Abstract
Several crosslinking methods have been developed to identify interacting RNAs for proteins of interest. Here, we describe variants of the UV crosslinking and analysis of cDNA (CRAC) method that allow target identification of RNA methyltransferases on a genome-wide scale. We present a detailed protocol for the application of CRAC in human cells that stably express the protein of interest fused to a tandem affinity tag. After the introduction of a covalent link between the protein and its target RNAs, protein-RNA complexes are purified and bound RNAs trimmed, ligated to adapters, reverse transcribed, and amplified. Sequences obtained from next-generation sequencing are then mapped onto the human genome allowing the identification of possible substrates. For some RNA methyltransferases, e.g., m5C MTases, their catalytic mechanism can be exploited for chemical crosslinking approaches instead of UV based crosslinking.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1784: BO3442/2-1 to M.T.B.), the Alexander von Humboldt Foundation (K.E.S. and M.T.B.), and the Faculty of Medicine, Georg-August-University Göttingen (M.T.B. and “Startförderung” to S.H.).
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Haag, S., Kretschmer, J., Sloan, K.E., Bohnsack, M.T. (2017). Crosslinking Methods to Identify RNA Methyltransferase Targets In Vivo. In: Lusser, A. (eds) RNA Methylation. Methods in Molecular Biology, vol 1562. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6807-7_18
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DOI: https://doi.org/10.1007/978-1-4939-6807-7_18
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