During the CLIP protocol, crosslinked protein–RNA complexes are purified and the RNA fragments are released by digesting the protein, resulting in RNAs with a covalently bound peptide at the crosslink site. To understand the mechanisms of their action, it is essential to identify the endogenous sites of protein–RNA interactions, which has been aided by the development of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP). RNA-binding proteins (RBPs) play crucial roles in all aspects of post-transcriptional gene regulation. We demonstrate the advantage of iCLIP and related methods that can amplify cDNAs that truncate at crosslink sites and we show that computational analyses based on cDNAs-starts are appropriate for such methods. ![]() In contrast, we show that a broad size range of cDNAs in iCLIP allows the cDNA-starts to efficiently delineate the complete RNA-binding sites. ![]() Our study also shows that if RNase does not efficiently cut within the binding sites, the original CLIP method is less capable of identifying the longer binding sites of RBPs. These constraints are overcome when fragmentation by RNase I is efficient and when a broad cDNA size range is obtained. As previously noted, the positions of complementary DNA (cDNA)-starts depend on cDNA length in several iCLIP experiments and we now find that this is caused by constrained cDNA-ends, which can result from the sequence and structure constraints of RNA fragmentation. We perform experiments for PTBP1 and eIF4A3 using individual-nucleotide resolution CLIP (iCLIP), employing either UV-C or photoactivatable 4-thiouridine (4SU) combined with UV-A crosslinking and compare the results with published data. Here, we produce data with multiple variants of CLIP and evaluate the data with various computational methods to better understand their suitability. This variety of approaches can create challenges for a novice user and can hamper insights from multi-study comparisons. Several variants of CLIP exist, which require different computational approaches for analysis. ![]() Ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) identifies the sites on RNAs that are in direct contact with RNA-binding proteins (RBPs).
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