The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of 'cross-linking and immunoprecipitation' (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (chi CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the chi CRAC method and presents recent results obtained with the Nrd1 RBP.
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NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
NYU, Sch Med, Dept Biochem, New York, NY 10016 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Beck, David B.
Narendra, Varun
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NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
NYU, Sch Med, Dept Biochem, New York, NY 10016 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Narendra, Varun
Drury, William J., III
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NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
NYU, Sch Med, Dept Biochem, New York, NY 10016 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Drury, William J., III
Casey, Ryan
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NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
NYU, Sch Med, Dept Biochem, New York, NY 10016 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Casey, Ryan
Jansen, Pascal W. T. C.
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Univ Med Ctr Utrecht, Dept Mol Canc Res, NL-3584 CG Utrecht, NetherlandsNYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Jansen, Pascal W. T. C.
Yuan, Zuo-Fei
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Univ Penn, Perelman Sch Med, Epigenet Program, Dept Biochem & Biophys, Philadelphia, PA 19103 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Yuan, Zuo-Fei
Garcia, Benjamin A.
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Univ Penn, Perelman Sch Med, Epigenet Program, Dept Biochem & Biophys, Philadelphia, PA 19103 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Garcia, Benjamin A.
Vermeulen, Michiel
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Univ Med Ctr Utrecht, Dept Mol Canc Res, NL-3584 CG Utrecht, NetherlandsNYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
Vermeulen, Michiel
Bonasio, Roberto
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Univ Penn, Perelman Sch Med, Epigenet Program, Dept Cell & Dev Biol, Philadelphia, PA 19103 USANYU, Sch Med, Howard Hughes Med Inst, New York, NY 10016 USA
机构:
GAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes InstituteGAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes Institute
Ka-Yun Ban
Yong-woo Na
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Seoul National University,School of Biological SciencesGAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes Institute
Yong-woo Na
Juhan Song
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GAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes InstituteGAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes Institute
Juhan Song
Jong-Seo Kim
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Seoul National University,School of Biological SciencesGAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes Institute
Jong-Seo Kim
Jimi Kim
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GAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes InstituteGAIHST,Department of Health Science and Technology, Lee Gil Ya Cancer and Diabetes Institute