YEAST RNA VIROLOGY

我们在酿酒酵母中发现了两种双链RNA病毒(L-A和L-BC)和两种单链RNA复制子(20S和23S)。MdsRNA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，SKI1，2，3，4，6，7和8，通过它们阻止这些复制子对酵母细胞致病的能力。这四个RNA复制子都产生无帽的mRNAs，并且缺乏3‘聚(A)结构。SKI1是一种外切核糖核酸酶，我们发现SKI2、SKI3、SKI6、SKI7和SKI8基因产物可以阻断非Poly(A)mRNAs的翻译。我们发现Ski2p是一种RNA解旋酶，Ski6p与tRNA加工RNase同源，Ski7p与翻译因子EF1pha相似。我们发现，导致M dsRNA丢失的20个染色体基因的突变在60S核糖体亚基中存在缺陷。这些突变被SKI突变抑制，但不能修复60S亚基缺失。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，Ski2slh1双突变体对非Poly(A)mRNA的处理方式与对Poly(A)+mRNA的处理方式相同，具有相同的翻译速率和翻译持续时间(反映相同的mRNA周转率)。Ski2slh1双突变体在30℃下正常生长，dsRNA病毒拷贝数显著降低。该双突变体的mRNA转换率无明显差异。这些结果表明，由于Ski2p和Slh1p(以及与它们合作的其他蛋白质)的协同作用，mRNA的3‘聚(A)结构只需要翻译。核糖体和翻译因子完全能够使用非Poly(A)mRNAs，即使在竞争翻译装置的全部Poly(A)+mRNAs存在的情况下也是如此。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·约翰逊博士已经使L-A病毒结晶，并正在通过X射线结晶学确定其结构。

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.