Methylation individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) was introduced by Hussain et al., (2013) and is a specialized version of iCLIP. miCLIP is designed to determine which RNA nucleotides are methylated by the RNA methyltransferase Nsun2. Nsun2 forms a covalent link with RNA as part of its enzymatic function. Nsun2 cysteine 271 is required to break that link after methylation takes place.
Hussain et al. used a mutant Nsun2 with cysteine converted to alanine. This forms a covalent link between the protein and its specific RNA targets. After these bonds are formed, a typical iCLIP protocol is performed identify the specific binding site down to the nucleotide. This biggest advantage of miCLIP is that it relies on an actual biological interaction, therefore the data it generates are much more reliable. The downside of miCLIP is that it is only useful in specific applications. Only some model systems are amenable to introduction of a mutated protein and only Nsun2-RNA interactions can be detected.
miCLIP Additional Reading
Tuorto, F., Liebers, R., Musch, T., Schaefer, M., Hofmann, S., Kellner, S., Frye, M., Helm, M., Stoecklin, G., and Lyko, F. (2012). RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat. Struct. Mol. Biol. 19, 900-905.
The function of Nsun2 as an RNA methylase is described in this paper.
- Hussain, S., Sajini, A.A., Blanco, S., Dietmann, S., Lombard, P., Sugimoto, Y., Paramor, M., Gleeson, J.G., Odom, D.T., Ule, J., and Frye, M. (2013). NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell. Rep. 4, 255-261.