All or nothing: protein complexes flip essentiality between distantly related eukaryotes.
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ABSTRACT:
SUBMITTER: Ryan CJ
PROVIDER: S-EPMC3698920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.
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Project description:Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.
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Project description:We lack an understanding of how the full range of genetic variants that occur in individuals can interact. To address this shortcoming, here we combine diverse mutations between genes in a model regulatory network, the galactose (GAL) switch of budding yeast. The effects of thousands of pairs of mutations fall into a limited number of phenotypic classes. While these effects are mostly predictable using simple rules that capture the 'stereotypical' genetic interactions of the network, some double mutants have unexpected outcomes including constituting alternative functional switches. Each of these 'harmonious' genetic combinations exhibits altered dependency on other regulatory genes. These cases illustrate how both pairwise and higher epistasis determines gene essentiality and how combinations of mutations rewire regulatory networks. Together, our results provide an overview of how broad spectra of mutations interact, how these interactions can be predicted, and how diverse genetic solutions can achieve 'wild-type' phenotypic behavior.
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Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.
2018-07-05 | GSE116570 | GEO
Project description:BackgroundThe replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best.Methodology/principal findingsWhile the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea.Conclusion/significanceThis domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.
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Project description:Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1alpha (EF-1alpha) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1alpha gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1alpha gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential "informational" genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing.
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Project description:BackgroundThe larval nervous system of the solitary tunicate Ciona is a simple model for the study of chordate neurodevelopment. The development and connectivity of the Ciona motor ganglion have been studied in fine detail, but how this important structure develops in other tunicates is not well known.Methods and resultsBy comparing gene expression patterns in the developing MG of the distantly related tunicate Molgula occidentalis, we found that its patterning is highly conserved compared to the Ciona MG. MG neuronal subtypes in Molgula were specified in the exact same positions as in Ciona, though the timing of subtype-specific gene expression onset was slightly shifted to begin earlier, relative to mitotic exit and differentiation. In transgenic Molgula embryos electroporated with Dmbx reporter plasmids, we were also able to characterize the morphology of the lone pair of descending decussating neurons (ddNs) in Molgula, revealing the same unique contralateral projection seen in Ciona ddNs and their putative vertebrate homologs the Mauthner cells. Although Dmbx expression labels the ddNs in both species, cross-species transgenic assays revealed significant changes to the regulatory logic underlying Dmbx transcription. We found that Dmbx cis-regulatory DNAs from Ciona can drive highly specific reporter gene expression in Molgula ddNs, but Molgula sequences are not active in Ciona ddNs.ConclusionsThis acute divergence in the molecular mechanisms that underlie otherwise functionally conserved cis-regulatory DNAs supports the recently proposed idea that the extreme genetic plasticity observed in tunicates may be attributed to the extreme rigidity of the spatial organization of their embryonic cell lineages.
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Project description:The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally2. However, although most of the cellular proteome forms oligomeric assemblies3, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences-such as the rarity of polycistronic mRNAs and different chaperone constellations-raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.
| S-EPMC6372068 | biostudies-literature
Project description:BACKGROUND:Elongation factor-1? (EF-1?) and elongation factor-like (EFL) proteins are functionally homologous to one another, and are core components of the eukaryotic translation machinery. The patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a 'differential loss' hypothesis that assumes that EF-1? and EFL were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. To date, however, just one diatom and one fungus have been found to have both EF-1? and EFL (dual-EF-containing species). RESULTS:In this study, we characterized 35 new EF-1?/EFL sequences from phylogenetically diverse eukaryotes. In so doing we identified 11 previously unreported dual-EF-containing species from diverse eukaryote groups including the Stramenopiles, Apusomonadida, Goniomonadida, and Fungi. Phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-EF lineages. In the dual-EF-containing species we identified, the EF-1? genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring EFL genes. CONCLUSIONS:According to the known EF-1?/EFL distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-EF-containing species remain unidentified. We predict that dual-EF-containing species retain the divergent EF-1? homologues only for a sub-set of the original functions. As the dual-EF-containing species are distantly related to each other, we propose that independent re-modelling of EF-1? function took place in multiple branches in the tree of eukaryotes.
| S-EPMC3699394 | biostudies-literature
Project description:BackgroundIdentifying all protein complexes in an organism is a major goal of systems biology. In the past 18 months, the results of two genome-scale tandem affinity purification-mass spectrometry (TAP-MS) assays in yeast have been published, along with corresponding complex maps. For most complexes, the published data sets were surprisingly uncorrelated. It is therefore useful to consider the raw data from each study and generate an accurate complex map from a high-confidence data set that integrates the results of these and earlier assays.ResultsUsing an unsupervised probabilistic scoring scheme, we assigned a confidence score to each interaction in the matrix-model interpretation of the large-scale yeast mass-spectrometry data sets. The scoring metric proved more accurate than the filtering schemes used in the original data sets. We then took a high-confidence subset of these interactions and derived a set of complexes using MCL. The complexes show high correlation with existing annotations. Hierarchical organization of some protein complexes is evident from inter-complex interactions.ConclusionWe demonstrate that our scoring method can generate an integrated high-confidence subset of observed matrix-model interactions, which we subsequently used to derive an accurate map of yeast complexes. Our results indicate that essentiality is a product of the protein complex rather than the individual protein, and that we have achieved near saturation of the yeast high-abundance, rich-media-expressed "complex-ome."
| S-EPMC1940025 | biostudies-literature