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Chromatin Protocols pp 413–428Cite as

Transcriptome-Wide Identification of In Vivo Interactions Between RNAs and RNA-Binding Proteins by RIP and PAR-CLIP Assays

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1288))

Abstract

Comprehensive genomic and computational studies in the era of high-throughput sequencing revealed that the major proportion of the human genome is transcribed. This novel insight confronted the scientific community with new questions concerning the expanded role of RNA, especially noncoding RNA (ncRNA), in cellular pathways. In recent years, there has been mounting evidence that ncRNAs and RNA binding proteins (RBPs) are involved in a wide range of biological processes, such as developmental transitions, cell differentiation, stress response, genome organization, and regulation of gene expression. In particular, in the chromatin field long noncoding RNAs (lncRNAs) have drawn increasing attention to their function in epigenetic regulation due to the fact that they were found to interact with multiple chromatin regulators and modifiers. Recently, techniques to study the extent of RNA–protein interactions have been developed in many research laboratories. Here we describe protocols for RNA Immunoprecipitation-Sequencing (RIP-Seq) and Photoactivatable-Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation combined with deep sequencing (PAR-CLIP-Seq) to identify RNA targets of RNA-binding proteins (RBPs) on a transcriptome-wide level, discussing advantages and drawbacks.

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Acknowledgements

We acknowledge the technical assistance of Georgina Guerrero Avendaño and Fernando Suaste Olmos. This work was supported by the DGAPA, UNAM (IN209403, IN203811 and IN201114), and CONACyT (42653-Q, 128464 and 220503); Ph.D. fellowship from CONACyT and Dirección General de Estudios de Posgrado-Universidad Nacional Autónoma de México (DGEP) (EG-B and RS-M). Additional support was provided by the PhD Graduate Program, “Doctorado en Ciencias Biomédicas,” to the Instituto de Fisiología Celular and the Universidad Nacional Autónoma de México.

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    Authors and Affiliations

    1. Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, Ciudad de México, DF, 04510, Mexico

      Edgar González-Buendía, Ricardo Saldaña-Meyer, Karin Meier & Félix Recillas-Targa

    2. Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, 10016, USA

      Ricardo Saldaña-Meyer

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    1. Edgar González-Buendía
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    Correspondence to Félix Recillas-Targa .

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    1. H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA

      Srikumar P. Chellappan

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    González-Buendía, E., Saldaña-Meyer, R., Meier, K., Recillas-Targa, F. (2015). Transcriptome-Wide Identification of In Vivo Interactions Between RNAs and RNA-Binding Proteins by RIP and PAR-CLIP Assays. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_24

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    • DOI: https://doi.org/10.1007/978-1-4939-2474-5_24

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-2473-8

    • Online ISBN: 978-1-4939-2474-5

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