YEAST RNA VIROLOGY

我们在酿酒酵母中描述了两种 dsRNA 病毒（L-A 和 L-BC）和两种 ssRNA 复制子（20S RNA 和 23S RNA）。 M dsRNA 是编码杀手毒素的 L-A 的卫星。 我们发现了 7 个染色体基因：SKI1、2、3、4、6、7 和 8，因为它们能够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，并且缺乏 3' 聚 (A) 结构。 SKI1 是一种对未加帽 mRNA 具有特异性的外核糖核酸酶，而我们发现 SKI2、SKI3、SKI6、SKI7 和 SKI8 基因产物可阻断非 Poly(A) mRNA 的翻译。 我们发现 Ski2p 是一种 RNA 解旋酶，Ski6p 与 tRNA 加工 RNAse 具有同源性，而 Ski7p 与翻译因子 EF1alpha 相似。 我们发现，导致 M dsRNA 丢失的 20 个染色体基因突变导致 60S 核糖体亚基缺陷。 这些突变被ski突变抑制，但不恢复60S亚基缺陷。 我们现在正在研究另一种酵母基因，称为 SLH1，它与 SKI2 同源。 我们发现ski2 slh1双突变体对待非poly(A) mRNA的方式与对待poly(A) mRNA的方式相同，具有相同的翻译速率和相同的翻译持续时间（反映相同的mRNA周转率）。 Ski2 slh1 双突变体在 30°C 下以正常速率生长，并且 dsRNA 病毒拷贝数大大降低。 在该双突变体中未检测到 mRNA 周转率存在差异。 这些结果表明，由于 Ski2p 和 Slh1p（以及与它们一起工作的其他蛋白质）的协作，mRNA 的 3' Poly(A) 结构仅在翻译时才需要。 即使存在与翻译装置竞争的全部 Poly(A) mRNA ，核糖体和翻译因子也完全能够使用非 Poly(A) mRNA。 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 John E. Johnson 博士已使 L-A 病毒结晶，并正在通过 X 射线晶体学确定其结构。

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.