RESUMO
The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5' untranslated region (5' UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3' UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as â¼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.
Assuntos
Loci Gênicos/efeitos dos fármacos , Gentamicinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Sequência , Transcrição Gênica/efeitos dos fármacos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Bases , Códon , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fases de Leitura Aberta , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.
Assuntos
Amiloide/metabolismo , Códon sem Sentido , Príons/genética , Príons/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Príons/química , Agregados Proteicos , Splicing de RNARESUMO
The [PSIâº] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSIâº] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSIâº], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSIâº] formation, while Sla2 can prevent [PSIâº] toxicity. Here, we identify two more non-chaperone proteins involved in [PSIâº] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSIâº] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSIâº] detoxification system.
Assuntos
Proteínas de Ligação a Poli(A)/metabolismo , Príons/toxicidade , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , Códon sem Sentido/genética , Deleção de Genes , Modelos Biológicos , Fatores de Terminação de Peptídeos/metabolismo , Plasmídeos/metabolismo , Polimerização , Saccharomyces cerevisiae/efeitos dos fármacos , Mutações Sintéticas LetaisRESUMO
A common problem in engineering industrial yeasts, and wine yeasts in particular, is the lack or scarcity of selective markers for introducing desired genetic changes. Almost all such markers, which are usually auxotrophic mutations, would reduce the growth characteristics of yeast strains. However, a potentially useful marker could be the CAR1 gene encoding arginase, the deletion of which reduces the accumulation of the carcinogen ethyl carbamate in wine, making such a deletion beneficial for wine production and maintainable in wine yeast strains. Here we demonstrate the use of the CAR1 gene as a selective marker. First, we observe that complete deletion of CAR1 in a triploid wine strain of Saccharomyces cerevisiae causes strong growth inhibition on a medium containing arginine as the only nitrogen source. Then, we show that strains with CAR1 deletion can be reliably transformed using CAR1 as a plasmid marker. Thus, the CAR1 gene can be used as a convenient selective marker in genetic engineering of wine yeasts, in particular using CRISPR/Cas9 technology.
Assuntos
Proteínas de Saccharomyces cerevisiae , Vinho , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vinho/análise , Engenharia Genética , Uretana , Fermentação , Leveduras/genéticaRESUMO
In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Biossíntese de Proteínas , RNA-Seq , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In eukaryotes, termination of translation is controlled by polypeptide chain release factors eRF1 and eRF3, of which the former recognizes nonsense codons, while the latter interacts with eRF1 and stimulates polypeptide release from the ribosome in a GTP- dependent manner, and ABCE1, which facilitates ribosome recycling. In this work, we demonstrate that Pub1, a yeast protein known to be involved in stress granule formation, regulation of gene expression, and organization of the tubulin cytoskeleton, also plays a role in translation termination. Pub1 was shown to bind to ribosomes independent of eRF1 and eRF3 and to interact with the N-terminal glutamine-/asparagine-rich prion domain of eRF3 via its short C-terminal glutamine-rich tract. High velocity sedimentation in sucrose gradient demonstrated that Pub1 was preferentially associated with heavy polysomes enriched with terminating ribosomes. Lack of Pub1 decreased efficiency of nonsense readthrough at a majority but not all tetranucleotide stop signals. This distinguishes Pub1 from most other known binding partners of the release factors which were shown to modulate readthrough of all nonsense codons uniformly. The obtained data show that Pub1 can act as an accessory translation factor involved in fine-tuning of translation termination.
Assuntos
Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Códon de Terminação , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Proteínas de Ligação a Poli(A)/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
BACKGROUND: Termination of translation in eukaryotes requires two release factors, eRF1, which recognizes all three nonsense codons and facilitates release of the nascent polypeptide chain, and eRF3 stimulating translation termination in a GTP-depended manner. eRF3 from different organisms possess a highly conservative C region (eRF3C), which is responsible for the function in translation termination, and almost always contain the N-terminal extension, which is inessential and vary both in structure and length. In the yeast Saccharomyces cerevisiae the N-terminal region of eRF3 is responsible for conversion of this protein into the aggregated and functionally inactive prion form. RESULTS: Here, we examined functional importance of the N-terminal region of a non-prion form of yeast eRF3. The screen for mutations which are lethal in combination with the SUP35-C allele encoding eRF3C revealed the sup45 mutations which alter the N-terminal domain of eRF1 and increase nonsense codon readthrough. However, further analysis showed that synthetic lethality was not caused by the increased levels of nonsense codon readthrough. Dominant mutations in SUP35-C were obtained and characterized, which remove its synthetic lethality with the identified sup45 mutations, thus indicating that synthetic lethality was not due to a disruption of interaction with proteins that bind to this eRF3 region. CONCLUSION: These and other data demonstrate that the N-terminal region of eRF3 is involved both in modulation of the efficiency of translation termination and functioning of the eRF1/eRF3 complex outside of translation termination.
Assuntos
Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alelos , Códon de Terminação/genética , Mutação/genética , Terminação Traducional da Cadeia Peptídica/fisiologia , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Príons/genética , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência/genéticaRESUMO
In eukaryotic cells amyloid aggregates may incorporate various functionally unrelated proteins. In mammalian diseases this may cause amyloid toxicity, while in yeast this could contribute to prion phenotypes. Insolubility of amyloids in the presence of strong ionic detergents, such as SDS or sarcosyl, allows discrimination between amorphous and amyloid aggregates. Here, we used this property of amyloids to study the interdependence of their formation in yeast. We observed that SDS-resistant polymers of proteins with extended polyglutamine domains caused the appearance of SDS or sarcosyl-insoluble polymers of three tested chromosomally-encoded Q/N-rich proteins, Sup35, Rnq1 and Pub1. These polymers were non-heritable, since they could not propagate in the absence of polyglutamine polymers. Sup35 prion polymers caused the appearance of non-heritable sarcosyl-resistant polymers of Pub1. Since eukaryotic genomes encode hundreds of proteins with long Q/N-rich regions, polymer interdependence suggests that conversion of a single protein into polymer form may significantly affect cell physiology by causing partial transfer of other Q/N-rich proteins into a non-functional polymer state.