Usage of alternative cleavage and polyadenylation sites (APA) is emerging as a critical gene regulatory mechanism, and it is known to play important roles in proliferation and development. Recent studies have shown that 3? untranslated region (UTR) lengthening occurs during mammalian embryonic development and skeletal muscle differentiation. Lengthening occurs through alternative polyadenylation, although the mechanisms that regulate this process are not completely understood. Furthermore, the mechanisms that link cleavage and polyadenylation to RNA polymerase II (PolII) termination are not well understood. We have investigated the basis for these key transcriptional mechanisms by focusing on the PAF complex (Paf1C) in mammalian muscle cells. Paf1C acts as a platform to orchestrate a multitude of activities during the transcription cycle, from transcriptional elongation to 3? end processing. Our combined studies have revealed novel roles for Paf1C in suppression of alternate polyadenylation (pA) sites as well as upstream and intragenic antisense transcription. This proposal will leverage the complementary expertise of investigators who will use a combination of state-of-the-art genomics, computational biology, and biochemistry to rigorously investigate the role of the Paf1C complex in transcript processing and alternative polyadenylation, testing the role of these critical events in myogenic differentiation. In two aims, we will explore potential physical and functional connections between Paf1C and 3? end processing factors, chromatin modifications, PolII progression, and regulation of pA usage, and we will place their coordinated activities in the context of myogenic differentiation. We will determine how Paf1C suppresses upstream anti-sense transcription and proximal pA usage, investigating potential involvement of cleavage and polyadenylation factors. Lastly, we will determine whether Paf1C serves a surveillance function by suppressing intergenic and intragenic transcription through interactions with another elongation factor. Altogether, these studies will attempt to integrate diverse roles for Paf1C in chromatin modifications with regulation of alternative polyadenylation, readthrough and antisense transcription, and changes in gene expression underlying myogenic differentiation. More broadly, our studies will reveal roles for Paf1C in genome surveillance that regulate production of lncRNAs and suppression of antisense transcription. Our studies are relevant to human health because loss of function mutations in a Paf1C component lead to parathyroid tumors, and mutations in a cleavage and polyadenylation factor lead to a type of muscular dystrophy. Thus, our proposal will contribute fundamental new insights into basic transcriptional mechanisms as well as potentially important information regarding human disease.
We have found novel roles for the Paf1C complex in the control of transcription and processing of RNA transcripts, in particular, the regulation of bidirectional and read-through transcription, as well as alternative poly-adenlyation, which has emerged as an important new layer in gene regulation. Given the importance of these processes in mammalian development and the observation that defects are associated with a number of diseases?from cancer to muscle disease--our proposal is highly relevant to human health.