The double life of the ribosome: When its protein folding activity supports prion propagation.

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.

Genetic interactions show the importance of rRNA modification machinery for the role of Rps15p during ribosome biogenesis in S. cerevisiae.

Rps15p, an essential ribosomal protein, was previously shown to be critical for nuclear export of small subunit pre-particles. We have designed a synthetic lethal screen in Saccharomyces cerevisiae to identify its genetic partners and further elucidate its role during ribosomal biogenesis. Our screen revealed interactions with mutants affected at various stages during ribosome biogenesis, from early nucleolar steps to nuclear export. Mutations were identified in genes encoding proteins involved in early ribosome biogenesis steps, like the small subunit processome component Utp15p, the 90S pre-ribosome factor Slx9p and the H/ACA snoRNP core protein Nhp2p. In addition, we found a synthetic lethality with BUD23, a gene encoding a methyltransferase involved both in rRNA modification and small subunit nuclear export. Interestingly, deletion of snR36 or snR85, two H/ACA snoRNAs that direct modifications close to Rps15p's binding site on the rRNA, produces mild and opposite effects on growth in an rps15 hypomorphic background. These data uncover an unreported link between a ribosomal protein and rRNA modification machinery.

An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation.

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.

Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae.

When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.

Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting.

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.

Gene overexpression as a tool for identifying new trans-acting factors involved in translation termination in Saccharomyces cerevisiae.

In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.

Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding.

The ribosome is a molecular machine that converts genetic information in the form of RNA, into protein. Recent structural studies reveal a complex set of interactions between the ribosome and its ligands, mRNA and tRNA, that indicate ways in which the ribosome could avoid costly translational errors. Ribosomes must decode each successive codon accurately, and structural data provide a clear indication of how ribosomes limit recruitment of the wrong tRNA (sense errors). In a triplet-based genetic code there are three potential forward reading frames, only one of which encodes the correct protein. Errors in which the ribosome reads a codon out of the normal reading frame (frameshift errors) occur less frequently than sense errors, although it is not clear from structural data how these errors are avoided. Some mRNA sequences, termed programmed-frameshift sites, cause the ribosome to change reading frame. Based on recent work on these sites, this article proposes that the ribosome uses the structure of the codon-anticodon complex formed by the peptidyl-tRNA, especially its wobble interaction, to constrain the incoming aminoacyl-tRNA to the correct reading frame.