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Two critical steps in mRNA processing are 3’ processing and splicing, both of which diversify the human genome and create mRNA isoforms with unique regulatory properties including mRNA stability, translation efficiency or intracellular location or even create distinct proteins. In addition to being essential steps in mRNA processing, polyadenylation and splicing are highly alternatively regulated, with approximately 70% and 95% of genes producing alternative isoforms respectively. Regulation of these isoforms have important biological consequences as mis-regulation is associated with many human diseases including cancer and neurological disorders (Tian and Manley 2017; S. Chan, Choi, and Shi 2011; Q. Pan et al. 2008; Lukong et al. 2008; Y. Zhang et al. 2021). While the core regulatory machineries for 3’ processing and splicing have been identified, there is still limited ability to predict how alternative regulation will occur, making this a critical field of study. One area of particular interest for the study of alternative RNA processing is the role of RNA binding proteins (RBPs). To better understand how RNA binding proteins regulate alternative 3’ processing and splicing, I carried out three projects.First, to investigate the roles of RNA binding proteins in 3’ processing regulation, a dual luciferase reporter system was utilized for a large-scale screen of RBPs for polyadenylation site regulation. In addition to validating several known regulators of APA, we identify several novel inhibitors of polyA site selection including hnRNP A0, hnRNP G, and Musashi1. Strikingly, we also demonstrate that the SR family of proteins are polyA site position-independent repressors of polyadenylation sites, indicating that their role in polyadenylation may be unique from their role in splicing regulation where they act as position dependent activators. This screen may also be used to identify other families of RBPs with the capability to regulate polyadenylation and make predictions about other new regulators.Next, genome-wide sequencing approaches were used to characterize the role of the core polyadenylation complex cleavage factor I (CFIm) in APA regulation. In addition to its known role in enhancing distal polyA sites, we demonstrate that CFIm promotes intronic polyadenylation, most notably within one member of each of the other core polyadenylation machinery components. We also propose a model by which CFIm regulates both 3’ UTR APA and intronic polyadenylation to modulate global protein production and therefore link 3’ processing with cell fate determination. Finally, we interrogate the role of CFIm in splicing regulation through a combination of genome-wide sequencing and biochemical analyses. We demonstrate that CFIm is a general alternative splicing regulator that binds 3’ splice site and interacts with U2AF through RS-RS domain interactions. In addition, CFIm promotes U2AF-RNA interaction at the 3’ splice sites of CFIm activated cassette exons. Our data supports a revised model for 3’ splice site selection by U2AF: CFIm and other RNA binding proteins compete for interaction with U2AF and each regulate the alternative splicing of a specific subset of cassette exons.

The 3′ processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps. The precursor of M1 RNA has an rne-dependent site downstream of the processing site, whose sequence variation affects the processing efficiency. In this study, we showed that the sequence itself of the rne-dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites.

Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3' processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing. The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p.

M1 RNA, the catalytic component of Escherichia coli RNase P, is derived from the 3′-end processing of precursor M1 RNA, a major transcript of the rnpB gene. In this study, we investigated the mechanism of 3′-end processing of M1 RNA using the recombinant N-terminal half RNase E. The cleavage site preference of RNase E differed from that of the 40% ammonium sulfate precipitate (ASP-40), a partially purified cell extract containing processing activity. However, the addition of a trace amount of ASP-40 changed the cleavage site preference of RNase E to that of ASP-40 suggesting the involvement of a soluble factor in cleavage site preference.