Yeast Rna Virology

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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.