RESUMEN
Mitochondrial genomes code for several core components of respiratory chain complexes. Thus, mitochondrial translation is of great importance for the organelle as well as for the whole cell. In yeast, mitochondrial translation initiation factor 3, Aim23p, is not essential for the organellar protein synthesis; however, its absence leads to a significant quantitative imbalance of the mitochondrial translation products. This fact points to a possible specific action of Aim23p on the biosynthesis of some mitochondrial protein species. In this work, we examined such peculiar effects of Aim23p in relation to yeast mitochondrial COX2 mRNA translation. We show that Aim23p is indispensable to this process. According to our data, this is mediated by Aimp23p interaction with the known specific factor of the COX2 mRNA translation, Pet111p. If there is no Aim23p in the yeast cells, an increased amount of Pet111p ensures proper COX2 mRNA translation. Our results demonstrate the additional non-canonical function of initiation factor 3 in yeast mitochondrial translation.
Asunto(s)
Ciclooxigenasa 2/genética , Factores Eucarióticos de Iniciación/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Regiones no Traducidas 5'/genética , Ciclooxigenasa 2/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Genéticos , Mutación , Factores de Iniciación de Péptidos/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.
Asunto(s)
Ciclooxigenasa 2/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Factores de Iniciación de Péptidos/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind RNA and influence gene expression in mitochondria and chloroplasts. Several PPR proteins in plants harbor a carboxy-terminal small-MutS-related (SMR) domain, but the functions of the SMR appendage are unknown. To address this issue, we studied a maize PPR-SMR protein denoted PPR53 (GRMZM2G438524), which is orthologous to the Arabidopsis protein SOT1 (AT5G46580). Null ppr53 alleles condition a chlorotic, seedling-lethal phenotype and a reduction in plastid ribosome content. Plastome-wide transcriptome and translatome analyses revealed strong defects in the expression of the ndhA and rrn23 genes, which were superimposed on secondary effects resulting from a decrease in plastid ribosome content. Transcripts with processed 5'-ends mapping approximately 70 nucleotides upstream of rrn23 and ndhA are absent in ppr53 mutants, and the translational efficiency of the residual ndhA mRNAs is reduced. Recombinant PPR53 binds with high affinity and specificity to the 5' proximal region of the PPR53-dependent 23S rRNA, suggesting that PPR53 protects this RNA via a barrier mechanism similar to that described for several PPR proteins lacking SMR motifs. However, recombinant PPR53 did not bind with high affinity to the ndhA 5' untranslated region, suggesting that PPR53's RNA-stabilization and translation-enhancing effects at the ndhA locus involve the participation of other factors.
Asunto(s)
Proteínas de Plantas/genética , Biosíntesis de Proteínas/genética , ARN del Cloroplasto/genética , Zea mays/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Genoma del Cloroplasto/genética , Immunoblotting , Mutación , Proteínas de Plantas/metabolismo , Unión Proteica , Estabilidad del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transcriptoma/genética , Zea mays/metabolismoRESUMEN
RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility.
