YEAST RNA VIROLOGY

酵母RNA病毒学

• 批准号: 6161903
• 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/6161903
• 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 and SKI8 gene products block the translation of non-poly(A) mRNAs. 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 proposed that SKI2, SKI3 and SKI8 block translation of non-poly(A) mRNA by an effect on ribosome biogenesis affecting the interaction of 60S subunits with the 3' poly(A) structure of the mRNA. We have now cloned the SKI6 and SKI7 genes. Ski6p has homology with RNase PH, an enzyme that removes 3' nucleotides from tRNA precursors in a phosphorolytic reaction (like polynucleotide phosphorylase). Ski6-2 mutants are hypersensitive to hygromycin, an indication of involvement of Ski6p in translation. These ski6-2 strains translate non-poly(A) mRNAs 10x better than isogenic wild type cells. They also accumulate a 38S particle with a partially degraded 25S rRNA, providing direct evidence for altered 60S subunits as the cause of the derepressed translation of non-poly(A) mRNAs. Ski7p is highly homologous to translation factor EF1alpha. The ski7 mutants are derepressed for translation of non-poly(A) mRNAs and overexpression of Ski7p results in loss of M dsRNA.

我们描述了两种 dsRNA 病毒（L-A 和 L-BC）和两种 ssRNA 酿酒酵母中的复制子（20S RNA 和 23S RNA）。 M dsRNA 是编码杀手毒素的 L-A 的卫星。 我们发现 7 个染色体基因，SKI1、2、3、4、6、7 和 8，按其能力 防止这些复制子对酵母细胞产生致病性。 这四个 RNA 复制子均产生无帽 mRNA，并且缺少 3' 聚 (A) 结构。 SKI1 是一种特异于无帽 mRNA 的外切核糖核酸酶， 同时我们证明 SKI2、SKI3 和 SKI8 基因产物阻断 非 Poly(A) mRNA 的翻译。 我们发现 20 个基因中的突变 导致 M dsRNA 丢失的染色体基因在 60S 中存在缺陷 核糖体亚基。 这些突变被ski突变抑制 不恢复 60S 亚基缺陷。 我们建议 SKI2、SKI3 和 SKI8 通过效应阻断非多聚腺苷酸 mRNA 的翻译 影响 60S 亚基相互作用的核糖体生物合成 mRNA 的 3' Poly(A) 结构。 我们现在已经克隆了 SKI6 并 SKI7基因。 Ski6p 与 RNase PH 具有同源性，RNase PH 是一种酶，可去除 磷酸解反应中 tRNA 前体的 3' 核苷酸（例如 多核苷酸磷酸化酶）。 Ski6-2突变体对以下物质过敏 潮霉素，表明 Ski6p 参与翻译。 这些 Ski6-2 菌株翻译非 Poly(A) mRNA 的效果比同基因野生菌株好 10 倍 类型细胞。 他们还积累了一个 38S 粒子，其部分 降解的 25S rRNA，为 60S 亚基改变提供直接证据 作为非多聚腺苷酸 mRNA 翻译抑制的原因。 滑雪7p 与翻译因子 EF1alpha 高度同源。 滑雪7突变体 非 Poly(A) mRNA 翻译和过度表达的抑制 Ski7p 的缺失会导致 M dsRNA 的丢失。