Abstract
With the many advances in genome-wide sequencing, it has been discovered that much more of the genome is transcribed into RNA than previously appreciated. These nonprotein-coding RNAs (ncRNAs) come in many different forms, and they have been shown to have a variety of functions within the cell, influencing processes such as gene expression, mRNA splicing, and transport, just as a few examples. As we delve deeper into studying their mechanisms of action, it becomes important to understand how they play these roles, in particular by understanding what proteins these ncRNAs interact with. This protocol describes one technique that can be used to study this, ultra-violet light cross-linking RNA immunoprecipitation (UV-RIP), which uses an antibody to pull down a specific protein of interest and then detects RNA that is bound to it. This technique utilizes UV light to cross-link the cells, which takes advantage of the fact that UV light will only cross-link proteins and nucleic acids that are directly interacting. This approach can provide key mechanistic insight into the function of these newly identified ncRNAs.
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References
Hockensmith JW, Kubasek WL, Vorachek WR et al (1986) Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system. J Biol Chem 261(8):3512–3518
Greenberg JR (1979) Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res 6(2):715–732
Brimacombe R, Stiege W, Kyriatsoulis A et al (1988) Intra-RNA and RNA-protein cross-linking techniques in Escherichia coli ribosomes. Methods Enzymol 164:287–309
Darnell RB (2010) HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip Rev RNA 1(2):266–286
Kim TK, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465(7295):182–187
Schaukowitch K, Joo JY, Liu X et al (2014) Enhancer RNA facilitates NELF release from immediate early genes. Mol Cell 56(1):29–42
Lai F, Orom UA, Cesaroni M et al (2013) Activating RNAs associate with mediator to enhance chromatin architecture and transcription. Nature 494(7438):497–501
Ule J, Jensen K, Mele A, Darnell RB (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37(4):376–386
Licatalosi DD, Mele A, Fak JJ et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456(7221):464–469
Huppertz I, Attig J, D'Ambrogio A et al (2014) iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65(3):274–287
Sei E, Conrad NK (2014) UV cross-linking of interacting RNA and protein in cultured cells. Methods Enzymol 539:53–66
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Schaukowitch, K., Joo, JY., Kim, TK. (2017). UV-RNA Immunoprecipitation (UV-RIP) Protocol in Neurons. In: Ørom, U. (eds) Enhancer RNAs. Methods in Molecular Biology, vol 1468. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4035-6_4
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DOI: https://doi.org/10.1007/978-1-4939-4035-6_4
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-4035-6
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