Partial purification and characterization of mRNA (guanine-7-) methyltransferase from the yeast Saccharomyces cerevisiae.

As a tool for the study of the capping-methylation process of yeast mRNA, we developed a procedure for the purification of the mRNA (guanine-7-)methyltransferase using the commercial cap analog guanosine(5')triphospho(5')guanosine as a substrate and radioactive S-adenosylmethionine (AdoMet) as the methyl group donor. The osmotic-sensitive yeast strain VY 1160 was used as the enzyme source. Little methyltransferase activity was detectable in a crude lysate obtained after osmotic shock. We showed that this was due to the presence of a low-molecular-weight inhibitor which could easily be eliminated by Sephadex G-25 gel filtration. The 10000 X g supernatant from the crude lysate was submitted to DEAE-cellulose and DNA-agarose chromatography. The resulting preparation was enriched about 450-fold in specific activity. Under standard assay conditions, the incorporation rate remained constant for at least 6 h at 30 degrees C. Transmethylation was not stimulated by KCl nor NaCl. Divalent cations were strong inhibitors. The partially purified enzyme was able to methylate undermethylated poly(A)-rich mRNA isolated from an AdoMet auxotrophic yeast strain briefly exposed to AdoMet-free medium.