title: Crosslinking Methods to Identify RNA Methyltransferase Targets In Vivo.
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.
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Sara
authors.initials: S
authors.lastname: Haag
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Kretschmer
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Sloan
authors.affiliation: Centre for Biochemistry and Molecular Cell Biology, Institute for Molecular Biology, Georg-August-University, Humboldtallee 23, 37073, Göttingen, Germany. markus.bohnsack@med.uni-goettingen.de.|Göttingen Centre for Molecular Biosciences, Georg-August-University, 37073, Göttingen, Germany. markus.bohnsack@med.uni-goettingen.de.
authors.forename: Markus T
authors.initials: MT
authors.lastname: Bohnsack
mesh_terms: D001374:Azacitidine / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D019295:Computational Biology / Q000379:methods; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013223:Statistics as Topic / Q000379:methods; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D003562:Cytidine; D012313:RNA; D008780:Methyltransferases; D001374:Azacitidine; C016568:5-methylcytidine
keywords: 5-Azacytidine; 5-Methylcytosine; CLIP; CRAC; Methyltransferase; UV crosslinking