PROJECT SUMMARY This proposal’s overall goal is to understand the molecular mechanism of telescripting, a new and major gene expression process that is crucial for full-length transcription of most protein-coding genes and regulates messenger RNAs (mRNAs) isoforms and mRNA length in humans and other complex organisms. mRNAs are processed from nascent RNA polymerase II (PolII) transcripts, which generally includes the removal of introns (splicing) and transcription-terminating 3’-end cleavage and polyadenylation (CPA). Splicing and CPA are specified by splice sites and CPA signals (PASs), respectively. However, numerous PASs indistinguishable from the ultimate, gene ends’ PASs are scattered throughout pre-mRNAs, especially in introns and 3’ untranslated regions (3’UTRs), and can trigger premature CPA (PCPA). PCPA is suppressed by U1 snRNP (U1), human cells’ most abundant small non-coding nuclear RNA-protein particle. For brevity, and to distinguish it from U1’s role in splicing, we call U1 suppression of PCPA, telescripting (as it is necessary for long-distance transcription). Like U1 function in splicing, telescripting also depends on U1 snRNA base-pairing to nascent transcripts, which can be abrogated with U1 antisense oligonucleotides (U1 AMO), causing PCPA. Recent studies revealed that even slight changes in the balance between U1 and PASs has great impact on gene expression and can profoundly alter the mRNAs and proteins cells produce. Such changes occur naturally, for example rapid transcription up-regulation during cell stimulation, and create transient U1 deficit relative to transcription output, causing PCPA that produces shorter mRNA isoforms needed to respond to acute environmental changes. Importantly, U1 AMO recapitulates the same mRNA isoform shifts and U1 over- expression can prevent their production in stimulated cells. U1 AMO also elicits widespread 3'UTR shortening, which occurs in and contributes to cell proliferation and cancer. U1 telescripting’s overarching role in transcriptome regulation impacts transcription, splicing, CPA and thereby all downstream events in the life of mRNAs. It has numerous potential applications in biology and medicine. Realizing them requires detailed understanding of the molecular mechanism by which U1 suppresses PASs and the factors that regulate it, which remain unknown. We have made significant progress towards that, including mapping the transcriptome binding locations of U1 and cleavage and polyadenylation factors (CPAFs), and interpreted them in relation to PCPA locations. We have also captured U1 and CPAFs complexes in cells, determined their compositions and stoichiometries, and determined how cells produce the great U1 abundance required for telescripting. These advances lay the foundation for future studies. I anticipate the mechanistic studies and new information will identify potential points of intervention, including druggable targets, and will advance the prospects of harnessing them f...