Abstract
Recent advances in next-generation sequencing have revealed that majority of the human genome is transcribed into long and short RNA (ncRNA) transcripts. Many ncRNAs function by interacting with proteins and forming regulatory complexes. RNA–protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore identifying proteins that interact with ncRNAs is central to deciphering ncRNA functions. Here we describe an RNA–protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example we describe pull-down of proteins interacting with ncRNA XIST, which assists in the recruitment of the polycomb-repressive complex-2 (PRC2) and drives X-chromosomal inactivation.
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References
Carninci P, Kasukawa T, Katayama S et al (2005) The transcriptional landscape of the mammalian genome. Science 309:1559–1563
Zhao J, Sun BK, Erwin J et al (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X-chromosome. Science 322:750–756
Rinn JL, Kertesz M, Wang JK et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323
Hung T, Wang Y, Lin MF, Koegel AK et al (2011) Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nat Genet 43:621–629
Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798
Zhao J, Ohsumi TK, Johnny T et al (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953
Chu C, Zhang QC, da Rocha ST et al (2015) Systematic discovery of Xist RNA binding proteins. Cell 161:404–416
McHugh CA, Chen CK, Chow A et al (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521:232–236
Minajigi A, Froberg JE, Wei C et al (2015) A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science 349 aab2276
Acknowledgements
This work was supported by University of Birmingham and BBSRC MIBTP program.
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Barnes, C., Kanhere, A. (2016). Identification of RNA–Protein Interactions Through In Vitro RNA Pull-Down Assays. In: Lanzuolo, C., Bodega, B. (eds) Polycomb Group Proteins. Methods in Molecular Biology, vol 1480. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6380-5_9
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DOI: https://doi.org/10.1007/978-1-4939-6380-5_9
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6380-5
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