RESUMEN
Epistatic interactions play a fundamental role in molecular evolution, but little is known about the spatial distribution of these interactions within genes. To systematically survey a model landscape of intragenic epistasis, we quantified the fitness of ~60,000 Saccharomyces cerevisiae strains expressing randomly mutated variants of the 333-nucleotide-long U3 small nucleolar RNA (snoRNA). The fitness effects of individual mutations were correlated with evolutionary conservation and structural stability. Many mutations had small individual effects but had large effects in the context of additional mutations, which indicated negative epistasis. Clusters of negative interactions were explained by local thermodynamic threshold effects, whereas positive interactions were enriched among large-effect sites and between base-paired nucleotides. We conclude that high-throughput mapping of intragenic epistasis can identify key structural and functional features of macromolecules.
Asunto(s)
Epistasis Genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , ARN Nucleolar Pequeño/genética , Saccharomyces cerevisiae/genética , Evolución Molecular , Redes Reguladoras de Genes , Variación Genética , Mutagénesis , Mutación , Pliegue del ARN , ARN Nucleolar Pequeño/química , TermodinámicaRESUMEN
Pentatricopeptide repeat (PPR) proteins are the largest known RNA-binding protein family, and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly to mitochondria and chloroplasts, and many were shown to modulate organellar genome expression on the posttranscriptional level. Although the genomes of land plants encode hundreds of PPR proteins, only a few have been identified in Fungi and Metazoa. As the current PPR motif profiles are built mainly on the basis of the predominant plant sequences, they are unlikely to be optimal for detecting fungal and animal members of the family, and many putative PPR proteins in these genomes may remain undetected. In order to verify this hypothesis, we designed a hidden Markov model-based bioinformatic tool called Supervised Clustering-based Iterative Phylogenetic Hidden Markov Model algorithm for the Evaluation of tandem Repeat motif families (SCIPHER) using sequence data from orthologous clusters from available yeast genomes. This approach allowed us to assign 12 new proteins in Saccharomyces cerevisiae to the PPR family. Similarly, in other yeast species, we obtained a 5-fold increase in the detection of PPR motifs, compared with the previous tools. All the newly identified S. cerevisiae PPR proteins localize in the mitochondrion and are a part of the RNA processing interaction network. Furthermore, the yeast PPR proteins seem to undergo an accelerated divergent evolution. Analysis of single and double amino acid substitutions in the Dmr1 protein of S. cerevisiae suggests that cooperative interactions between motifs and pseudoreversion could be the force driving this rapid evolution.
Asunto(s)
Algoritmos , Evolución Molecular , Genómica/métodos , Cadenas de Markov , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Análisis por Conglomerados , Genoma Mitocondrial , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to rho(-)/rho(0) petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Delta mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Secuencia de Bases , Respiración de la Célula , Citoplasma/metabolismo , Exorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Mitocondriales/química , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
The mitochondrial degradosome (mtEXO) of S. cerevisiae is the main exoribonuclease of yeast mitochondria. It is involved in many pathways of mitochondrial RNA metabolism, including RNA degradation, surveillance, and processing, and its activity is essential for mitochondrial gene function. The mitochondrial degradosome is a very simple example of a 3' to 5'-exoribonucleolytic complex. It is composed of only two subunits: Dss1p, which is an RNR (RNase II-like) family exoribonuclease, and Suv3p, which is a DExH/D-box RNA helicase. The two subunits form a tight complex and their activities are highly interdependent, with the RNase activity greatly enhanced in the presence of the helicase subunit, and the helicase activity entirely dependent on the presence of the ribonuclease subunit. In this chapter, we present methods for studying the function of the yeast mitochondrial degradosome in vivo, through the analysis of degradosome-deficient mutant yeast strains, and in vitro, through heterologous expression in E. coli and purification of the degradosome subunits and reconstitution of a functional complex. We provide the protocols for studying ribonuclease, ATPase, and helicase activities and for measuring the RNA binding capacity of the complex and its subunits.
Asunto(s)
Mitocondrias/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , Northern Blotting , Cromatografía en Capa Delgada , Cartilla de ADN , Hidrólisis , Cinética , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción GenéticaRESUMEN
The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3'-to-5' exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Delta) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Delta strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.