Publications

• HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA.

  Keith J.M., Ensinger M.J., Moss B.

  A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmeth ... >> More

  A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm-represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm-leads to AdoHcy + m7G(5')pppm6A7-Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000. << Less

  J Biol Chem 253:5033-5039(1978) [PubMed] [EuropePMC]

• Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

  Bokar J.A., Shambaugh M.E., Polayes D., Matera A.G., Rottman F.M.

  The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known ... >> More

  The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism. << Less

  RNA 3:1233-1247(1997) [PubMed] [EuropePMC]

• Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

  Akichika S., Hirano S., Shichino Y., Suzuki T., Nishimasu H., Ishitani R., Sugita A., Hirose Y., Iwasaki S., Nureki O., Suzuki T.

  <i>N</i> ⁶-methyladenosine (m⁶A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m⁶A, <i>N</i> ⁶, 2'-<i>O</i>-dimethyladenosine (m⁶Am) is present at the transcri ... >> More

  <i>N</i> ⁶-methyladenosine (m⁶A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m⁶A, <i>N</i> ⁶, 2'-<i>O</i>-dimethyladenosine (m⁶Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for <i>N</i> ⁶-methylation of m⁶Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m⁶A formation. A transcriptome-wide analysis revealed that <i>N</i> ⁶-methylation of m⁶Am promotes the translation of capped mRNAs. Thus, a cap-specific m⁶A writer promotes translation of mRNAs starting from m⁶Am. << Less

  Science 0:0-0(2018) [PubMed] [EuropePMC]