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.
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Plant Sci
December 2024
Laboratory of molecular biology of tomato, Bioengineering College, Chongqing University, Chongqing 400030, China. Electronic address:
Members of the MT-A70 family are key catalytic proteins involved in mA methylation modifications in plants. They play diverse roles at the posttranscriptional level by regulating RNA secondary structure, selective splicing, stability, and translational efficiency, which collectively affect plant growth, development, and stress responses. In this study, we explored the function of the gene SlMTC, a Class C member of the MT-A70 family, in tomatoes by using CRISPR/Cas9 technology.
View Article and Find Full Text PDFPlant J
October 2024
Basic Forestry and Proteomics Research Center, School of Future Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
Front Plant Sci
July 2024
College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
N-methyladenosine (mA), a well-characterized RNA modification, is involved in regulating multiple biological processes; however, genome-wide identification and functional characterization of the mA modification in legume plants, including soybean ( (L.) Merr.), remains lacking.
View Article and Find Full Text PDFGenome Res
June 2024
Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA;
Although DNA -adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated.
View Article and Find Full Text PDFmSphere
January 2024
Korean Lichen Research Institute, Sunchon National University, Suncheon, South Korea.
In eukaryotes, -methyladenosine (mA) RNA modification plays a crucial role in governing the fate of RNA molecules and has been linked to various developmental processes. However, the phyletic distribution and functions of genetic factors responsible for mA modification remain largely unexplored in fungi. To get insights into the evolution of mA machineries, we reconstructed global phylogenies of potential mA writers, readers, and erasers in fungi.
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