Global profiling of protein–RNA interactions has been successful in elucidating principles of post-transcriptional regulation. Over the past years, CLIP was proven as a powerful method to determine protein–RNA interactions in vivo
on a global scale9-12
. However, the resolution of this method is limited due to the inability to directly identify the cross-linked nucleotides. Moreover, CLIP suffers from the inherent problem that most cDNAs truncate at the cross-link site and are thus lost during the amplification process. Here, we developed iCLIP, which overcomes these obstacles and identifies the positions of cross-link sites at nucleotide resolution. iCLIP also introduces a random barcode to mark individual cDNA molecules, thereby solving an inherent problem of all current high-throughput sequencing methods that suffer from PCR artefacts. Therefore, exploiting the random barcode strongly improves the quality of quantitative information. Due to the low abundance of introns, the obtained sequence coverage is at present insufficient to quantitatively compare individual binding sites at single nucleotide resolution. However, the quantitative information could be exploited on a transcriptome-wide scale to show that hnRNP C binds longer uridine tracts with higher affinity, underlining the great potential of iCLIP’s quantitative nature. In order to identify clustered cross-link nucleotides, we applied a statistical algorithm to filter for enriched hnRNP C binding. Comparison of the clustered cross-link nucleotides with the complete dataset showed that both datasets generate consistent results, suggesting that real binding sites constitute a major proportion of both. This observation underlines the high quality of iCLIP data, achieved by high stringency of purification and library preparation. Thus, iCLIP allows the transcriptome-wide analysis of protein–RNA interactions at individual nucleotide resolution.
We used iCLIP to show that hnRNP C binds to uridine tracts in nascent transcripts with a defined spacing of 165 and 300 nucleotides. These data agree with past findings that the hnRNP C tetramer binds in repetitive units of approximately 150 – 300 nucleotides6,23,24
. Whereas some studies suggested that this binding occurs in a sequence-independent manner6,23,24
, other studies proposed that the sequence-specific RRM domains critically contribute to high-affinity RNA binding of the hnRNP C tetramer17-19
. iCLIP data agree with the latter model that hnRNP C is positioned on pre-mRNAs via sequence-specific binding of its RRM domains (). In addition, the precise spacing between the hnRNP C cross-link sites suggests that in accordance with the former model the basic leucine zipper-like RNA-binding motif (bZLM) domains guide the intervening RNA along the axis of the hnRNP C tetramer via sequence-independent electrostatic interactions22,29
. Thus, by measuring the spacing between distant binding sites, iCLIP can yield structural insights into ribonucleoprotein complexes.
Figure 6 A model of hnRNP C tetramer binding at silenced and enhanced alternative exons. hnRNP C protein monomers are depicted in yellow with the RRM domains in grey. The schematic RNA molecule is shown to contact the RRM domains via uridine tracts and the bZLM (more ...)
Even though hnRNP particles were found to form on nuclear RNAs more than 30 years ago, their function in pre-mRNA processing remained unresolved4-8
. Here, we present nucleotide-resolution mapping of in vivo
hnRNP C cross-link sites, which reveals a role of hnRNP particles in splicing regulation. Importantly, we found that binding of hnRNP particles is guided by the pre-mRNA sequence to determine the splicing outcome in a position-dependent manner. In particular, alternative exons are silenced by incorporation into the hnRNP particles, whereas binding to the preceding intron enhances inclusion of alternative exons. Early studies had hypothesized that hnRNP particles might function to organize long introns for efficient splicing30
. This was based on the observation that long pre-mRNAs are highly compacted in hnRNP particles. In accordance with this hypothesis, we propose that hnRNP particles might act as ‘RNA nucleosomes’ that bind long regions of pre-mRNA, but maintain the correct splice sites accessible to the splicing machinery. The ability of iCLIP to study protein–RNA interactions with high resolution and in a quantitative manner holds promise for future studies of the structure and function of ribonucleoprotein complexes.