RESUMO
In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Técnicas do Sistema de Duplo-HíbridoRESUMO
In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.
Assuntos
Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Exorribonucleases/metabolismo , Substâncias Macromoleculares , Proteínas Nucleares/fisiologia , Ligação Proteica , Precursores de RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Proteínas de Saccharomyces cerevisiae/fisiologia , TemperaturaRESUMO
Rrp43p is a Saccharomyces cerevisiae exosome subunit involved in pre-rRNA processing which is found both in the nucleus and in the cytoplasm. So far, no function has been assigned to the cytoplasmic fraction of Rrp43p. We have addressed Rrp43p function by analyzing mRNA stability in three rrp43 temperature-sensitive (ts) strains, which carry different ts alleles (rrp43-1, rrp43-2 and rrp43-3), and by analyzing Rrp43p interactions with the remaining exosome subunits. In the ts strains, endogenous mRNAs (ACT1 and PAB1), as well as a heterologous reporter mRNA (CATpG) showed longer half-lives, relative to a control strain carrying wild-type RRP43. The mutants also accumulated a degradation intermediate of the reporter mRNA that is typical of defective mRNA decay. These results allow us to propose that Rrp43p is required for mRNA degradation. Rrp43p interacts with the exosome complex via Rrp46p, as determined by two-hybrid analyses. Interestingly, the rrp43 ts mutant proteins do not interact with Rrp46p, indicating that the ts phenotype may be caused by disruption of the Rrp43p- Rrp46p interaction. The ts strains also showed a pre-rRNA processing defect, which is consistent with previous studies on Rrp43p function.