Mouaikel John, Bujnicki Janusz M, Tazi Jamal, Bordonné Rémy
Institut de Génétique Moléculaire, IFR122 CNRS-UMR5535, 1919 route de Mende, 34000 Montpellier, France.
Nucleic Acids Res. 2003 Aug 15;31(16):4899-909. doi: 10.1093/nar/gkg656.
The Saccharomyces cerevisiae Tgs1 methyltransferase (MTase) is responsible for conversion of the m(7)G caps of snRNAs and snoRNAs to a 2,2,7- trimethylguanosine structure. To learn more about the evolutionary origin of Tgs1 and to identify structural features required for its activity, we performed a structure-function study. By using sequence comparison and phylogenetic analysis, we found that Tgs1 shows strongest similarity to Mj0882, a protein related to a family comprised of bacterial rRNA:m(2)G MTases RsmC and RsmD. The structural information of Mj0882 was used to build a homology model of Tgs1p which allowed us to predict the range of the minimal globular MTase domain and the localization of other residues that may be important for enzyme function. To further characterize functional domains of Tgs1, mutants were constructed and tested for their effects on cell viability, subcellular localization and binding to the small nuclear ribonucleoproteins (snRNPs) and small nucleolar RNPs (snoRNPs). We found that the N-terminal domain of the hypermethylase is dispensable for binding to the common snRNPs and snoRNPs proteins but essential for correct nucleolar localization. Site- directed mutagenesis of Tgs1 allowed also the identification of the residues likely to be involved in the formation of the m7G-binding site and the catalytic center.
酿酒酵母Tgs1甲基转移酶（MTase）负责将snRNA和snoRNA的m（7）G帽转化为2,2,7-三甲基鸟苷结构。为了更深入了解Tgs1的进化起源并确定其活性所需的结构特征，我们进行了一项结构-功能研究。通过序列比较和系统发育分析，我们发现Tgs1与Mj0882具有最强的相似性，Mj0882是一种与由细菌rRNA:m（2）G MTases RsmC和RsmD组成的家族相关的蛋白质。Mj0882的结构信息被用于构建Tgs1p的同源模型，这使我们能够预测最小球状MTase结构域的范围以及其他可能对酶功能重要的残基的定位。为了进一步表征Tgs1的功能结构域，构建了突变体并测试了它们对细胞活力、亚细胞定位以及与小核核糖核蛋白（snRNP）和小核仁核糖核蛋白（snoRNP）结合的影响。我们发现，高甲基化酶的N末端结构域对于与常见的snRNP和snoRNP蛋白结合并非必需，但对于正确的核仁定位至关重要。Tgs1的定点诱变还使我们能够鉴定可能参与m7G结合位点和催化中心形成的残基。
