The more than 780,000 individual manually dissected wild-type yeast daughter cells in this project provide a high resolution for making accurate estimates of false positive and negative rates, allowing us to estimate the total fraction of viable yeast deletions likely to affect RLS. We generated sampling distributions from our wild-type cells ( Figure S3 , related to Figure 2 Supplemental Experimental Procedures ). Using these, we estimated our false positive and false negative rates as a function of the percent increase in RLS and sample size n ( Figure 2 and Table S3 , related to Figure 2 ). These results suggest that the estimated total number of additional viable deletions that extend RLS >50% relative to wild-type is likely <1. For a 40% increase in RLS, we estimate ∼10 additional viable deletions, and for a 30% increase, ∼58 additional viable deletions ( Table S3 , related to Figure 2 ). In considering false negative rates, it is worth stating explicitly that there is a class of S. cerevisiae genes whose effects cannot be reflected in this work: essential genes. Further, previous work () has suggested that essential genes may be more likely than nonessential genes to have a strong effect on lifespan, implying that the number of essential longevity genes remaining to be discovered could exceed a rough approximation based on extrapolation from nonessential genes.

(B) Estimated false negative rate at a threshold of 30% increased RLS, for given actual percent increased RLS and number of mother cells n. x axis indicates actual percent increase expected for the genotype, z axis indicates the estimated false negative proportion using a 30% screening cutoff, and point color indicates sample size n (black, n = 5; blue, n = 10; red, n = 40).

We performed a genome-wide analysis of viable S. cerevisiae single-gene deletions by measuring the RLS of 5 mother cells in the MATα mating type for 4,698 unique strains, based on the approach outlined previously (). For each strain that showed a mean RLS increase of >30% over control, or p < 0.05 for increased RLS, we measured RLS for 20 cells in the MATstrain carrying the same gene deletion. For all gene deletions that extended RLS significantly in both mating types, at least 20 mother cells total were scored in each mating type. In some cases of divergent mating type RLS, the difference may be due to the selection of slow-growth suppressors in the non-long-lived mating type, as has been observed for ribosomal protein mutants (). In cases where we have observed a changed RLS upon reconstruction of the strain, only reconstructed data is included. We have observed zero examples in this data where a significant difference between mating types survived reconstruction of the strains, and also note that the very large number of mother cells scored for wild-type MATand MATα show no significant difference in RLS. A graphical summary of all long-lived deletions found in this screen is shown in Figure 1 A.

(A) Summary of RLS data for long-lived deletion strains. Axes indicate percent increase in RLS relative to control in MAT a and MATα, respectively. Point size is proportional to number of mother cells scored, and point color indicates p value for increased RLS relative to control. Point outline indicates stringency for inclusion: high stringency cutoff was p < 0.05 for Wilcoxon rank-sum increased survival independently in both mating types, and low stringency was p < 0.05 for pooled data from both mating types with increased RLS shown in each mating type alone.

A biologically meaningful list of lifespan-extending genes is unlikely to be a random assortment, and the genes identified in our screen are not; many of these genes cluster into known functional groups. All of these groups are conserved from yeast to humans. Genes whose molecular function category is overrepresented among our long-lived deletions are summarized by category ( Table 1 ), and individual categories are described further. A network showing all long-lived deletions as nodes, and published physical protein-protein interactions as edges, shows that many of the genes not only cooperate in related functional processes, but are physically bound in protein complexes such as the cytosolic ribosome, mitochondrial ribosome, and SAGA complex ( Figure 1 B).

Although not overrepresented by automated gene ontology analysis, one of the longest-lived single deletions identified here is that of UBR2, a ubiquitin ligase that regulates the turnover of the transcription factor Rpn4, which positively regulates transcription of components of the yeast proteasome. As the extended RLS of ubr2Δ depends completely on Rpn4, we concluded that proteasomal activity is central to this phenotype (), and we have recently dissected the RLS effects of UBR2 and RPN4 in additional detail ().

