After purification and protein digestion, cross-linked peptides remain attached to RNA fragments. In addition to cross link, cDNA library preparation further decreases CLIP efficiency. However, incorporation in living cells of nucleoside analogs into RNA is likely to introduce a bias in the RNA sequences that interact with RBPs. The crosslinking efficiency per se can be strongly improved by using photoactivatable ribonucleosides combined with UV-A irradiation (PAR-CLIP) ( 10). The importance of CLIP methods prompted the community to further improve their efficiency, specificity, and accuracy, as reviewed recently by Lee and Ule ( 8).Ī major caveat of CLIP methods is the poor efficiency of UV-C crosslinking, which is estimated not to exceed a few percent ( 9). Coupled to high throughput sequencing, CLIP offers a transcriptome-wide snapshot of RNA-protein interactions in live cells as covalent links are formed before any disturbing purification step ( 7).
Once cross-linked, RNA digestion separates RNA-protein complexes before immunoprecipitation under stringent washing conditions. The basic principle of this strategy is the covalent binding of RBPs with their direct RNA targets by ultraviolet (UV) light irradiation. The development of the cross-linking and immunoprecipitation (CLIP) method represented a pioneering step in the quest of RBP mapping ( 6). Despite the physiological importance of RBPs evidenced by their implication in diverse pathologies ( 5), the precise function of most RBPs remains obscure. In the case of human messenger RNAs (mRNAs), literally covered by proteins, RNA is in direct contact with >800 different RBPs ( 2, 3), which modulate transcript processing and destiny ( 4). The repertoire of eukaryotic RBPs comprises over 1500 different RBPs in human ( 1). RBPs form ribonucleoprotein complexes with all kind of RNAs to function as genetic information support, structural scaffold, interaction guide, or enzyme. Post-transcriptional gene regulation is governed by hundreds of RNA binding proteins (RBPs).
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