Profiling the locations of U1 snRNP binding across the nuclear human and Drosophila transcriptomes. D. Rio ? P.I. Spliceosomal U1 snRNP functions in the nucleus to influence both pre-mRNA alternative splicing and polyadenylation site usage, which are two key gene expression mechanisms. The goal of this proposal is to develop a method to comprehensively map the locations of U1 snRNP across the nuclear transcriptomes of Drosophila and human cells and systematically categorize the function of the spliceosomal U1 snRNP at specific sites in modulating global pre-mRNA splicing and polyadenylation patterns. In order to address this question we will: 1) Develop a genome-wide mapping and profiling method for characterizing the binding sites of U1 snRNP across the nuclear transcriptomes of human and Drosophila cells. To do this, we will develop a novel and highly-specific two-step immunoaffinity selection strategy in combination with an RNase T1 nuclease protection assay to characterize the widespread targeting of U1 snRNP (a complex of 10 proteins and one non-coding, small RNA) to intron 5' splice sites, premature cleavage and polyadenylation (PCPA) sites and splicing silencer elements. We will develop novel computational methods to extensively and accurately profile significant U1 snRNP binding sites; 2) Categorize and define U1 snRNP binding sites as bone fide or cryptic 5' splice sites, telescripting sites or splicing silencer elements. For this purpose we will use state-of-the-art cDNA sequencing after perturbation of U1 snRNP activity in human and Drosophila cells. Wewillusetheaccurate U1 snRNP profile maps generated in Aim 1 to correlate with altered pre-mRNA splicing patterns, polyadenylation events and splicing control elements transcriptome-wide. Mapping U1 snRNP binding sites to the nuclear transcriptome will link pre-mRNA splicing patterns, splicing silencer elements and PCPA sites to decode the function of U1 snRNP-mediated post-transcriptional regulation at specific binding sites. These new methods will be transformative by allowing predictions about where and how U1 snRNP binding to nuclear pre-mRNA affects constitutive splicing, alternative splicing, surveillance by premature transcript cleavage and polyadenylation (PCPA) and alternative polyadenylation, all of which are profoundly perturbed in many disease states. The proposed research will reveal for the first time a bona-fide transcriptome-wide map of U1 snRNP binding to nuclear pre-mRNAs and allow the definition of molecular function of the U1 snRNP- mediated post-transcriptional regulatory pathways in both RNA surveillance and RNA processing of the human and Drosophila transcriptomes. This information has the potential to allow the development of new therapeutic strategies to treat disease through investigation and manipulation of U1 snRNP function.