RESUMO
Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Nutrientes/genética , Elongação Traducional da Cadeia Peptídica/genética , RNA de Transferência/genética , Schizosaccharomyces/genética , Transdução de Sinais/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação Fúngica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Mitose/genética , Fosforilação/genéticaRESUMO
More than a decade ago, the purification of the form of the RNA polymerase II (PolII) engaged in elongation led to the discovery of an associated, multi-subunit (Elp1-6) complex named "Elongator" by the Svejstrup lab. Although further evidence supported the original notion that Elongator is involved in transcription, Elongator lacked some of the expected features for a regulator of the elongating PolII. The discovery by the Byström lab, based on genetic dissection, that Elongator is pivotal for tRNA modifications, and that all the reported phenotypes of Elongator mutants are suppressed by the overexpression of two tRNAs added to the confusion. The increasing range of both potential substrates and biological processes regulated by Elongator in higher eukaryotes indicates that the major challenge of the field is to determine the biologically relevant function of Elongator. Our recent proteome-wide study in fission yeast supports a coordinated codon-dependent regulation of translation by Elongator. Here we provide additional analyses extending this hypothesis to budding yeast and worm.
Assuntos
Códon/genética , Histona Acetiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Histona Acetiltransferases/genética , Humanos , Modelos Biológicos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA de Transferência/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast-tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.
Assuntos
Proteômica/métodos , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Fases de Leitura Aberta , Análise Serial de Proteínas , Proteoma , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Elongator is required for the synthesis of the mcm(5)s(2) modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.
Assuntos
Divisão Celular/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/genética , Códon/genética , Códon/fisiologia , Citocinese/genética , Citocinese/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histona Acetiltransferases/genética , Histona Acetiltransferases/fisiologia , Lisina/genética , Lisina/fisiologia , Mitose/genética , Mitose/fisiologia , Biossíntese de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , RNA de Transferência/genética , RNA de Transferência/fisiologia , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Modified nucleosides close to the anticodon are important for the proper decoding of mRNA by the ribosome. Particularly, the uridine at the first anticodon position (U34) of glutamate, lysine, and glutamine tRNAs is universally thiolated (S(2)U34), which is proposed to be crucial for both restriction of wobble in the corresponding split codon box and efficient codon-anticodon interaction. Here we show that the highly conserved complex Ctu1-Ctu2 (cytosolic thiouridylase) is responsible for the 2-thiolation of cytosolic tRNAs in the nematode and fission yeast. In both species, inactivation of the complex leads to loss of thiolation on tRNAs and to a thermosensitive decrease of viability associated with marked ploidy abnormalities and aberrant development. Increased level of the corresponding tRNAs suppresses the fission yeast defects, and our data suggest that these defects could result from both misreading and frame shifting during translation. Thus, a translation defect due to unmodified tRNAs results in severe genome instability.
