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编码一种对无帽mrna特异性的外核糖核酸酶，而我们发现SKI2、SKI3、SKI6、SKI7和SKI8基因产物阻断非多聚(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。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。尽管体外实验中酵母的polyA结合蛋白是对poly(A)+ mRNA提取物偏好的必要因素，但我们发现，尽管PAB1基因缺失，电穿孔细胞仍然偏爱poly(A)+ mRNA，其因素与等基因野生型细胞相同。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。我们现在正在研究poly(A)在翻译中的作用以及与Ski蛋白的关系。

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 encodes 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. 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. Although the polyA binding protein of yeast is necessary for the preference of extracts for poly(A)+ mRNA in vitro, we find that in spite of deletion of the PAB1 gene, electroporated cells still prefer poly(A)+ mRNAs by the same factor as in isogenic wild-type cells. 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 suggests that the 3' poly(A) structure has a role in the 60S joining reaction. We are now pursuing an understanding of the role of poly(A) in translation and the relation to the Ski proteins. Mak3p is an N-acetyl transferase that acetylates the N-terminus of the L?A major coat protein (Gag). We find that a homologous protein from Arabidopsis is able to substitute for the yeast Mak3p. While yeast Mak10p is also necessary for this reaction in yeast, we find that the Arabidopsis Mak3p can substitute for Mak10p as well as Mak3p. Our collaborators, Drs. Hisashi Naitoh and John E. Johnson, determined the structure of the L-A virus at 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. 7methylGDP bound to viral particles shows a density in the trench near His154, the residue to which the cap is attached in this reaction. We have now shownn that Tyr150, Tyr 452 and Tyr 538 are essential for the decapping reaction. Positively charged residues in this region are also important. These experiments have permitted proposal of a mechanism of the decapping reaction.