paralyzer definition of rna binding sites from par-clip short-read sequence data阻滞剂rna结合位点的定义从par-clip短内容序列数据.pdf

Corcoran et al. Genome Biology 2011, 12:R79 /2011/12/8/R79 METHOD Open Access PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data 1† 1,2† 1 3,4 5 David L Corcoran , Stoyan Georgiev , Neelanjan Mukherjee , Eva Gottwein , Rebecca L Skalsky , Jack D Keene5 and Uwe Ohler1,6* Abstract Crosslinking and immunoprecipitation (CLIP) protocols have made it possible to identify transcriptome-wide RNA- protein interaction sites. In particular, PAR-CLIP utilizes a photoactivatable nucleoside for more efficient crosslinking. We present an approach, centered on the novel PARalyzer tool, for mapping high-confidence sites from PAR-CLIP deep-sequencing data. We show that PARalyzer delineates sites with a high signal-to-noise ratio. Motif finding identifies the sequence preferences of RNA-binding proteins, as well as seed-matches for highly expressed microRNAs when profiling Argonaute proteins. Our study describes tailored analytical methods and provides guidelines for future efforts to utilize high-throughput sequencing in RNA biology. PARalyzer is available at http:// /labs/ohler/research/PARalyzer/. Background RNA-protein interactions occur within a transcript. The RNA binding proteins (RBPs) play important roles in the second method, CLIP, typically uses short wave UV life cycle of a transcript, from its nascence by RNA poly- 254 nm crosslinking followed by immunoprecipitation and merase until its decay by RNases. All steps of RNA proces- partial RNase digestion of the bound transcript. Conver- sing and function, including splicing, nuclear export,