RESUMO
Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.
Assuntos
Adaptação Biológica/genética , Splicing de RNA/genética , Trans-Splicing/genética , Adaptação Biológica/fisiologia , Evolução Molecular , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Íntrons/genética , Mutação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/metabolismoRESUMO
Growing cells are subject to cycles of nutrient depletion and repletion. A shortage of nutrients activates a starvation program that promotes growth in limiting conditions. To examine whether nutrient-deprived cells prepare also for their subsequent recovery, we followed the transcription program activated in budding yeast transferred to low-phosphate media and defined its contribution to cell growth during phosphate limitation and upon recovery. An initial transcription wave was induced by moderate phosphate depletion that did not affect cell growth. A second transcription wave followed when phosphate became growth limiting. The starvation program contributed to growth only in the second, growth-limiting phase. Notably, the early response, activated at moderate depletion, promoted recovery from starvation by increasing phosphate influx upon transfer to rich medium. Our results suggest that cells subject to nutrient depletion prepare not only for growth in the limiting conditions but also for their predicted recovery once nutrients are replenished.
Assuntos
Crescimento Celular , Fosfatos/metabolismo , Meios de Cultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SaccharomycetalesRESUMO
Most of the phenotypes in nature are complex and are determined by many quantitative trait loci (QTLs). In this study we identify gene sets that contribute to one important complex trait: the ability of yeast cells to survive under alkali stress. We carried out an in-lab evolution (ILE) experiment, in which we grew yeast populations under increasing alkali stress to enrich for beneficial mutations. The populations acquired different sets of affecting alleles, showing that evolution can provide alternative solutions to the same challenge. We measured the contribution of each allele to the phenotype. The sum of the effects of the QTLs was larger than the difference between the ancestor phenotype and the evolved strains, suggesting epistatic interactions between the QTLs. In parallel, a clinical isolated strain was used to map natural QTLs affecting growth at high pH. In all, 17 candidate regions were found. Using a predictive algorithm based on the distances in protein-interaction networks, candidate genes were defined and validated by gene disruption. Many of the QTLs found by both methods are not directly implied in pH homeostasis but have more general, and often regulatory, roles.
Assuntos
Evolução Molecular Direcionada/métodos , Aptidão Genética/genética , Locos de Características Quantitativas , Saccharomyces cerevisiae/genética , Algoritmos , Antiporters/genética , Proteínas de Transporte de Cátions/genética , Mapeamento Cromossômico , Meios de Cultura/metabolismo , Homeostase/genética , Concentração de Íons de Hidrogênio , Mutação , Fenótipo , Reprodutibilidade dos Testes , Proteínas SLC31 , Proteínas de Saccharomyces cerevisiae/genética , Biologia de Sistemas/métodosRESUMO
Growing cells coordinate protein translation with metabolic rates. Central to this coordination is ribosome production. Ribosomes drive cell growth, but translation of ribosomal proteins competes with production of non-ribosomal proteins. Theory shows that cell growth is maximized when all expressed ribosomes are constantly translating. To examine whether budding yeast function at this limit of full ribosomal usage, we profiled the proteomes of cells growing in different environments. We find that cells produce excess ribosomal proteins, amounting to a constant ≈8% of the proteome. Accordingly, ≈25% of ribosomal proteins expressed in rapidly growing cells does not contribute to translation. Further, this fraction increases as growth rate decreases and these excess ribosomal proteins are employed when translation demands unexpectedly increase. We suggest that steadily growing cells prepare for conditions that demand increased translation by producing excess ribosomes, at the expense of lower steady-state growth rate.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Perfilação da Expressão Gênica , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Biossíntese de Proteínas , Proteoma/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Ribossomos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Depletion of essential nutrients triggers regulatory programs that prolong cell growth and survival. Starvation-induced processes increase nutrient transport, mobilize nutrient storage, and recycle nutrients between cellular components. This leads to an effective increase in intracellular nutrients, which may act as a negative feedback that downregulates the starvation program. To examine how cells overcome this potential instability, we followed the transcription response of budding yeast transferred to medium lacking phosphate. Genes were induced in two temporal waves. The first wave was stably maintained and persisted even upon phosphate replenishment, indicating a positive feedback loop. This commitment was abolished after 2 hr with the induction of the second expression wave, coinciding with the reduction in cell growth rate. We show that the overall temporal stability of the expression response depends on the sequential pattern of gene induction. Our results emphasize the key role of gene expression dynamics in optimizing cellular adaptation.