Comprehensive Identification of mRNA Polyadenylation Sites by PAPERCLIP

We discuss a newly developed method to profile mRNA polyadenylation (pA) sites in an unbiased manner, PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3'End Retrieval by CrossLinking ImmunoPrecipitation). Based on the well-established CLIP (crosslinking immunoprecipitation) technique, PAPERCLIP utilizes the poly(A) binding protein (PABP) as a biological filter to selectively retrieve mRNA 3' end fragments by immunoprecipitation from ultraviolet (UV) irradiated tissues or cultured cells. The mRNA fragments are subsequently extracted from the immunoprecipitated PABP:RNA complexes to generate a cDNA library, which goes through two rounds of purification before the final amplification by real-time polymerase chain reaction (PCR). The amplified cDNA library can then be read out by high-throughput sequencing to generate a transcriptomic profile and comprehensive alternative poly(A) (APA) site map from intact tissue or cultured cells.