While these RHFs could act indirectly or extrariboso mally, at least a few may influence they the translation of Ty1 RNA. These include ribosome biogenesis factors, Bud21, Hcr1, Loc1, and Puf6, whose absence resulted in decreased Ty1 Gag GFP fusion protein levels despite wild type or increased levels of Ty1 RNA. The RHF Bud21, also known as Utp16, is a component of the small ribosomal subunit processosome that con tains U3 snoRNA. The level of the 40 S subunit is mark edly decreased in a bud21 mutant. Hcr1 encodes eIF3j, a dual function protein involved in translation initiation as a component of translation initiation factor 3 and in processing of 20 S pre rRNA, a precursor of the 40 S subunit. When BUD21or HCR1 is deleted, Gag GFP fusion protein levels are reduced to 44 and 52% of the wild type level, respectively .

however, Ty1 RNA levels are increased 11 fold and 3 fold, re spectively. Thus, Ty1 RNA translation may be very sensitive to mutations that perturb 40 S riboso mal subunit formation because of stable secondary structure within the 5 UTR. Another ribosome biogen esis mutant with reduced 40 S subunit formation, bud22, also has a reduced level of Ty1 Gag protein. however, Ty1 RNA is not increased in bud22 mutants. Moreover, the ratio of p45 Gag to p49 Gag is sig nificantly decreased in a bud22 mutant, but we did not observe an obvious Gag processing defect in the bud21 or hcr1 mutant. Thus, the mechanism by which BUD21 and HCR1 affect Ty1 RNA translation is likely to be different from that of BUD22.

The simplest inter pretation of our findings is that Bud21 and Hcr1 are ne cessary for efficient of Ty1 RNA translation via their roles in ribosome biogenesis, although other models, in cluding indirect effects on Gag synthesis or stability are also consistent with our data. The RHFs Puf6 and Loc1 are required for biogenesis of the 60 S ribosomal subunit. Interestingly, both also bind ASH1 mRNA and mediate its translational repres sion and localization to the bud tip. Another RHF that is required for Ty1 cDNA accumulation, YDL124W, also binds to ASH1 RNA. In contrast to ASH1 mRNA, Ty1 RNA translation may be reduced in puf6 and loc1 mutants. Moreover, Ty1 mRNA is not loca lized to the bud tip like ASH1 mRNA, but it is localized to microscopically distinct cytoplasmic foci known as T bodies or retrosomes.

It is possible that Puf6 and Loc1 promote translation of Ty1 RNA simply via their effects on biogenesis of Batimastat the 60 S subunit. However, Loc1 and Puf6 have been implicated in the localization of spe cific ribosomal protein paralogs and the formation of specialized ribosomes that are required for the regu lated translation of ASH1 mRNA. Based on this model, it is also conceivable that Loc1 and Puf6 are involved in the formation of ribosomes containing spe cific ribosomal paralogs that are necessary for the regu lated translation of Ty1 RNA.