Comprehensive Maps of U1 snRNP Binding to Nascent RNA in Human Cells

项目总结/文摘

PROJECT SUMMARY/ABSTRACT We propose to develop new methods for the global identification of protein and RNP interactions with nascent unspliced RNA. We will use these methods to produce comprehensive maps of spliceosomal U1 snRNP binding across the human transcriptome. U1 functions in pre-mRNA splicing, in the suppression of premature cleavage/polyadenylation, and in the nuclear retention of RNA. However, information on its binding sites is very limited, and how these sites differ for the different functions of U1 is not understood. Unspliced introns in nascent RNA fractionate with the chromatin, and we recently showed that within the chromatin compartment, splicing factors engage in interactions not seen elsewhere. We developed methods to isolate proteins and RNP’s that allowed identification of new regulatory proteins interacting with the U2 snRNP, and the isolation of U2-bound pre-mRNA fragments encompassing the intronic branch points of HEK293 cells. We call this method fractionation/immunopurification/RNAse protection (FIRP) and find the FIRP maps of U2/pre-mRNA interactions to be more comprehensive than previous approaches for mapping branchpoints. We now propose to adapt FIRP to characterizing interactions of the U1 snRNP with pre-mRNA. We will optimize the extraction of material from chromatin to obtain U1 snRNP complexes bound to pre-mRNA. We will develop new anti-U1 antibodies that allow 5’ splice site binding analysis across all types of cells. These methods will be applied both to FIRP assays of U1 snRNP binding and to iCLIP analyses of U1 protein contacts on chromatin-associated RNA. By characterizing U1 binding sites on a global scale and developing methods for assaying its interactions in different cells, regulatory environments and genetic backgrounds, we can examine the processes of 5’ splice site recognition with new breadth and precision.