Abstract
UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are methods to study protein-RNA interactions in untreated cells and tissues. Here, we analyzed six published and two novel data sets to confirm that both methods identify protein-RNA cross-link sites, and to identify a slight uridine preference of UV-C-induced cross-linking. Comparing Nova CLIP and iCLIP data revealed that cDNA deletions have a preference for TTT motifs, whereas iCLIP cDNA truncations are more likely to identify clusters of YCAY motifs as the primary Nova binding sites. In conclusion, we demonstrate how each method impacts the analysis of protein-RNA binding specificity.
Highlights
To understand post-transcriptional regulation, it is crucial to study protein-RNA interactions in the cellular environment
To analyze if the proportion of truncated cDNAs in individual-nucleotide resolution CLIP (iCLIP) depends on the protein being studied, we evaluated iCLIP data from past studies of heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C), cytotoxic granule-associated RNA binding protein (TIA1), TIA1-like 1 (TIAL1) and TAR DNA binding protein (TDP-43; known as TARDBP) [7,23]
The proportion of cDNAs containing deletions in TIA1, TIAL1 and TDP-43 iCLIP was close to that of mRNA-Seq, indicating that over 95% of cDNAs in these iCLIP experiments truncated at crosslink sites (Table 1; Figures s2 and s3 in Additional file 1). To further consolidate this finding, we evaluated crosslinking of TIA1 and TIAL1 at positions +6 to +30 downstream of exon-intron junctions, which were shown by an independent study to be important for TIA-dependent splicing regulation [24,25]. cDNA truncations identified this region 291 and 457 times more frequently compared to cDNA deletions in TIA1 and TIAL1 iCLIP, respectively (Figure s4 in Additional file 1)
Summary
To understand post-transcriptional regulation, it is crucial to study protein-RNA interactions in the cellular environment. In combination with high-throughput sequencing, CLIP (or HITS-CLIP) identified RNA targets of RBPs in a transcriptome-wide manner [2,3,4,5] These studies showed that the precise position of protein binding sites on target RNAs is extremely important, since the effect of RBPs on the alternative splicing largely depends on their precise binding position. This was most clearly shown by genome-wide RNA maps of splicing regulation [6,7]
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