Hafner, M; Katsantoni, M; Köster, T; Marks, J; Mukherjee, J; Staiger, D; Ule, J; Hafner, M; Katsantoni, M; Köster, T; Marks, J; Mukherjee, J; Staiger, D; Ule, J; Zavolan, M; - view fewer (2021) CLIP and complementary methods. Nature Reviews Methods Primers , 1 (1) , Article 20. 10.1038/s43586-021-00018-1.

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Abstract

RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications.

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