RESUMEN
While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Edético/metabolismo , Regulación Fúngica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/metabolismo , Puromicina/metabolismo , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cells rapidly alter gene expression in response to environmental stimuli such as nutrients, hormones, and drugs. During the imposed "remodeling" of gene expression, changes in the levels of particular mRNAs do not necessarily correlate with those of the encoded proteins, which could in part rely on the differential recruitment of mRNAs to translating ribosomes. To systematically address this issue, we have established an approach to rapidly access the translational status of each mRNA in the yeast Saccharomyces cerevisiae by affinity purification of endogenously formed ribosomes and the analysis of associated mRNAs with DNA microarrays. Using this method, we compared changes in total mRNA levels (transcriptome) with ribosome associations (translatome) after the application of different conditions of cellular stress. Severe stresses, induced by amino acid depletion or osmotic shock, stimulated highly correlated responses affecting about 15% of both total RNA levels and translatome. Many of the regulated messages code for functionally related proteins, thus reflecting logical responses to the particular stress. In contrast, mild stress provoked by addition of Calcofluor-white and menadione altered the translatome of approximately 1% of messages with only marginal effects on total mRNA, suggesting largely uncorrelated responses of transcriptome and translatome. Among these putative translationally regulated messages were most components of the mitochondrial ATPase. Increased polysome associations of corresponding messages and higher mitochondrial ATPase activities upon treatment confirmed the relevance for regulation of this macromolecular complex. Our results suggest the presence of highly sensitive translational regulatory networks that coordinate functionally related messages. These networks are preferentially activated for rapid adaptation of cells to minor environmental perturbations.
Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacos , Aminoácidos/deficiencia , Bencenosulfonatos/farmacología , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Presión Osmótica , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/genética , Transcriptoma , Vitamina K 3/farmacologíaRESUMEN
We describe ribosome affinity purification (RAP), a method that allows rapid purification of ribosomes and associated messages from the yeast Saccharomyces cerevisiae. The method relies on the expression of protein A tagged versions of the ribosomal protein Rpl16, which is used to efficiently recover endogenously formed ribosomes and polysomes from cellular extracts with IgG-coupled spherical microbeads. This approach can be applied to profile reactions of the translatome, which refers to all messages associated with ribosomes, with those of the transcriptome using DNA microarrays. In addition, ribosomal proteins, their modifications, and/or other associated proteins can be mapped with mass spectrometry. Finally, application of this method in other organisms provides a valuable tool to decipher cell-type specific gene expression patterns.