Mechanism of U1 snRNPs suppression of premature cleavage & polyadenylation

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DESCRIPTION (provided by applicant): This proposal's overarching goal is to understand the mechanism of telescripting, a new and major gene expression process recently discovered in my laboratory. Telescripting is essential for full length RNA polymerase II (pol II) transcription from the majority of protein-coding genes in eukaryotes necessary for messenger RNA (mRNA) synthesis. It relies on U1 snRNP (U1), an abundant non-coding 11-subunit ribonucleoprotein particle, to protect nascent pol II transcripts from early termination by cleavage and polyadenylation (CPA) in introns, which is highly destructive. U1 snRNP is well characterized for its role in 5' splices site (ss) recognition, a key and the first step in splicing of introns. Howeer, we found that U1 has an additional non-splicing function as a suppressor of premature CPA (PCPA) from cryptic polyadenylation signals (PASs) that are stochastically present in large introns. We refer to U1's PCPA suppression also as telescripting, as it is necessary for allowing transcription to go farther. In contrast, nascent upstream antisense transcripts from divergent polII promoters are relatively unprotected due to an inverse PAS to U1 binding ratio and are rapidly degraded, indicating telescripting's general role in shaping the transcriptome. Furthermore, telescripting activity, which can be finely modulated by slight changes in U1 level also determines mRNA length. For example, slight U1 decrease causes widespread shortening due to usage of more proximal PASs in the 3' untranslated region (3'UTR), thereby removing mRNA-regulating elements such as translation repressing microRNA binding sites. Based on our previous studies we proposed a model to explain how U1 suppresses PASs in introns, including the hypothesis that it binds in introns and not only to 5'ss. However, basic information needed to test this hypothesis and for understanding telescripting mechanism is lacking. To address this, my laboratory established tools to precisely manipulate PCPA and its suppression by U1, and assays to probe it in detail. We propose to pursue three specific aims: 1) To generate genome wide map of U1 snRNP binding sites on nascent transcripts in live cells, which will be interpreted relative to genome wide map of PCPA sites that we recently completed. 2) To determine the role of U1 snRNP proteins and essential U1 snRNA domain(s) for telescripting. 3) To identify CPA complex and/or other targets of U1's PCPA suppression using a comprehensive RNP interactome discovery approach pioneered in my laboratory. Together, these aims will provide important insights into mechanism of U1 snRNP telescripting, and advance understanding and potential applications of this new dimension in gene regulation to biology and medicine.