Our biochemical analysis of the yeast DRG factors Rbg1 and Rbg2 indicates that they form two distinct complexes. Rbg1 interacts with Tma46 and this complex associates with translating ribosomes, while Rbg2 binds to Gir2. The latter complex does not appear to associate directly with ribosomes. However, it interacts with Gcn1, which itself has been shown to bind to ribosome in specific starvation conditions. Interaction of Rbg1 with Tma46 and Rbg2 with Gir2 in yeast was previously reported but our observation differs significantly from the conclusion reached earlier (26
). It is noteworthy that, in the latter study, an interaction between Rbg1 and Gir2 was detected in a two hybrid assays in which proteins fused to heterologous domains are overexpressed and targeted to the nuclear compartment. These authors confirmed this observation by co-precipitation of tagged proteins, which were again overexpressed in yeast. These data, obtained upon artificial expression conditions, together with the conservation of the DFRP domain, suggest that overexpressed Rbg1 is endowed with the capacity to interact with Gir2 even though this interaction is not detectable in cells probably because Rbg1 has higher affinity for Tma46 while Rbg2 associates preferentially with Gir2, especially when these factors are in competition. Consistent with this interpretation, we observed that joint deletions of rbg2
did not produce a slow growth phenotype when combined with a slh1
deletion (data not shown) supporting the possible formation of a functional Rbg1-Gir2 complex in the absence of competing factors (e.g. their natural partners). As our TAP purification results are consistent with the cellular co-fractionation of Rbg1 with Tma46 and Rbg2 with Gir2, our data leave little doubt that two distinct complexes are present in yeast cells. More importantly, all of our observations are in total agreement with the results obtained with human factors (16
). This is also in agreement with the extremely high conservation of these factors in eukaryotic cells.
Consistent with previous results, we observed association of Rbg1 and Tma46 with ribosomes (25
). Contradictory results had been reported for the association of Gir2 with Gcn1 and with ribosomes (26
). We observe that Gcn1 copurifies with Rbg2 probably by interacting with Gir2, but we do not detect a co fractionation of Gir2 with translating ribosomes. Again, the latter result parallel the one obtained in mammals (37
) supporting the high functional conservation of these factors. Although Gir2 and its binding partner Rbg2 do not appear to be associated with translating ribosomes in actively growing cells, it is likely that they can interact with ribosomes through Gcn1, which, itself, is known to bind directly to ribosomes together with its partner Gcn20 under specific conditions (49
). Overall, these observations were consistent with a role of the yeast DRGs Rbg1 and Rbg2 in translation. Yet, deletion of these two proteins, independently or simultaneously, did not generate strong growth phenotypes (Supplementary Figure S2
) nor alteration of polysome profiles (data not shown). Reduced cell fitness upon the simultaneous inactivation of rbg1
, or tma46
was only detected using a sensitive competitive assay (27
). Interestingly, this study also reported the same phenotype resulting from inactivation of rbg1
together with gcn1
. The latter observation supports the idea that Rbg2 acts through the Gcn1/Gcn20 complex, consistent with the observation that Rbg2/Gir2 interact with Gcn1. This association most likely occurs through the RWD domain of Gir2 that is highly similar to the homologous domain of Gcn2, which is known to mediate interaction with Gcn1 (19
). In standard laboratory conditions, the synthetic interactions detected between Rbg1/Tma46, on the one hand, and Rbg2/Gir2/Gcn1/Gcn20, on the other hand, have no significant impact on cell growth (Supplementary Figure S2
). The lack of clear function for DRG proteins, opposed to their strong sequence conservation, was highly surprising. Thus, we performed a genetic screen to identify factors that would act redundantly with Rbg1 and Rbg2, and identified Slh1. Synthetic interactions suggest that Rbg1, Rbg2 and Slh1 most likely act in parallel pathways. Indeed, synthetic interactions occur between cells carrying complete deletions of these genes, which is not consistent with the additive impairment resulting from combination of partially active alleles acting in the same pathway usually detected with conditional genes. Thus, it is likely that one pathway requires Rbg1 and Tma46, the second Rbg2, Gir2 and possibly Gcn1 and Gcn20, while the third would require Slh1. It is unlikely that additional factors specifically join Slh1 for this task as we recovered different alleles of SLH1
in our screen (judged from their growth phenotype) but failed to identify any other genes redundant with RBG1
. Moreover, genetic screens for gene dosage-dependent or genomic suppressors of Δrbg1
growth defect failed so far to uncover any additional factor (data not shown), suggesting again that the absence of additional players able to mediate the same function. Like for the Rbg-dependent pathways, the Slh1-dependent pathway is likely to be conserved in eukaryotes given the good sequence conservation of this factor across the eukaryotic kingdom. In this situation, it will be of interest to test whether the simultaneous inhibition of Drg1, Drg2 and Slh1 also result in translation inhibition in other eukaryotic species.
