YEAST RNA VIROLOGY

我们在酿酒酵母中描述了两个dsRNA病毒（L-A和L-BC）和两个ssRNA复制子（20S RNA和23S RNA）。M - dsRNA是L-A的卫星，编码杀伤毒素。我们发现了7个染色体基因，SKI1、2、3、4、6、7和8，它们能够阻止这些复制子对酵母细胞产生致病性。这四种RNA复制子都产生无帽mrna，并且缺乏3' poly(a)结构。SKI1是一种针对无帽mrna的外核糖核酸酶，而我们发现SKI2、SKI3、SKI6、SKI7和SKI8基因产物阻断了非聚(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，Ski7p与翻译因子EF1alpha相似。我们发现，导致M dsRNA缺失的20个染色体基因突变是60S核糖体亚基缺陷。这些突变被ski突变所抑制，而60S亚基缺陷无法恢复。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双突变体对非poly(a) mRNA的处理与对poly(a)+ mRNA的处理相同，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）。sk2slh1双突变体在30C条件下以正常速率生长，dsRNA病毒拷贝数显著降低。该双突变体的mRNA转换率无显著差异。这些结果表明，mRNA的3' poly(A)结构只需要翻译，因为协同的Ski2p和Slh1p（以及与它们一起工作的其他蛋白质）。核糖体和翻译因子完全能够使用非多聚(A) mrna，即使存在完整的多聚(A)+ mrna竞争翻译装置。我们目前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·约翰逊博士，已经将L-A病毒结晶并正在用x射线晶体学测定其结构。

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.