The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 801% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.The 5'-terminal cap structure m7GpppX (where X is any nucleotide) is required for efficient mRNA translation and plays a prominent role in translational control. This ubiquitous feature of eukaryotic mRNAs is also important for nuclear events. Precursor mRNA splicing (23, 46) and 3'-end processing (26, 34) are enhanced by the presence of a cap structure. In addition, the cap structure protects the mRNA against 5' exonucleolytic degradation in both the nucleus and the cytoplasm (25,30) and is implicated in nucleocytoplasmic transport (33). The best-characterized role of the cap structure is its stimulatory effect on ribosome binding (for reviews, see references 66 and 82).

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m 7 GMP from 5 capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 533 exoribonuclease specific for cap ؊ RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and ␤-glucuronidase mRNAs in vivo, but only if they lack the 3 poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5 uncapped ends, but translation of the L-A poly(A) ؊ mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap ؉ poly(A) ؊ mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.

The coat protein (Gag) of the double-stranded RNA virus L-A was previously shown to form a covalent bond with the cap structure of eukaryotic mRNAs. Here, we identify the linkage as a phosphoroimidazole bond between the alpha phosphate of the cap structure and a nitrogen in the Gag protein His-154 imidazole side chain. Mutations of His-154 abrogate the ability of Gag to bind to the cap structure, without affecting cap recognition, in vivo virus particle formation from an L-A cDNA clone, or in vitro specific binding and replication of plus-stranded single-stranded RNA. However, genetic analyses demonstrate that His-154 is essential for M1 satellite virus expression.

The eukaryotic mRNA 5' cap structure m7GpppX (where X is any nucleotide) interacts with a number of cellular proteins. Several of these proteins were studied in mammalian, yeast, and drosophila cells and found to be involved in translation initiation. Here we describe a novel cap-binding protein, the coat protein of L-A, a double-stranded RNA virus that is persistently maintained in many Saccharomyces cerevisiae strains. The results also suggest that the coat protein of a related double-stranded RNA virus (L-BC) is likewise a cap-binding protein. Strikingly, in contrast to the cellular cap-binding proteins, the interaction between the L-A virus coat protein and the cap structure is through a covalent bond.All eukaryotic cellular mRNAs (except for organellar mRNAs) possess the 5' structure m7GpppX (where X is any nucleotide), termed cap. The cap structure plays important roles in several cytoplasmic and nuclear processes.