Not only is mRNA 3’ processing essential for gene expression, it is crucial in gene regulation as well. The regulation of 3’ end processing is important in maintaining normal processes like neural activity, T-cell activity and stem cell differentiation and renewal, whereas its dysregulation can result in many diseases such as cancers. To study how 3’ processing factors regulate gene expression, comprehensive ChIP-seq (Chromatin Immunoprecipitation-Seq) analyses of subunits of the 3’ processing complex were performed. Unexpectedly, one subunit, CPSF100, showed minimal peaks at the 3’ ends of genes, with maximum signal at the promoters of a large subset of genes and another large subset of strong peaks at enhancers, both regulatory elements that can be dysregulated to precipitate aberrant gene expression patterns. To analyze the effect of CPSF100 and the CPSF complex on transcription and its termination at protein coding genes and enhancers, we performed RNA PolII ChIP-Seq, 4SU-Seq and PolyA Site (PAS)-Seq in HeLa cells where CPSF73 and CPSF100 were knocked down using an shRNA-mediated lentiviral delivery method. We found that knocking down CPSF factors led to bidirectional transcription

termination defects at enhancers and protein coding genes. In addition, it led to an aberrant response to transcription activation by reducing the ratio of PolII elongating on the gene body to the amount of PolII at the promoter. Finally, depletion of CPSF led to the formation of long and abundant noncoding RNAs from enhancers, promoters of genes, and at permissive intergenic regions. These findings advance the field of RNA 3’ end processing in several ways. We show that CPSF factors are important for facilitating productive elongation. In addition, the effect of depleting them on transcription termination far surpasses the effect of depleting the exonuclease that is thought to enable termination, suggesting that another exonuclease-independent model of termination may be the major mechanism. Finally, our findings show that CPSF is primarily responsible for mediating the cleavage and termination of eRNA transcripts, which was previously thought to occur via another factor. These findings open up new avenues of research and highlight the importance of the 3’end processing machinery in regulating transcription.

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.

Eukaryotic mRNA 3' processing is an essential step in the gene expression pathway and it is tightly coupled to many other cellular processes including transcription termination, splicing, mRNA export, and translation. As a result, it is under stringent regulation by many different factors such as viral infection, stress, and small molecule inhibitors. This study examines how mRNA 3' processing and transcription termination are regulated during herpes simplex virus 1 (HSV-1) infection and during treatment of JTE-607, a small molecule inhibitor. We found that HSV-1 and its immediate early factor ICP27 contribute to widespread changes in host mRNA alternative polyadenylation (APA) by activating intronic polyadenylation sites (PAS). We also discovered that the small molecule inhibitor JTE-607 inhibits mRNA 3' processing in a sequence-specific manner and the cleavage site region immediately following AAUAAA is the core sensitivity determinant of a PAS to JTE-607. A machine learning model that predicts the impact of JTE-607 on PAS selection and transcription termination genome-wide was developed. The findings of this study provide insights into the regulatory mechanism of mRNA 3' processing and transcription termination by viral infection and small molecule inhibitors, which shed light on targeting mRNA 3' processing as a novel strategy for broad spectrum anti-viral and anti-cancer therapeutics.