Since these highly abundant particles are likely to constitute a general platform for other splicing regulators, deciphering their function would greatly advance our understanding of splicing regulation. In particular, genome-wide mapping of hnRNP C positioning would provide critical information on how hnRNP particles control splicing. Resolving these seemingly contradictory observations was hindered by the inability to locate precisely hnRNP particles on nascent transcripts in vivo. Whereas some studies suggested that hnRNP particles generally facilitate splicing 5, 6, individual hnRNP proteins were thought to function as splicing silencers 7, 8. However, although hnRNP C is one of the most abundant proteins in the nucleus, its role in splicing regulation remained unresolved. Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) was identified over 30 years ago as a core component of hnRNP particles that form on all nascent transcripts 4. Splice-site selection is primarily mediated by RNA-binding proteins that bind regulatory elements within nascent transcripts 2, 3. In humans, it was recently estimated that 95-100% of all multi-exon transcripts undergo alternative splicing 1. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.Ī major source of proteomic diversity in multicellular eukaryotes is the production of multiple mRNA isoforms. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an ‘RNA map’ indicates how the positioning of hnRNP particles determines their effect on inclusion of alternative exons. hnRNP particles assemble on both introns and exons, but remain generally excluded from splice sites. iCLIP data demonstrate that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. Here, we developed individual-nucleotide resolution UV-cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. Despite their abundance however, it remained unclear whether these particles control pre-mRNA processing. In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C.
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