Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.
Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.
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发表时间：
1997-11
期刊：
影响因子：
4.5
通讯作者：
J. Bokar;M. E. Shambaugh;D. Polayes;A. Matera;F. Rottman
中科院分区：
文献类型：
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作者：
J. Bokar;M. E. Shambaugh;D. Polayes;A. Matera;F. Rottman
真核生物mRNA内部腺苷残基的甲基化，形成N6-甲基腺苷（m6 A），是由复杂的多组分酶催化的。先前的研究表明，m6 A影响mRNA加工或转运的效率，尽管这种情况发生的机制尚不清楚。作为更好地理解这种普遍存在的转录后修饰的机制和功能的一步，我们已经表明，HeLa mRNA（N6-腺苷）-甲基转移酶需要至少两个单独的蛋白质因子，MT-A和MT-B，MT-A包含一个70 kDa的亚基（MT-A70）上的C3 Met结合位点。MT-A70通过常规层析和电泳纯化，并进行微测序。肽序列用于设计简并寡脱氧核苷酸，该简并寡脱氧核苷酸又用于从HeLa cDNA文库中分离编码MT-A70的cDNA克隆。重组MT-A70在细菌中表达为融合蛋白，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识别HeLa细胞核提取物中的MT-A70，并且能够从HeLa细胞核提取物中消除（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。北方印迹分析表明，MT-A70 mRNA存在于各种各样的人类组织中，并可能经历选择性剪接。MT-A70 cDNA探针与从HeLa细胞中分离的2.0-腺苷酸酶（kb）聚腺苷酸化RNA杂交，而使用从六种不同人体组织中分离的mRNA，它与两种主要RNA种类（约2.0 kb和3.0 kb）杂交。cDNA序列的分析表明，它编码一个580个氨基酸的蛋白质，预测MW = 65 kDa。预测的蛋白质包含类似于在原核DNA（N6-腺苷）-甲基转移酶中鉴定的一致甲基化基序I和II的序列，这表明肽基序的功能保守。MT-A70还含有与酵母蛋白SPO 8同源的长区域，其通过未知机制参与孢子形成的诱导。
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.