YEAST RNA VIROLOGY

酵母RNA病毒学

• 批准号: 6432069
• 负责人: Reed B. WICKNER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432069
• 关键词: RNA virus; double stranded RNA; gene deletion mutation; gene mutation; genetic translation; messenger RNA; molecular cloning; nucleic acid sequence; virus RNA; virus genetics; virus protein; virus replication

We have described two dsRNA viruses (L-A and L-BC) and two ssRNA replicons (20S RNA and 23S RNA) in the yeast Saccharomyces cerevisiae. M dsRNA is a satellite of L-A encoding the killer toxin. We discovered 7 chromosomal genes, SKI1, 2, 3, 4, 6, 7, and 8, by their ability to prevent these replicons from causing pathogenicity to yeast cells. These four RNA replicons all make uncapped mRNAs and lack a 3' poly(A)structure. SKI1 is an exoribonuclease specific for uncapped mRNAs, while we showed that the SKI2, SKI3, SKI6, SKI7 and SKI8 gene products block the translation of non-poly(A) mRNAs. We showed that Ski2p is an RNA helicase, Ski6p has homology to a tRNA - processing RNAse, and Ski7p is similar to translation factor EF1alpha. We showed that mutations in 20 chromosomal genes resulting in loss of M dsRNA are deficient in 60S ribosomal subunits. These mutations are suppressed by ski mutations without restoration of the 60S subunit deficiency. We are now studying another yeast gene, called SLH1, that is homologous to SKI2. We find that a ski2 slh1 double mutant treats non-poly(A) mRNA the same as it treats poly(A)+ mRNA, with the same rate of translation and the same duration of translation (reflecting the same mRNA turnover rate). The ski2 slh1 double mutant grows at a normal rate at 30C, and is greatly derepressed for dsRNA virus copy number. No difference was detected in mRNA turnover rate in this double mutant. These results imply that the 3' poly(A) structure of mRNA is only needed for translation because of the cooperating Ski2p and Slh1p (and other proteins that work with them). The ribosomes and translation factors are fully able to use non-poly(A) mRNAs even in the presence of a full complement of poly(A)+ mRNAs competing for the translation apparatus. We are now studying the mechanism of the effects of these genes on translation. Our collaborator, Dr. John E. Johnson, has crystallized the L-A virus and is determining its structure by X-ray crystallography.

我们已经描述了两个dsRNA病毒（L-A和L-BC）和两个ssRNA复制子（20 S RNA和23 S RNA）在酵母酿酒酵母。 M dsRNA是L-A的一个卫星，编码杀手毒素。 我们发现了7个染色体基因，SKI 1，2，3，4，6，7和8，通过它们的能力，以防止这些复制子引起致病性的酵母细胞。 这四种RNA复制子都产生未加帽的mRNA，并且缺乏3' poly（A）结构。 SKI 1是一种对未加帽mRNA具有特异性的核糖核酸外切酶，而我们发现SKI 2、SKI 3、SKI 6、SKI 7和SKI 8基因产物阻断了非poly（A）mRNA的翻译。 我们发现Ski 2 p是一种RNA解旋酶，Ski 6p与tRNA加工RNA酶具有同源性，Ski 7 p与翻译因子EF 1 α相似。 我们发现，20个染色体基因突变导致的损失M双链RNA是缺乏60 S核糖体亚基。 这些突变被ski突变抑制，而不恢复60 S亚基缺陷。 我们现在正在研究另一个酵母基因，称为SLH 1，它与SKI 2同源。 我们发现，一个ski 2 slh 1双突变体处理非poly（A）mRNA与它处理poly（A）+ mRNA相同，具有相同的翻译速率和相同的翻译持续时间（反映了相同的mRNA周转率）。ski 2 slh 1双突变体在30 ℃下以正常速率生长，并且对dsRNA病毒拷贝数大大去抑制。 在这种双突变体中，mRNA周转率没有差异。 这些结果意味着mRNA的3' poly（A）结构只需要翻译，因为Ski 2 p和Slh 1 p（以及与它们一起工作的其他蛋白质）相互协作。 核糖体和翻译因子完全能够使用非poly（A）mRNA，即使在存在竞争翻译装置的poly（A）+mRNA的完整互补物的情况下。 我们现在正在研究这些基因对翻译的影响机制。 我们的合作者，约翰·E。约翰逊已经使L-A病毒结晶，并正在用X射线晶体学确定其结构。