PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step.

Abstract

RNA binding proteins (RBPs) are responsible for facilitating a wealth of post-transcriptional gene regulatory functions. The role of an RBP on regulated transcripts can be investigated through a pull-down of the RBP and high-throughput sequencing (HTS) of the associated transcripts. Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), is one such pull-down method that isolates, detects, and sequences the cDNA of RBP-associated transcripts. PAR-CLIP relies on a photoactivatable ribonucleoside analogue, 4-thiouridine, to facilitate covalent RNA-protein crosslinks at 365 nm. These crosslinks permit stringent wash conditions and result in T to C mismatch incorporations during reverse transcription, a unique parameter for the computational analysis of high-confidence binding sites. However, until now, RBPs that bind at the 5'-termini of RNAs have been uniquely restricted from the full potential bandwidth of autoradiographic detection and HTS library preparation. The 5'-termini of RNAs are highly modified, including the most common Pol-II derived modification: the 7-methylguanosine (m7G) cap. In the conventional PAR-CLIP protocol, cap-binding proteins protect the m7G cap from the RNase treatment that generates the necessary substrate for autoradiographic detection and 5' adapter ligation-thus occluding entire populations of RNA from visualization and HTS. Here, we introduce decapping-PAR-CLIP or PAR-dCLIP. We incorporate a decapping step into the PAR-CLIP protocol to generate the necessary substrate to sequence m7G capped transcripts. While PAR-dCLIP was originally targeted towards known m7G-cap binding proteins, we argue that all RBP inquiries, and particularly those suspected to regulate translation, should incorporate this decapping step to ensure that all possible populations of bound transcripts are identified.