Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex.

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.