Ja. Bokar et al., PURIFICATION AND CDNA CLONING OF THE ADOMET-BINDING SUBUNIT OF THE HUMAN MESSENGER-RNA (N-6-ADENOSINE)-METHYLTRANSFERASE, RNA, 3(11), 1997, pp. 1233-1247
The methylation of internal adenosine residues in eukaryotic mRNA, for
ming N-6-methyladenosine (m(6)A), is catalyzed by a complex multicompo
nent enzyme. Previous studies suggested that m(6)A affects the efficie
ncy of mRNA processing or transport, although the mechanism by which t
his occurs is not known. As a step toward better understanding the mec
hanism and function of this ubiquitous posttranscriptional modificatio
n, we have shown that HeLa mRNA (N-6-adenosine)-methyltransferase requ
ires at least two separate protein factors, MT-A and MT-B, and MT-A co
ntains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 wa
s 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 c
oding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expr
essed as a fusion protein in bacteria and was used to generate anti-MT
-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nucl
ear extracts by western blot and are capable of depleting (N-6-adenosi
ne)-methyltransferase activity from HeLa nuclear extract, confirming t
hat MT-A70 is a critical subunit of (N-6-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 isolat
ed from HeLa cells, whereas it hybridizes to two predominant RNA speci
es (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six diff
erent human tissues. Analysis of the cDNA sequence indicates that it c
odes for a 580-amino acid protein with a predicted MW = 65 kDa. The pr
edicted protein contains sequences similar to consensus methylation mo
tifs I and II identified in prokaryotic DNA (NG-adenosine)-methyltrans
ferases, 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
.