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.
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
J A
JA
Bokar
M E
ME
Rath-Shambaugh
R
R
Ludwiczak
P
P
Narayan
F
F
Rottman
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger; D008780:Methyltransferases; C022556:mRNA (2'-O-methyladenosine-N6-)-methyltransferase