(c) Reproducibility of sequence composition at cross-link nucleotides. The percentage of reproduced cross-link nucleotides increased with the incidence of hnRNP C cross-linking (cDNA count). Percentage of cross-link nucleotides with a given cDNA count that were identified in at least two (circles) or all three experiments (triangles) are shown. (b) Reproducibility of cross-link nucleotide positions. The first nucleotides of each sequence contain the barcode followed by the nucleotide where cDNAs truncated during reverse transcription. Resulting cDNA molecules are circularized, linearized, PCR-amplified and subjected to high-throughput sequencing. The red bar indicates the last nucleotide added during reverse transcription. Proteinase K digestion leaves a covalently bound polypeptide fragment on the RNA that causes premature truncation of reverse transcription (RT) at the cross-link site. After UV irradiation, the covalently linked RNA is co-immunoprecipitated with the RNA-binding protein (RBP) and ligated to an RNA adapter at the 3′ end. (a) Schematic representation of the iCLIP protocol. ICLIP identifies hnRNP C cross-link nucleotides on RNAs. Taken together, iCLIP enables precise mapping of protein–RNA interactions in intact cells. We successfully applied individual-nucleotide resolution CLIP (iCLIP) to study hnRNP C-dependent splicing regulation in human cells. This allowed us to discriminate between unique cDNA products and PCR duplicates. In order to quantify cDNA molecules that truncate at the same nucleotide, we added a random barcode to the DNA adapter. Here, we exploited this apparent limitation to achieve single nucleotide resolution by capturing these truncated cDNAs through the introduction of a second adapter after reverse transcription via self-circularization ( Fig. Primer extension assays have shown that the vast majority of cDNAs prematurely truncate immediately before the ‘cross-link nucleotide’ 13. An additional disadvantage of CLIP is the requirement of reverse transcription to pass over residual amino acids that remain covalently attached to the RNA at the cross-link site. However, since identification of binding sites relied on the analysis of overlapping sequence clusters, distances of less than 30 nucleotides were not resolved. UV-cross-linking and immunoprecipitation (CLIP) combined with high-throughput sequencing was previously used to generate transcriptome-wide binding maps of several RNA-binding proteins 9 - 12. 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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