TY  - JOUR
TI  - RNA Cap Methyltransferase Activity Assay
AU  - Trotman, Jackson 
AU  - Schoenberg, Daniel R.
VL  - 8
IS  - 6
PY  - 2018
DA  - 2018/03/20
SP  - e2767
C1  - Bio-protocol 2018;8:e2767
DO  - 10.21769/BioProtoc.2767
UR  - https://doi.org/10.21769/BioProtoc.2767
AB  - Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.
KW  - RNA
KW  - 5’ Cap
KW  - Cap methyltransferase
KW  - RNMT
KW  - Enzyme activity assay
KW  - Subcellular fractionation
KW  - P1 nuclease
KW  - Thin-layer chromatography
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False