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To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA cross-linking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare complementary DNA (cDNA) libraries for sequencing. Here we describe the core steps of CLIP, its primary variations, and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice and discuss the mechanistic and physiologic insights that have already been gained from studies using CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms while providing important biologic and clinically relevant insights., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

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.

Accurate and precise annotation of 3' UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding protein-mediated mRNA 3' end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3' ends and compares favorably with existing 3' end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3' UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)