Bokar J A, Shambaugh M E, Polayes D, Matera A G, Rottman F M
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
RNA. 1997 Nov;3(11):1233-47.
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.
真核生物mRNA内部腺苷残基的甲基化形成N6-甲基腺苷（m6A），这一过程由一种复杂的多组分酶催化。先前的研究表明，m6A会影响mRNA加工或转运的效率，尽管其发生机制尚不清楚。作为朝着更好地理解这种普遍存在的转录后修饰的机制和功能迈出的一步，我们已经表明，HeLa mRNA（N6-腺苷）-甲基转移酶至少需要两个独立的蛋白质因子，MT-A和MT-B，并且MT-A在一个70 kDa的亚基（MT-A70）上含有AdoMet结合位点。MT-A70通过常规色谱法和电泳进行纯化，并进行了微量测序。该肽序列被用于设计简并寡脱氧核苷酸，进而用于从HeLa cDNA文库中分离编码MT-A70的cDNA克隆。重组MT-A70在细菌中作为融合蛋白表达，并用于在兔中产生抗MT-A70抗血清。这些抗血清通过蛋白质印迹法识别HeLa核提取物中的MT-A70，并且能够从HeLa核提取物中耗尽（N6-腺苷）-甲基转移酶活性，证实MT-A70是（N6-腺苷）-甲基转移酶的关键亚基。Northern印迹分析显示，MT-A70 mRNA存在于多种人类组织中，并且可能经历可变剪接。MT-A70 cDNA探针与从HeLa细胞中分离的2.0千碱基（kb）聚腺苷酸化RNA杂交，而使用从六种不同人类组织中分离的mRNA时，它与两种主要的RNA种类（约2.0 kb和3.0 kb）杂交。对cDNA序列的分析表明，它编码一个580个氨基酸的蛋白质，预测分子量= 65 kDa。预测的蛋白质包含与原核DNA（N6-腺苷）-甲基转移酶中鉴定的共有甲基化基序I和II相似的序列，表明肽基序的功能保守性。MT-A70还包含与酵母蛋白SPO8的一个长同源区域，SPO8通过未知机制参与孢子形成的诱导。
