S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Journal Papers (저널논문_생명과학부)
fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA
- Kim, Baekgyu; Kim, V. Narry
- Issue Date
- Methods, Vol.152, pp.3-11
- CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease DROSHA initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of DROSHA in miRNA maturation and sequence determination, accurate mapping of DROSHA cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo DROSHA cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of DROSHA, which allows precise mapping of the DROSHA processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of DROSHA. fCLIP-seq is a useful tool to obtain transcriptome-wide information on DROSHA activity and can be applied further to investigate other dsRNA-protein interactions.
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