Characterization of human, Schizosaccharomyces pombe, and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes
Characterization of human, Schizosaccharomyces pombe, and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes
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DOI：
10.1074/jbc.274.23.16553
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发表时间：
1999-06-04
影响因子：
4.8
通讯作者：
Shuman, S
中科院分区：
文献类型：
--
作者：
Saha, N;Schwer, B;Shuman, S
基于人（Hcm 1 p; 476个氨基酸）和粟酒裂殖酵母（Pcm 1 p; 389个氨基酸）多肽与酿酒酵母（Saccharomyces cerevisiae）的帽甲基转移酶（Abd 1 p）的相似性，鉴定了编码mRNA（鸟嘌呤-N7）甲基转移酶的人和裂殖酵母cDNA。酿酒酵母补充了ABD 1基因缺失导致的致死表型，正如NH 2-末端缺失突变体PCM 1（94-389）和HCM 1（121-476）的表达一样。（Ccm1p; 474个氨基酸）通过选择S的条件生长表型的互补从白色念珠菌基因组文库中分离，酿酒酵母abd 1-ts突变体。人帽甲基转移酶在细菌中表达，纯化和表征。重组Hcm 1 p催化S-腺苷甲硫氨酸依赖性的GpppA-封端的poly（A）向m7 GpppA-封端的poly（A）的定量转化，我们通过丙氨酸扫描诱变鉴定了8个氨基酸（Asp-203、Gly-207、Asp-211、Asp-227、Arg-239、Tyr-289、Phe-291和Phe-354），其对于人cap甲基转移酶在体内的功能是必需的，所有八个残基在其他细胞帽甲基转移酶中是保守的。在细菌中表达了五种突变的人类蛋白质（D203 A、R239 A、Y289 A、F291 A和F354 A），并发现其在体外帽甲基化中存在缺陷。Hcm 1 p、Abd 1 p和牛痘加帽酶突变效应的一致性强调了DNA病毒、酵母和后生动物中帽甲基化的保守结构基础，这与酵母和后生动物加帽系统的RNA三磷酸酶组分的结构和机制分歧形成对比。然而，我们证明了整个三组分酵母帽装置，包括RNA 5 '-三磷酸酶（Cet 1 p），RNA鸟苷酰转移酶（Ceg 1 p），Abd 1 p在体内可被双功能三磷酸酶-鸟苷酰转移酶Mce 1 p和甲基转移酶Hcm 1组成的双组分哺乳动物装置所取代（121-476）具有真菌与哺乳动物帽系统的同基因酵母菌株应有助于合理筛选靶向体内帽形成的抗真菌药物。
Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p), Expression of PCM1 or HCM1 in S. cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH2-terminal deletion mutants PCM1(94-389) and HCM1(121-476), The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C, albicans genomic library by selection for complementation of the conditional growth phenotype of S, cerevisiae abd1-ts mutants. Human cap methyltransferase was expressed in bacteria, purified, and characterized. Recombinant Hcm1p catalyzed quantitative S-adenosylmethionine-dependent conversion of GpppA-capped poly(A) to m7GpppA-capped poly(A), We identified by alanine-scanning mutagenesis eight amino acids (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, Phe-291, and Phe-354) that are essential for human cap methyltransferase function in vivo, All eight residues are conserved in other cellular cap methyltransferases. Five of the mutant human proteins (D203A, R239A, Y289A, F291A, and F354A) were expressed in bacteria and found to be defective in cap methylation in vitro. Concordance of mutational effects on Hcm1p, Abd1p, and vaccinia capping enzyme underscores a conserved structural basis for cap methylation in DNA viruses, yeast, and metazoans, This is in contrast to the structural and mechanistic divergence of the RNA triphosphatase components of the yeast and metazoan capping systems. Nevertheless, we demonstrate that the entire three-component yeast capping apparatus, consisting of RNA 5'-triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and Abd1p could be replaced in vivo by the two-component mammalian apparatus consisting of a bifunctional triphosphatase-guanylyltransferase Mce1p and the methyltransferase Hcm1(121-476)p. Isogenic yeast strains with fungal versus mammalian capping systems should facilitate rational screens for antifungal drugs that target cap formation in vivo.