We identified seven genes encoding proteins central to the tricarboxylic acid pathway: isocitrate dehydrogenase subunits IDP1, IDH1, and IDH2; lipoamide dehydrogenase LPD1; and succinate dehydrogenase subunits SDH1, SDH2, and EMI5/SDH5. All of these genes are involved in a very short section of the TCA cycle: the conversion of isocitrate to alpha-ketoglutarate (IDP1, IDH1, IDH2), on to succinyl-CoA (LPD1), and from there to succinate and then fumarate (SDH1, SDH2, EMI5). It is interesting to note that this pathway converges on many events linked to longevity, including production of amino acids that regulate TOR activity, control of metabolite levels including NAD(+), and use of different substrates for ATP production ().

Recently, Labunskyy et al. reported that yeast lacking ALG12 and BST1, which controls ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins (), were long-lived because they activate the unfolded protein response, which promotes multiple forms of stress resistance (). It remains to be determined whether other long-lived deletions of mannosyltransferase-related genes enhance RLS by this mechanism.

Protein O-mannosylation is an essential modification that occurs at the endoplasmic reticulum and is conserved from yeast to humans (). In yeast, three mannosyltransferase subfamilies, PMT1 (consisting of Pmt1 and Pmt5), PMT2 (Pmt2 and its paralog Pmt3), and PMT4, are thought to mannosylate distinct protein targets (). In our screen, PMT1, PMT3, and PMT5 all significantly extended yeast RLS. Closer inspection of the remaining individual PMT gene deletions revealed additional interesting results: pmt2Δ just missed the cutoff for inclusion, exhibiting a 26.3% increase in RLS (p < 0.038) in MAT, and a 15.6% increase (p < 0.054) in MATα. pmt6Δ and pmt7Δ each extended RLS in one mating type when tested (22.6% and 31.3%, respectively), but not in the other mating type, leaving open the possibility that the nonextended mating type in each case may harbor a second mutation which masks the RLS phenotype of the deletion. We also identified the two yeast mannosyltransferases involved in O-linked glycosylation, MNN1 and MNN2; the N-linked mannosyltransferases YUR1, ALG6, and ALG12; and EOS1, which has been implicated in protein N-glycosylation.

The yeast SAGA and SLIK chromatin remodeling complexes and the orthologous human complexes STAGA and SALSA are histone-modifying complexes which contain at least two enzymatic activities: a protein acetyltransferase (in yeast, Gcn5) and a deubiquitinase (in yeast, Ubp8) (). The Ubp8 deubiquitinase acts as part of a deubiquitinating module, or DUBm, alongside Sgf73, Sgf11, and Sus1. Deletions of genes encoding three of the four DUBm components, Ubp8, Sgf73, and Sgf11, increase yeast RLS significantly, and sgf73Δ has one of the largest RLS increases seen in our screen. These genes were also identified early in our screen, and a parallel project has now looked in more detail at the mechanisms by which disruption of the SAGA/SLIK DUBm may increase yeast RLS ().

Overall, ribosome and cytosolic ribosome are detected independently as overrepresented categories, due to the fact that alongside the components of the cytosolic ribosome, we identified genes encoding six components of the large subunit of mitochondrial ribosome: IMG1, IMG2, MRPL33, MRPL40, MRPL49, and YDR115W. We also saw increased RLS upon deletion of the genes encoding two mitochondrial tRNA synthetases, MSK1 and MSW1, and three mitochondrial translation control (MTC) genes, SOV1, SUV3, and CBS1, which themselves affect levels of mitochondrial ribosomal proteins. Deletion of these three MTC genes has been shown previously to extend yeast RLS (). The bias toward the large subunit seen in the RLS-extending cytosolic ribosome deletions is repeated in the mitochondrial ribosome. It will be interesting to learn how much the mechanisms of RLS extension by deletion of cytosolic ribosomal components and mitochondrial ribosomal components have in common.

Ribosomal gene deletions were one of the first categories noted to be long-lived during the progress of this project. Four previous publications, three from our group () and one other (), have investigated the mechanism by which deletion of large subunit genes extends RLS, leading to the identification of the nutrient-responsive transcription factor Gcn4 as an important downstream mediator of longevity ().

The largest functional category of long-lived deletions is the cytosolic ribosome. These 32 genes include 26 paralogs encoding 20 protein components of the large ribosomal subunit, 4 paralogs encoding 2 proteins in the ribosomal stalk, and 2 paralogs encoding 2 proteins in the small ribosomal subunit. This function is conserved; several components of the large subunit of the cytosolic ribosome have also been shown to affect lifespan in C. elegans ().

