Nova proteins, encoded by Nova1 and Nova2 genes, contain three KH RNA-binding domains. Due to the well-characterized sequence preference of Nova proteins and the available CLIP data, we performed iCLIP with Nova proteins in order to compare the two methods. Our second goal was to compare the cross-link sites identified by CLIP and iCLIP. If the read-through cDNAs dominated the iCLIP libraries, they could impair the ability of iCLIP to identify the cross-link sites with nucleotide resolution. However, these studies did not evaluate the proportion of cDNAs that truncate at the cross-link sites, as compared to the cDNAs that read through the cross-link sites. The ability of iCLIP to provide nucleotide-resolution information about the cross-link sites was initially demonstrated by determining the positions within uridine tracts that cross-link to heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C), and the positions downstream of 5' splice sites that cross-link to cytotoxic granule-associated RNA binding proteins (TIA1 and TIAL1). The position of cDNA truncation therefore enables iCLIP to identify the cross-link sites. iCLIP employs a different cDNA cloning protocol from CLIP and PAR-CLIP, which enables identification of the cDNAs that truncate at the cross-link sites. However, the peptide or amino acid left on the RNA after treatment with proteinase K can obstruct the reverse transcriptase, and therefore primer extension studies showed that a significant proportion of cDNAs truncate at the cross-link sites. CLIP and PAR-CLIP protocols identify only the cDNAs that have read through the cross-link site. Our first goal was to determine the proportion of truncated cDNAs in the iCLIP cDNA libraries. Recently, individual-nucleotide resolution CLIP (iCLIP) was developed to identify cross-link sites independently of cDNA mutations. The cDNA deletions in HITS-CLIP data were then used to identify cross-link sites of Neuro-oncological ventral antigen 1 and 2 (Nova1 and Nova2, which will be together referred to as Nova) and Argonaute (Ago) proteins in a genome-wide manner. However, a study by Zhang and Darnell compared the frequency and distribution of deletions and point mutations in CLIP and mRNA-Seq cDNAs, and found that CLIP cDNA deletions were a more reliable signature of cross-link sites compared to point mutations. Methods that identify cross-link sites without the need of photo-reactive nucleosides are therefore required.Īs originally described by Granneman and colleagues, cross-link sites induced by UV-C light are associated with point mutations and deletions in CLIP cDNAs, which was supported by Kishore and colleagues. The efficiency of nucleoside uptake, and the potential toxicity of these nucleosides, might vary between cell lines and tissues. However, application of PAR-CLIP requires pre-incubation of cells with photoreactive ribonucleoside analogs, and therefore cannot be performed with untreated cells and tissues. One such approach, Photoactivatable Ribonucleoside-Enhanced CLIP (PAR-CLIP), uses photo-reactive nucleotides and UV-A light for the cross-linking reaction, which increases the incidence of point mutations at the cross-link sites. All of these approaches exploit the effect of cross-linked nucleotides during the reverse transcription reaction. To understand the precise position of protein-RNA cross-linking, several modifications of CLIP were developed. This was most clearly shown by genome-wide RNA maps of splicing regulation. 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. Especially in combination with high-throughput sequencing, CLIP (or HITS-CLIP) identified RNA targets of RBPs in a transcriptome-wide manner. Cross-linking and immunoprecipitation (CLIP) was therefore developed to identify RNA sites in direct contact with RNA-binding proteins (RBPs). Irradiation with UV-C light creates a covalent bond between proteins and RNAs that are in direct contact in vivo without requiring pre-incubation of cells with photoreactive ribonucleoside analogs.
To understand post-transcriptional regulation, it is crucial to study protein-RNA interactions in the cellular environment.
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