RESUMO
Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot-dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis , Complexo I de Transporte de Elétrons/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica de Plantas , Íntrons/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Proteínas de Plantas/genética , RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The Arabidopsis genome encodes >450 proteins containing the pentatricopeptide repeat (PPR) motif. The PPR proteins are classified into two groups, termed as P and P Long-Short (PLS) classes. Typically, the PLS subclass proteins are mainly involved in the RNA editing of mitochondrial and chloroplast transcripts, whereas most of the analyzed P subclass proteins have been mainly implicated in RNA metabolism, such as 5' or 3' transcript stabilization and processing, splicing and translation. Mutations of PPR genes often result in embryogenesis and altered seedling developmental defect phenotypes, but only a limited number of ppr mutants have been characterized in detail. In this report, we show that null mutations in the EMB2794 gene result in embryo arrest, due to altered splicing of nad2 transcripts in the Arabidopsis mitochondria. In angiosperms, nad2 has five exons that are transcribed individually from two mitochondrial DNA regions. Biochemical and in vivo analyses further indicate that recombinant or transgenic EMB2794 proteins bind to the nad2 pre-mRNAs in vitro as well as in vivo, suggesting a role for this protein in trans-splicing of nad2 intron 2 and possibly in the stability of the second pre-mRNA of nad2. Homozygous emb2794 lines, showing embryo-defective phenotypes, can be partially rescued by the addition of sucrose to the growth medium. Mitochondria of rescued homozygous mutant plants contain only traces of respiratory complex I, which lack the NADH-dehydrogenase activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/metabolismo , RNA Mensageiro/metabolismo , Arabidopsis/enzimologia , Perfilação da Expressão Gênica , Potencial da Membrana Mitocondrial , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Sementes/metabolismo , TranscriptomaRESUMO
Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.
Assuntos
Brassicaceae/enzimologia , Endorribonucleases/metabolismo , Íntrons/genética , Nucleotidiltransferases/metabolismo , Proteínas de Plantas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Brassicaceae/genética , Brassicaceae/metabolismo , Endorribonucleases/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Nucleotidiltransferases/genética , Proteínas de Plantas/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , DNA Polimerase Dirigida por RNA/genéticaRESUMO
Plant mitochondria are remarkable with respect to the presence of numerous group II introns which reside in many essential genes. The removal of the organellar introns from the coding genes they interrupt is essential for respiratory functions, and is facilitated by different enzymes that belong to a diverse set of protein families. These include maturases and RNA helicases related proteins that function in group II intron splicing in different organisms. Previous studies indicate a role for the nMAT2 maturase and the RNA helicase PMH2 in the maturation of different pre-RNAs in Arabidopsis mitochondria. However, the specific roles of these proteins in the splicing activity still need to be resolved. Using transcriptome analyses of Arabidopsis mitochondria, we show that nMAT2 and PMH2 function in the splicing of similar subsets of group II introns. Fractionation of native organellar extracts and pulldown experiments indicate that nMAT2 and PMH2 are associated together with their intron-RNA targets in large ribonucleoprotein particle in vivo. Moreover, the splicing efficiencies of the joint intron targets of nMAT2 and PMH2 are more strongly affected in a double nmat2/pmh2 mutant-line. These results are significant as they may imply that these proteins serve as components of a proto-spliceosomal complex in plant mitochondria.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , RNA Helicases/metabolismo , Splicing de RNA/fisiologia , DNA Polimerase Dirigida por RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Íntrons , Mutação , RNA Helicases/genética , RNA de Plantas/metabolismo , DNA Polimerase Dirigida por RNA/genética , Spliceossomos/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.
Assuntos
Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas/genética , Mitocôndrias/metabolismo , Nucleotidiltransferases/metabolismo , Trans-Splicing/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Respiração Celular , Técnicas de Inativação de Genes , Íntrons/genética , Metaboloma , Proteínas Mitocondriais/genética , Mutação , Nucleotidiltransferases/genética , Fenótipo , RNA/genética , RNA Mitocondrial , RNA de Plantas/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
Plant mitochondrial genomes (mtDNAs) are large and undergo frequent recombination events. A common phenotype that emerges as a consequence of altered mtDNA structure is cytoplasmic-male sterility (CMS). The molecular basis for CMS remains unclear, but it seems logical that altered respiration activities would result in reduced pollen production. Analysis of tobacco (Nicotiana tabacum) mtDNAs indicated that CMS-associated loci often contain fragments of known organellar genes. These may assemble with organellar complexes and thereby interfere with normal respiratory functions. Here, we analyzed whether the expression of truncated fragments of mitochondrial genes (i.e. atp4, cox1 and rps3) may induce male sterility by limiting the biogenesis of the respiratory machinery. cDNA fragments corresponding to atp4f, cox1f and rps3f were cloned in-frame to a mitochondrial localization signal and a C-termini HA-tag under a tapetum-specific promoter and introduced to tobacco plants by Agrobacterium-mediated transformation. The constructs were then analyzed for their effect on mitochondrial activity and pollen fertility. Atp4f, Cox1f and Rps3f plants demonstrated male sterility phenotypes, which were tightly correlated with the expression of the recombinant fragments in the floral meristem. Fractionation of native organellar extracts showed that the recombinant ATP4f-HA, COX1f-HA and RPS3f-HA proteins are found in large membrane-associated particles. Analysis of the respiratory activities and protein profiles indicated that organellar complex I was altered in Atp4f, Cox1f and Rps3f plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes Mitocondriais/genética , Nicotiana/anatomia & histologia , Nicotiana/genética , Infertilidade das Plantas/genética , Respiração Celular/genética , Espaço Intracelular/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese Insercional/genética , Fases de Leitura Aberta/genética , Fenótipo , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/genética , Transporte Proteico , Proteínas Recombinantes/metabolismo , Recombinação Genética/genética , Nicotiana/citologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0201631.].
RESUMO
Mitochondria are key sites for cellular energy metabolism and are essential to cell survival. As descendants of eubacterial symbionts (specifically α-proteobacteria), mitochondria contain their own genomes (mtDNAs), RNAs and ribosomes. Plants need to coordinate their energy demands during particular growth and developmental stages. The regulation of mtDNA expression is critical for controlling the oxidative phosphorylation capacity in response to physiological or environmental signals. The mitochondrial transcription termination factor (mTERF) family has recently emerged as a central player in mitochondrial gene expression in various eukaryotes. Interestingly, the number of mTERFs has been greatly expanded in the nuclear genomes of plants, with more than 30 members in different angiosperms. The majority of the annotated mTERFs in plants are predicted to be plastid- or mitochondria-localized. These are therefore expected to play important roles in organellar gene expression in angiosperms. Yet, functions have been assigned to only a small fraction of these factors in plants. Here, we report the characterization of mTERF22 (At5g64950) which functions in the regulation of mtDNA transcription in Arabidopsis thaliana. GFP localization assays indicate that mTERF22 resides within the mitochondria. Disruption of mTERF22 function results in reduced mtRNA accumulation and altered organelle biogenesis. Transcriptomic and run-on experiments suggest that the phenotypes of mterf22 mutants are attributable, at least in part, to altered mitochondria transcription, and indicate that mTERF22 affects the expression of numerous mitochondrial genes in Arabidopsis plants.