RLS-Extending Single Gene Deletions in Yeast Overlap at High Significance with Known Lifespan-Extending Hypomorphs in C. elegans

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Sonnhammer E.L. InParanoid 7: new algorithms and tools for eukaryotic orthology analysis. Table 2 Lifespan Phenotypes of Yeast Deletions Are Significantly Conserved Worm Orthologs Yeast Orthologs Worm Orthologs Yeast Orthologs ant-1.1 AAC3 rpl-1 RPL1B cyc-2.1 CYC1 rpl-19 RPL19A RPL19B dld-1 LPD1 rpl-23 RPL23A dod-18 YOR111W rpl-31 RPL31A idh-1 IDP1 rpl-6 RPL6A RPL6B idha-1 IDH2 rpl-7 RPL7A ifg-1 TIF4631 rpl-9 RPL9A inf-1 TIF1 TIF2 rps-6 RPS6A jmjd-2 RPH1 rsks-1 SCH9 let-363 TOR1 sams-1 SAM1 nac-2 nac-3 PHO87 spg-7 AFG3 pos-1 CTH1 TIS11 spt-4 SPT4 raga-1 GTR1 unc-26 INP53 Listed are genes whose deletion in S. cerevisiae was shown to increase RLS in this screen, and whose C. elegans orthologs have been published to have increased lifespan upon deletion, hypomorphic mutation, or RNAi knockdown. p overlap <4E-11; if all ribosomal genes are excluded p overlap <2E-6. We examined whether these data would provide further evidence of evolutionary conservation of longevity pathways. To test this, we asked whether our RLS-extending deletions overlapped significantly with genes whose knockdown or deletion was reported to extend the lifespan of the distantly related nematode C. elegans. We compiled a list of 428 genes whose deletion, hypomorphic or null allele mutation, or RNAi knockdown had been reported to extend C. elegans lifespan, taking as our starting point previous work where a portion of the yeast genome was similarly examined () ( Table S4 , related to Table 2 ). We then used paralog-ortholog groups generated by InParanoid () to find genes whose lifespan-extending phenotype was shared between S. cerevisiae and C. elegans, and calculated the probability of an overlap of the observed magnitude. Long-lived yeast deletions whose hypomorphic lifespan extension has also been reported in C. elegans are shown in Table 2 . The probability of this overlap absent functional conservation of lifespan phenotypes (i.e., at random) is p < 4.4E-11. Because of the large number of ribosomal proteins present in both species’ lifespan-altering orthologs, we asked whether they alone were responsible for the significance of the overlap we observed, and found that the remaining list still exhibited a significant overlap (p < 2.0E-6) with ribosomal proteins omitted.

We note that the genes affecting both yeast and C. elegans lifespan contain a similar proportion of genes from our functionally overrepresented pathways as the screen as a whole. Specifically, 0.255 of the overall screen results fall into the functionally overrepresented groups such as the ribosome, TCA cycle, SAGA, etc., while 0.423 of the conserved genes do so, a higher proportion by gene. Nevertheless, not all five functionally overrepresented pathways we describe here in yeast are represented among the conserved genes; only two (ribosome and TCA cycle) are. We propose at least two possible factors that may contribute to the absence of the other pathways. First, although we here quantify the false negative rate of our screen with high precision, our list of C. elegans genes is generated by consideration of aggregate data from multiple studies, most of which are not whole-genome studies, whose aggregate false negative rate cannot be easily estimated. We suspect that some absences from our list of conserved genes may be because the phenotype has not been observed in C. elegans although it may exist. Further, interspecies ortholog prediction is an imperfect science. In the example of the SAGA group, although both the individual genes and the overall protein complexes are conserved at the sequence and functional level between yeast, mice, and humans, we were not able to identify putative C. elegans orthologs for any of the RLS-extending SAGA deletions. We do not know whether this is due to sequence divergence, complete loss of this functionality from C. elegans, or other reasons, but it may offer an additional explanation for the fact that not every pathway we have uncovered in yeast is represented in the smaller subset of identified conserved genes.

Taken in context, the highly significant overlap between the final results of our screen of viable yeast deletions and published C. elegans genes suggests a high degree of general conservation in lifespan genotype-phenotype relationships between very distantly related species. This leaves open the possibility that these genes may be enriched for orthologs that can alter lifespan and aging in other organisms, such as humans.