Abstract
The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput sequencing followed by bioinformatic analysis, the exact RBP-binding sites can be identified at a single nucleotide resolution. Here, we describe in detail a PAR-CLIP protocol to prepare cDNA libraries for high-throughput sequencing from RNA fragments that are bound to RBPs not only in the nucleus but also in the cytoplasm.
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Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research (B) (25293201), for Scientific Research on Innovative Areas (23112712, 25110719), for Challenging Exploratory Research (25670419), and for Genome Science (221S0002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Y.K.). Q.L. was supported by a JSPS postdoctoral fellowship for foreign researchers from MEXT.
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Li, Q., Uemura, Y., Kawahara, Y. (2015). Cross-Linking and Immunoprecipitation of Nuclear RNA-Binding Proteins. In: Nakagawa, S., Hirose, T. (eds) Nuclear Bodies and Noncoding RNAs. Methods in Molecular Biology, vol 1262. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2253-6_15
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DOI: https://doi.org/10.1007/978-1-4939-2253-6_15
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