PROJECT SUMMARY: Precise control of gene expression in response to external signals and cues is essential for organismal development, growth and homeostasis. In metazoan cells, the regulated pausing of RNA polymerase II (RNAPII) in early transcript elongation is pivotal for defining gene output. However, the mechanisms that govern pausing and release of RNAPII into productive elongation remain poorly defined. Our work demonstrates that the Integrator complex (INT) targets paused RNAPII to attenuate messenger RNA (mRNA) expression. Moreover, we find that INT is a central regulator of gene expression in Drosophila cells, through its selective recruitment to specific gene promoters and subsequent INT-mediated termination of transcription. INT is known to be required for 3'-end formation of small nuclear RNAs involved in splicing (snRNAs), and has been implicated in cleavage and processing of other non-coding RNAs. Accordingly, the Integrator 11 subunit (IntS11) was found to possess an RNA endonuclease activity. Interestingly, INT was recently shown to also associate with RNAPII at protein- coding genes, however, our understanding of INT function on mRNAs is very limited. As described below, our preliminary data and proposed research will address fundamental, unanswered questions about gene regulation, and aim to elucidate the roles of INT in human disease. For example, paused RNAPII is very stable at some mRNA gene promoters, yet highly unstable at others. This diversity of behaviors for paused RNAPII has generated considerable interest concerning its underlying causes. In particular, the evidence for rapid turnover of paused RNAPII suggests the presence of a promoter-proximal termination factor. However, no such factor has been described in metazoan cells. Our work demonstrates that INT is this ‘missing’ termination factor and suggests a role for IntS11-mediated RNA cleavage in transcript termination. In addition, we discovered that INT recruits a protein phosphatase, PP2A, to RNAPII leading to de-phosphorylation of the RNAPII C-terminal domain (CTD). This finding is highly significant, since RNAPII CTD phosphorylation is involved in the release of paused RNAPII into productive mRNA elongation. The proposed work expands on this preliminary data. Aim 1 will determine how INT is targeted to specific gene promoters, pursuing in particular evidence for an interaction between INT and the cohesin complex. Notably, INT has already been shown to interact with cohesin in mammalian neurons, and implicated together with cohesin in Cornelia de Lange Syndrome. Aim 2 will probe the impact of the IntS11 RNA endonuclease activity on RNAPII elongation properties. Aim 3 will define the substrates and consequences of INT-mediated recruitment of the PP2A phosphatase to the early elongation complex. Critically, mutations in INT have been associated with a large number of diseases, with every one of the 14 subunits in the INT complex being implicated a pathophysiologica...