Comprehensive Maps of Early Spliceosome and Regulator Binding to Nascent RNA in Human Cells

PROJECT SUMMARY/ABSTRACT We propose to develop new methods for the global identification of protein and RNP interactions with nascent unspliced RNA in human cells. We will use these methods to produce comprehensive binding site maps for several important molecules in splicing. In previous work, we isolated proteins and RNP’s from the chromatin

fraction of cells to identify a new complex of splicing regulatory proteins, Rbfox/LASR, as well as a new U2 snRNP complex containing novel regulatory proteins. We further found that sequencing the pre-mRNA fragments bound within both these complexes allows the comprehensive mapping of their transcriptome-wide

interactions. This “IPseq” mapping of U2/pre-mRNA interactions provides sensitive detection of intron branchpoints across the expressed transcriptome. We now propose to apply IPseq to develop binding maps of several key splicing components. We will continue ongoing studies of Rbfox/LASR and the U1 snRNP, and we

will apply the method to the early spliceosome component: U2AF2. In preliminary data, we find that IPseq generates precise maps of the binding of both Rbfox1 and the LASR subunits on pre-mRNAs. Analyses of these data will yield important understanding of how the large Rbfox/LASR complex interacts with RNA, and

how individual bound elements cooperate to generate a splicing regulatory code. The U1 snRNP recognizes the 5’ splice during early spliceosome assembly, binds within splicing silencer elements, and also acts to suppress premature cleavage/polyadenylation events in nascent RNA transcripts. Genomewide information on

U1 snRNP binding is very limited, and the global analysis of its many RNA interactions will provide new insights into its multitude of functions. The U2AF heterodimer binds the 3’ splice site during early spliceosome assembly and is essential for the formation of an exon definition complex. A combined map of U1 snRNP and

U2AF binding sites will allow their assessment as parts of possible cryptic exons and allow better prediction of mutational activation of cryptic splicing. By building a comprehensive map of the key factors for exon definition, our goal is to enable better prediction of pathogenic mutations affecting splicing. Finally, we believe that

chromatin RNA IPseq can be broadly applied to many other cellular components.