RESUMO
tRNA precursors, which are transcribed by RNA polymerase III, undergo end-maturation, splicing, and base modifications. Hypomodified tRNAs, such as tRNA(Val(AAC)), lacking 7-methylguanosine and 5-methylcytidine modifications, are subject to degradation by a rapid tRNA decay pathway. Here we searched for genes which, when overexpressed, restored stability of tRNA(Val(AAC)) molecules in a modification-deficient trm4Δtrm8Δ mutant. We identified TEF1 and VAS1, encoding elongation factor eEF1A and valyl-tRNA synthetase respectively, which likely protect hypomodified tRNA(Val(AAC)) by direct interactions. We also identified MAF1 whose product is a general negative regulator of RNA polymerase III. Expression of a Maf1-7A mutant that constitutively repressed RNA polymerase III transcription resulted in increased stability of hypomodified tRNA(Val(AAC)). Strikingly, inhibition of tRNA transcription in a Maf1-independent manner, either by point mutation in RNA polymerase III subunit Rpc128 or decreased expression of Rpc17 subunit, also suppressed the turnover of the hypomodified tRNA(Val(AAC)). These results support a model where inhibition of tRNA transcription leads to stabilization of hypomodified tRNA(Val(AAC)) due to more efficient protection by tRNA-interacting proteins.
Assuntos
RNA Polimerase III/antagonistas & inibidores , Estabilidade de RNA/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Regulação para Baixo/genética , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Proteínas Mutantes/fisiologia , Organismos Geneticamente Modificados , Plasmídeos/genética , RNA Polimerase III/metabolismo , RNA Polimerase III/fisiologia , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , TransfecçãoRESUMO
Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner.
Assuntos
Núcleo Celular/metabolismo , Modelos Biológicos , RNA Polimerase III/metabolismo , Precursores de RNA/biossíntese , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/biossíntese , RNA de Transferência/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/genética , Deleção de Genes , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Polimerase III/genética , Precursores de RNA/genética , RNA Fúngico/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
Assuntos
Genes Reporter , Processamento de Terminações 3' de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Saccharomyces cerevisiae , Algoritmos , Estudos de Avaliação como Assunto , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente/métodos , Cinética , Modelos Biológicos , Modelos Genéticos , Processamento de Terminações 3' de RNA/fisiologia , Precursores de RNA/análise , Precursores de RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/biossíntese , Precursores de RNA/biossíntese , RNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
The Krr1 protein of Saccharomyces cerevisiae is involved in processing of pre-rRNA and assembly of pre-ribosomal 40S subunits. To further investigate the function of Krr1p we constructed a conditional cold sensitive mutant krr1-21, and isolated seven genes from Schizosaccharomyces pombe whose products suppressed the cold sensitive phenotype of krr1-21 cells. Among the multicopy suppressors we found genes coding for translation elongation factor EF-1alpha, a putative ribose methyltransferase and five genes encoding ribosomal proteins. Using the tandem affinity purification (TAP) method we identified thirteen S. cerevisiae ribosomal proteins interacting with Krr1p. Taken together, these results indicate that Krr1p interacts functionally as well as physically with ribosomal proteins. Northern blot analysis revealed that changes in the level of krr1-21 mRNA were accompanied by similar changes in the level of mRNAs of genes encoding ribosomal proteins. Thus, Krr1p and the genes encoding ribosomal proteins it interacts with seem to be coordinately regulated at the level of transcription.
