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。SLH1是一种与SKI2同源的RNA解旋酶。我们发现Ski2p和Slh1p具有重叠功能，并共同阻断非poly(A) mRNA的翻译。ski2 slh1双突变体处理非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。ski2 slh1双突变体在mRNA转换率上没有可检测到的差异。因此，由于Ski2p和Slh1p（以及与它们协同作用的其他蛋白质）的协同作用，mRNA的3' poly(A)结构仅用于翻译。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。翻译中3' poly(A)作用的标准模型是poly(A)结合蛋白（Pab1p）与起始因子4G （eIF4G）相互作用，通过使mRNA循环，促进40S亚基募集。然而，我们发现消除Pab1p - eIF4G相互作用并不影响对3' poly(A)的翻译要求，表明该模型是不正确的。然而，Fun12p（一种参与60S核糖体亚基连接的蛋白）的消除会优先损害poly(a)+ mRNA的翻译，而不会显著影响poly(a)- mRNA的翻译。这说明3′聚(A)结构在60S连接反应中起作用。我们的合作者，博士。Hisashi Naitoh和John E. Johnson已经将L-A病毒结晶，并通过x射线晶体学确定了其结构，分辨率为3.4埃。L A dsRNA病毒直径为400埃，包含60个不对称外壳蛋白二聚体的单个蛋白壳，这是dsRNA病毒内部蛋白壳的共同特征，可能与它们独特的转录和复制模式有关。每个二聚体中的两个相同的Gag分子处于不相等的环境中，并且在特定的表面区域显示出本质上不同的构象。这种病毒使细胞mRNA脱帽以表达其自身的未脱帽mRNA。我们的结构揭示了脱帽反应活性部位的沟槽，并表明附近残基在反应中的作用。

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. SLH1 is an RNA helicase homologous to SKI2. We showed that Ski2p and Slh1p have overlapping function, and together block the translation of non-poly(A) mRNA. 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. The ski2 slh1 double mutant has no detectable difference in mRNA turnover rate. Thus the 3' poly(A) structure of mRNA is only needed for translation because of the cooperating action of 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. The standard model of 3' poly(A) action in translation is that interaction of the poly(A) binding protein (Pab1p) with initiation factor 4G (eIF4G), by circularizing the mRNA, promotes 40S subunit recruitment. However, we find that elimination of the Pab1p - eIF4G interaction does not affect the requirement of translation for the 3' poly(A), showing that this model is incorrect. However, elimination of Fun12p, a protein involved in 60S ribosomal subunit joining, preferentially impairs translation of poly(A)+ mRNA without significantly affecting translation of poly(A)- mRNA. This indicates that the 3' poly(A) structure has a role in the 60S joining reaction. Our collaborators, Drs. Hisashi Naitoh and John E. Johnson, have crystallized the L-A virus and determined its structure to 3.4 angstroms resolution by X-ray crystallography. The L A dsRNA virus is 400 angstroms in diameter, and contains a single protein shell of 60 asymmetric dimers of the coat protein, a feature common among the inner protein shells of dsRNA viruses, and probably related to their unique mode of transcription and replication. The two identical Gag molecules in each dimer are in non-equivalent environments, and show substantially different conformations in specific surface regions. This virus decaps cellular mRNA in order to express its own uncapped mRNA. Our structure reveals a trench at the active site of the decapping reaction and suggests a role for nearby residues in the reaction.