![]() ![]() Computationally scoring these transitions determines the precise location and intensity of the RBP interaction and importantly, removes background from contaminating non-crosslinked RNAs derived from fragments of abundant transcripts ( 11, 12).Ī typical challenge for CLIP methods is the low yield of recovered RNA footprints that hovers in the nanogram range and complicates cDNA library construction efforts. Crosslinked 4SU preferentially pairs with guanosine during reverse transcription, which results in a characteristic T-to-C transition (or G-to-A when using 6SG) in the sequenced cDNA. In the case of 4SU, the photoaddition of reactive amino acids leaves a lesion at position 4 of the base and changes its base-pairing properties ( 14). The exocyclic thione group increases the photoreactivity of the base, allowing lower energy UV light (312 ≤ λ ≤ 365 nm) to be used for crosslinking. In PAR-CLIP, cells are incubated prior to crosslinking with modified nucleosides, 4-thiouridine (4SU) or 6-thioguanisine (6SG) that get incorporated into nascent RNAs. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) ( 13). (iv) Sequenced reads are aligned to the genome and high-confidence RBP binding sites are computationally separated from noise derived from fragments of abundant cellular RNAs ( 11, 12). (iii) RNA footprints are converted into cDNA suitable for next generation sequencing. (ii) The crosslinked ribonucleoprotein (RNP) of interest is immunopurified and RNA footprints are isolated. SELEX ( 9, 10), RNA Bind’n'seq ( 5), RNAcompete ( 4).Īll CLIP protocols share the same basic workflow: (i) RNA and interacting proteins are irreversibly crosslinked in intact cells by UV light and the RNA is digested to short footprints by RNases. This in turn reveals critically important details of RBP function, such as the location of its binding site relative to other cis-acting elements or its recognized sequence motifs, which often resemble but may not be identical to high-affinity motifs determined in vitro by e.g. In CLIP approaches, protected footprints of RNA irreversibly crosslinked to interacting RBPs are sequenced to precisely identify RBP binding sites on a target RNA. Crosslinking and Immunoprecipitation (CLIP) ( 7) and its many variants ( 8) address this need by allowing to identify not only the RNA bound by the RBP in living cells, but also its binding sites. RBPs typically recognize their targets at short, degenerate sequence-motifs ( 4–6), complicating prediction of RBP–RNA interactions and calling for systems-wide experimental methods to dissect the complex posttranscriptional gene regulatory networks controlled by RBPs. The importance of posttranscriptional gene regulatory networks is highlighted by the fact that >1500 human genes encode for proteins known or predicted to bind RNA ( 2) and that multiple diseases are associated with RBPs ( 3). RNA binding proteins (RBPs) participate in all processes involving RNA, including RNA biogenesis, processing, modification, translation and finally turnover. The fate of all eukaryotic RNAs is controlled by their protein partners which accompany them from transcription to turnover ( 1). These improvements cut the experimentation by half to 2 days and increases sensitivity by 10–100-fold. It is based on direct ligation of a fluorescently labeled adapter to the 3′end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. ![]() Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks.
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