Gene function prediction from congruent synthetic lethal interactions in yeast

Abstract

We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae . Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein–dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans. ### Synopsis With the completion of the genome sequence for human and model organisms, the next phase is to understand how genes and gene products function together in pathways. Assays for physical interactions between proteins reveal how protein subunits assemble into larger machines and how protein–protein interactions provide the mechanism for regulation. Distinct from assays for physical interactions are assays for genetic interactions. Two genes have a genetic interaction if a double mutant (including null alleles, other mutant alleles, and dosage‐dependent effects) has a phenotype distinct from the phenotype of the individual single mutants. Unlike a physical interaction, however, a genetic interaction does not provide direct evidence for the pathway wiring underlying the observation. This manuscript describes a method for reverse‐engineering a pathway wiring diagram underneath genetic interaction data and applies the method to high‐throughput screens in yeast, which has ∼6000 total genes and ∼5000 non‐essential genes. While each of the 5000 non‐essential gene deletions yields a viable phenotype, pairwise deletions of non‐essential genes may be lethal. A lethal pairwise mutation is termed a synthetic lethal genetic interaction and indicates how gene functions buffer or compensate each other. The metaphor we employ for an essential biological process is an electric circuit where nodes represent genes or gene products and wires represent physical interactions between biomolecules ([Figure 1][1]). Deleting a gene corresponds to cutting the wires it connects. Robustness arises from multiple pathway branches connected in parallel. If one branch is cut, current still flows, but if both are cut the process fails and the cell dies. In this picture, synthetic lethal interactions should be observed between pathway branches, but not within branches. Synthetic lethal interactions between pathways are orthogonal to physical interactions within pathways. Two genes that share synthetic lethal interaction partners are therefore likely to function within the same pathway branch. The genes that share synthetic lethal partners, termed congruent genes, should have greater functional similarity than genes with direct synthetic lethal interactions. The congruence score provides a numerical ranking of the degree of partner sharing. It is defined as the −log10 of the P ‐value for the number of shared genetic interaction partners of two genes. We find that genes with significant congruence score have more similar database annotations than genes with direct synthetic lethal interactions. Products of congruent genes are also more likely to have direct physical interactions or to share protein complex membership than products of synthetic lethal genes. We conducted unbiased, genome‐scale tests of the concept of congruence by identifying landmark genes whose mutants have a distinct phenotype, ranking the rest of the genome by congruence to the landmarks, and scoring the phenotypes of the mutants in rank order ([Figure 3][2]). Genes congruent to known members of the dynein–dynactin spindle orientation pathway exhibit a nuclear migration defect rate that increases with increasing congruence score ([Figure 3A][2]). One of these genes is YLL049w, an uncharacterized ORF. Pathway membership for YLL049w is further defined by the observation that the temperature dependence of its defect rate matches JNM1, a component of dynactin, rather than KIP2, a kinesin‐like motor protein involved in delivering dynein to the cell cortex. We have also independently validated a physical interaction between Yll049w and Jnm1p. Taken together, these data indicate a role for YLL049w in a dynactin‐related activity within the dynein–dynactin spindle orientation pathway. In a second test, we identified genes congruent to CIN1 , a microtubule biogenesis gene, whose deletion mutant confers sensitivity to the anti‐microtubule drug benomyl. Genes congruent to CIN1 are enriched for benomyl sensitivity ([Figure 3B][2]). Furthermore, the quantitative LD50 benomyl concentration is correlated with the congruence score to seven benomyl‐sensitive landmarks ([Figure 3C][2]). Finally, a predictor based on the number of direct synthetic lethal interactions with benomyl‐sensitivity landmarks, rather than on the congruence score, fails to predict benomyl sensitivity ([Figure 3D][2]). This result is consistent with a metric that successfully identifies within‐pathway gene pairs, which are expected to exhibit more phenotypic similarity than between‐pathway gene pairs. Beyond providing...