Comprehensive Identification of mRNA Polyadenylation Sites by PAPERCLIP

Methods Mol Biol. 2017:1648:79-93. doi: 10.1007/978-1-4939-7204-3_7.

Abstract

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.

Keywords: Crosslinking immunoprecipitation; Poly(A)-binding protein; mRNA polyadenylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Library*
  • Humans
  • Immunoprecipitation / methods*
  • Poly(A)-Binding Proteins / chemistry*
  • Polymerase Chain Reaction / methods*
  • RNA 3' Polyadenylation Signals*
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • Sequence Analysis, RNA / methods*

Substances

  • Poly(A)-Binding Proteins
  • RNA, Messenger