Analyses of polysomes assembled in vivo
demonstrate that Rbg1, Rbg2 and Slh1 are required for efficient translation. Taken together with their association with ribosomes, this new result indicates that these proteins are new translation factors with redundant functions. However, given that the triple mutant still grows, albeit slowly, this function is not essential for protein production. A possibility is that Rbg1, Rbg2 and Slh1 are involved in quality control pathways. Indeed, such a process would not be essential but its inactivation could result in impaired translation. Polysome profiles of the triple mutant strain are highly altered, indicating that in this context the translation of most or all mRNA is affected. This suggests that Rbg1, Rbg2 and Slh1 do not control the expression of a subset of specific mRNAs but rather that they have a more general function. The profiles that we observed are consistent with altered translation initiation defects. Interestingly, Slh1 was first identified by its role in the inhibition of translation of mRNAs lacking poly(A) tails (43
) and the effect of the poly(A) tail on translation was proposed to be mediated during initiation (51
), at least in part by favoring 60S subunit joining (42
). Further analyses will be required, however, to characterize the precise molecular function of Rbg1, Rbg2 and Slh1 in translation.
Although DRGs have long been known to contain GTPase signature sequences, the role of this domain remained unclear in the absence of a functional test. The observation that triple mutant altering Rbg1, Rbg2 and Slh1 (or partners thereof) grow slowly offered the possibility to assay this in vivo
. Hence, it appears that the GTP-binding site of Rbg1 is required for its function. Similarly, mutant analysis supports the idea that RNA binding by Tma46 and association of Gir2 with Gcn1 and Rbg2 is necessary for their activity. Finally, the function of Slh1 appears to require both of its helicase domains and most likely its ability to hydrolyze ATP. GTPases and helicases have been implicated as switches, or through their capacities to remodel RNPs, at many steps of the translation process. If some GTPase and helicase have been shown to cooperate in translation, as for Vasa and eIF5B (52
), they had up-to-now distinct dedicated functions, including partners and binding sites. How can we imagine Rbg1, Rbg2 and Slh1 being redundant? While it is easy to envisage that the two Rbg factors have similar molecular mechanisms of action, it is more difficult to conceive that Slh1 would act through the same binding site and partners. It is more likely that these proteins act on related substrates through different molecular mechanisms. Hence, if Rbg1, Rbg2 and Slh1 are involved in quality control reactions, it is easy to imagine that an aberrant substrate may be recognized through different means, and redirected to a normal status or discarded through different pathways. In this vein, it is noteworthy that both some GTPases and helicases have been shown to participate to quality control processes. This includes, for example, the precise selection of cognate tRNA during translation (54
) or the targeting of mRNA containing premature stop codons to decay (55
While further experiments will be required to delineate the functions of yeast DRGs and Slh1, an important orthogonal conclusion of our work is that cellular processes are very robust owing to the presence of redundant factors that protect cells from aggression through their buffering effects. Indeed, the simultaneous inactivation of Rbg1, Rbg2 and Slh1 is required to produce a clear growth inhibition phenotype; in standard laboratory conditions strains containing any combination of double mutant behaved as wild type. In this context, it is interesting to note that even for an extensively analyzed organism such as S. cerevisiae, many genes remain classified as orphan for function. It is not unlikely that some of those have redundant roles with several other proteins. Such a situation would however not be detected by current large-scale analyses that only address effects of binary interactions. These analyses indeed suffer from technical limitations as the number of combinations to be tested to assay for triple interactions is currently far beyond our capacity.