PROJECT SUMMARY/ABSTRACT For a protein-coding gene to perform its cellular function, it must first generate RNA transcripts that are expressed at the appropriate level and properly processed. This is no small feat when one considers that RNA polymerase II can prematurely terminate and that nascent transcripts can be acted upon by a variety of RNA processing machines, including ones that yield mature transcripts lacking a canonical 5' cap or 3' poly(A) tail. A major focus of our laboratory has thus been to identify and characterize novel “non-canonical” processing pathways that can act on nascent RNAs. Here, we propose to build upon our recent work to study two such mechanisms that are widely employed across eukaryotic genomes. First, we will mechanistically dissect how the Integrator (Int) complex catalyzes premature transcription termination at hundreds of protein-coding genes. Integrator was long known to be critical for the biogenesis of small nuclear RNAs (snRNAs), but we recently showed that Integrator also binds to many protein-coding loci and attenuates production of their full- length mRNAs, in some cases by more than 100-fold. This is because the IntS11 RNA endonuclease directly cleaves nascent mRNAs, triggering degradation of the transcripts and premature transcription termination. Nevertheless, it remains poorly understood how Integrator is assembled, regulated, and recruited to protein- coding genes as most of the other subunits in the complex have no known function and lack obvious paralogs or known protein domains. Our preliminary data indicate that non-catalytic Integrator subunits have distinct roles at snRNA vs. protein-coding gene loci, and thus we will characterize in detail how these subunits are recruited and function. Crosslinking mass spectrometry will further be used to define physical interfaces between Integrator subunits, thereby revealing novel insights into how Integrator is globally assembled and controlled. Second, we will investigate why many protein-coding genes generate circular RNAs with covalently linked ends. Some of these non-canonical transcripts are greater than 10-fold more abundant than their associated linear mRNAs. This suggests the main function of these genes may be to produce circular RNAs, but the physiological functions of almost all mature circular RNAs remain unknown. We thus will use high-throughput screening coupled to detailed biochemical studies to identify critical functions for circular RNAs and their underlying molecular mechanisms. We further will systematically identify factors that modulate circular RNA levels, especially post-transcriptionally, as very little is currently known about how the fate and decay rates of these transcripts are controlled. Characterization of these key regulatory mechanisms will not only provide important insights into endogenous circular RNAs, but may also ultimately enable circular RNAs to become novel long-lasting therapeutic modalities. In total, these in...