Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Bioinformatic Analysis

Protein–RNA interactions are integral components of posttranscriptional gene regulatory processes including mRNA processing and assembly of cellular architectures. Dysregulation of RNA-binding protein (RBP) expression or disruptions in RBP–RNA interactions underlie a variety of human pathologies and genetic diseases including cancer and neurodegenerative diseases (reviewed in (Cooper et al., Cell 136(4):777–793, 2009; Darnell, Cancer Res Treat 42(3):125–129, 2010; Lukong et al., Trends Genet 24 (8):416–425, 2008)). Recent studies have uncovered only a small proportion of the extensive RBP–RNA interactome in any organism (Baltz et al., Mol Cell 46(5):674–690, 2012; Castello et al., Cell 149(6):1393–1406, 2012; Freeberg et al., Genome Biol 14(2):R13, 2013; Hogan et al., PLoS Biol 6(10):e255, 2008; Mitchell et al., Nat Struct Mol Biol 20(1):127–133, 2013; Tsvetanova et al. PLoS One 5(9): pii: e12671, 2010; Schueler et al., Genome Biol 15(1):R15, 2014; Silverman et al., Genome Biol 15(1):R3, 2014). To expand our understanding of how RBP–RNA interactions govern RNA-related processes, we developed gPAR-CLIP-seq (global photoactivatable-ribonucleoside-enhanced cross-linking and precipitation followed by deep sequencing) for capturing and sequencing all regions of the Saccharomyces cerevisiae transcriptome bound by RBPs (Freeberg et al., Genome Biol 14(2):R13, 2013). This chapter describes a pipeline for bioinformatic analys...
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