Mao X, Schwer B, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Mol Cell Biol. 1995 Aug;15(8):4167-74. doi: 10.1128/MCB.15.8.4167.
RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.
RNA（鸟嘌呤-7-）甲基转移酶是一种负责对真核生物mRNA的5'帽结构进行甲基化的酶，它是从酿酒酵母提取物中分离得到的。这种酵母酶催化甲基基团从S-腺苷-L-甲硫氨酸转移到带帽的未甲基化多聚腺苷酸的鸟嘌呤碱基上。帽甲基化受到低浓度盐的刺激，并被反应的推定产物S-腺苷-L-高半胱氨酸抑制，但不受S-腺苷-D-高半胱氨酸抑制。甲基转移酶在甘油梯度中沉降为一个单一的离散组分，沉降系数为3.2S。基于系统发育学原因，筛选出了一个可能编码酵母帽甲基转移酶的基因。位于酵母II号染色体上的ABD1基因编码一种436个氨基酸（50 kDa）的多肽，该多肽与痘苗病毒帽甲基转移酶的催化结构域具有区域相似性。通过在细菌中表达ABD1蛋白、将该蛋白纯化至同质，并对重组ABD1固有的帽甲基转移酶活性进行表征，确定了ABD1基因产物确实是RNA（鸟嘌呤-7-）甲基转移酶。重组ABD1甲基转移酶的物理和生化特性与从全细胞酵母提取物中分离和部分纯化得到的帽甲基转移酶的特性无法区分。我们发现ABD1基因是酵母生长所必需的，这提供了首个遗传学证据，表明帽甲基转移酶（以及由此推断的帽甲基基团）在体内细胞功能中起着至关重要的作用。
