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

Abstract

Ns-Methyladenosine is found at internal positions of mRNA in higher eukaryotes.This post-transcriptional modification occurs at a frequency of one to three methylationslaverage 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 NE-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 NEadenosine 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 ZOO-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit.The 875- kDa component has affinity for single-stranded DNAagarose, suggesting that it may cqntain the mRNAbinding site.NE-Adenosine methyl; .*ansferase 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.Post-transcriptional modification of eukaryotic pre-mRNA includes formation of a 5"terminal cap, splicing of introns, polyadenylation, and methylation of specific internal adenosine residues.Enzymes which catalyze cap formation, including both N7-guanosine and 2'-O-methyltransferases, and the complex systems required for splicing and polyadenylation, have been extensively studied (1-6).In contrast, relatively little is known about the enzymatic formation of N6-methyladenosine (m6A)' in mRNA.CA31810 from the National Cancer Institute (to F. M. R.).The costs of