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.

我们已经描述了两种DSRNA病毒（L-A和L-BC）和两个酿酒酵母中的两个SSRNA复制子（20S RNA和23S RNA）。 M dsRNA是L-A编码杀手毒素的卫星。 我们通过防止这些复制子引起致病性到酵母细胞的能力，发现了7种染色体基因SKI1、2、2、3、4、6、7和8。 这四个RNA复制子都形成了未封闭的mRNA，并且缺乏3'poly（a）结构。 SKI1是一种针对非封闭mRNA的驱齿核酸酶，而我们表明SKI2，SKI3，SKI6，SKI7，SKI7和SKI8基因产品阻止了非Poly（a）mRNA的翻译。 我们表明SKI2P是一种RNA解旋酶，SKI6P与tRNA的RNase具有同源性，而SKI7P类似于翻译因子EF1alpha。 我们表明，在60s核糖体亚基中，导致M dsRNA损失的20种染色体基因的突变缺乏。 这些突变被滑雪突变抑制，而没有恢复60年代亚基缺乏症。 我们现在正在研究另一个称为SLH1的酵母基因，该基因与SKI2同源。 我们发现SKI2 SLH1双重突变体处理非Poly（a）mRNA与处理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转换率）。 SKI2 SLH1双重突变体以正常速率在30℃下生长，并且对DSRNA病毒拷贝数大大消除。 该双突变体中的mRNA周转率未检测到差异。 这些结果表明，由于SKI2P和SLH1P（以及与它们一起使用的其他蛋白质），仅需要翻译mRNA的3'poly（a）结构。 核糖体和翻译因子也完全能够使用非聚（a）mRNA，即使在有完整的poly（a）+ mRNA竞争转化设备的互补的情况下。 我们现在正在研究这些基因对翻译的影响的机制。 我们的合作者John E. Johnson博士已经结晶了L-A病毒，并通过X射线晶体学确定了其结构。