basis of alternative splicing by 4 identifying and characterizing cis elements that direct differential spliceosome assembly in S. pombe . Investigating alternative splicing regulation will enhance our understanding of this important process as it pertains to diversity, gene expression, developmental processes, and disease. 5 BIOGRAPHICAL SKETCH Rachel Sandman received her undergraduate degree in subcellular biology from the University of Louisville and entered Cornell’s Genetics, Genomics and Development PhD program in 2017. During this time, she completed course and teaching requirements, as well as her A exam. In 2020, she graduated with her master’s in Genetics, Genomics and Development and got a job as a Territory Business Manager with Pathline Labs. In her free time, Rachel enjoys playing soccer, crossfit, and gardening. 6 ACKNOWLEDGEMENTS I would like to acknowledge the Jeff Pleiss and members of the Pleiss lab, my committee members, John Lis and Maureen Hanson , and Andrew Grimson, the director of graduate studies, for helping further this work and publish my thesis. I would also like to acknowledge the National Institute of Health and American Cancer Society for funding this research. 7 TABLE OF CONTENTS Introduction……………................................................................................................page 8 Methods………………………………………………………………………………………page 13 Results………………………………………………………………………………………..page 16 Discussion……………………………………………………………………………………page 34 Supplementary Material…………………………………………………………………….page 36 References……………………………………………………………………………………page 37 8 Introduction An estimated 95% of human genes undergo alternative splicing, where a single pre- mRNA is spliced into multiple mRNA isoforms 1. This process is a major source of proteome diversity in eukaryotes and its regulation is vital to cellular processes that depend on precise ratios of mRNA isoforms 2. While advances in sequencing technology have enabled cataloging of genetic variation, the functional consequences of these phenotypes, such as alternative splicing, remain poorly predictable. Enhancing our understanding of alternative splicing regulation is crucial to understanding proteomic diversity, gene expression, developmental processes, and disease. Exon skipping, a predominant form of alternative splicing, involves differential spliceosome assembly upon cis elements in the pre-mRNA transcript 2. In exon skipping, a degenerate, conserved set of cis acting elements known as the core splicing signals (5’ splice site, branch point, and 3’ splice site) guide interactions between spliceosomal components and pre-mRNA 3. However, core signals are not sufficient to ensure correct splice site selection and to regulate alternative splicing 4. Spliceosome assembly decisions are regulated by the combinatorial activities of short, degenerate RNA motifs known as sequence regulatory elements (SREs). SREs have been identified via minigene analyses, genome editing analyses, sequence conservation between species, or by computational analysis of motif enrichment near splice sites, enabling classification of these elements into four classes: exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs) 5-10 . Experiments with individual SREs have shown they bind trans factors which influence splice site recognition and/or alter the secondary structure of a pre- mRNA to modulate accessibility to splice sites and other SREs 10 . Studies have also shown that 9 changing a single base pair an SRE and/or transferring an SRE to a new location can alter splicing of a transcript 9,10,11 . Despite these advances, the identity and prevalence of these SREs throughout most pre-mRNA transcripts, as well as their influence on splice site selection, are mostly unknown. A logical first step to investigate the mechanism by which SREs modulate alternative splicing is to understand how they direct spliceosome assembly. Currently, two models exist for spliceosome assembly upon a transcript: intron definition and exon definition 12 . In intron definition, U1 assembles upon a 5’ splice site and pairs with a downstream U2, assembled upon the branch point. In exon definition, U1 assembles on a 5’ splice site and pairs with an upstream U2, across the exon. This is followed by a rearrangement to form interactions between U1 and the downstream U2 (Figure 1). Subsequent steps of splicing are thought to proceed in standard fashion regardless of the assembly mode. Since the initial proposal of exon definition in 1995, the splicing field is trying to understand how spliceosome assembly modes influence and/or regulate alternative splice site selection. A major breakthrough occurred in 2005, Hertel et al. noticed that Drosophila melanogaster exons flanked by long introns have a 90-fold-higher probability of being alternatively spliced when compared with exons flanked by short introns 13 . From these results, his group proposed that intron/exon architecture in Drosophila melanogaster is a major determinant governing the frequency of alternative splicing. This work was followed by a 2008 proposal which stated that alternative splice site selection occurs in the transition from exon definition to intron definition in in vitro splicing assays using human RNAs 14 . The increased availability of high throughput sequencing techniques also allowed Lawrence Chasin’s group to propose spliceosome assembly outcomes are mediated by protein interactions that vary according to exon size, splice site strength, and the additive effects of splicing silencers and enhancers in 2015 15 ; and for Burge et al. to propose that transcripts with exon definition-like or intron definition-like architectures exhibit different splicing kinetics, leading 10 Figure 1 11 to changes in the rate and/or accuracy of splice site selection and splicing in 2017 16 . In 2019, Rui Zhao’s group used cryo-EM and in vivo splicing of a mini gene to propose a unified mechanism for intron definition and exon definition and to claim that these two spliceosome assembly modes can occur through the same spliceosomal structure in both higher and lower eukaryotes 17 . Despite this progress, there are still significant discrepancies in knowledge about the degree of mutual exclusivity/overlap between spliceosome assembly modes, the prevalence of exon definition in lower eukaryotes and intron definition in higher eukaryotes, the auxiliary trans factors associated with each, and the biological significance of these two assembly modes. Most importantly, a set of general principles that allow distinction and differential activation of splice sites via spliceosome assembly still eludes us. Recently, our lab has discovered three environmentally regulated alternative splicing events in the fission yeast Schizosaccharomyces pombe (S. pombe) 18,19 . S. pombe is genetically tractable and high throughput sequencing technologies have been developed to query splicing events in this organism 20 . In one

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