RESUMO
RNA C-to-U editing in organelles is essential for plant growth and development; however, the underlying mechanism is not fully understood. Here, we report that pentatricopeptide repeat (PPR)-E subclass proteins carry out RNA C-to-U editing by recruiting the trans deaminase PPR motifs, coiled-coil, and DYW domain-containing protein 1 (PCW1) in maize (Zea mays) mitochondria. Loss-of-function of bZIP and coiled-coil domain-containing PPR 1 (bCCP1) or PCW1 arrests seed development in maize. bCCP1 encodes a bZIP and coiled-coil domain-containing PPR protein, and PCW1 encodes an atypical PPR-DYW protein. bCCP1 is required for editing at 66 sites in mitochondria and PCW1 is required for editing at 102 sites, including the 66 sites that require bCCP1. The PCW1-mediated editing sites are exclusively associated with PPR-E proteins. bCCP1 interacts with PCW1 and the PPR-E protein Empty pericarp7 (EMP7). Two multiple organellar RNA editing factor (MORF) proteins, ZmMORF1 and ZmMORF8, interact with PCW1, EMP7, and bCCP1. ZmMORF8 enhanced the EMP7-PCW1 interaction in a yeast three-hybrid assay. C-to-U editing at the ccmFN-1553 site in maize required EMP7, bCCP1, and PCW1. These results suggest that PPR-E proteins function in RNA editing by recruiting the trans deaminase PCW1 and bCCP1, and MORF1/8 assist this recruitment through protein-protein interactions.
Assuntos
Edição de RNA , Zea mays , Zea mays/metabolismo , Edição de RNA/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Organelas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNARESUMO
The essential role of plastid translation in embryogenesis has been established in many plants, but a retrograde signal triggered by defective plastid translation machinery that may leads to embryogenesis arrest remains unknown. In this study, we characterized an embryo defective27 (emb27) mutant in maize (Zea mays), and cloning indicates that Emb27 encodes the plastid ribosomal protein S13. The null mutant emb27-1 conditions an emb phenotype with arrested embryogenesis; however, the leaky mutant emb27-2 exhibits normal embryogenesis but an albino seedling-lethal phenotype. The emb27-1/emb27-2 trans-heterozygotes display varying phenotypes from emb to normal seeds but albino seedlings. Analysis of the Emb27 transcription levels in these mutants revealed that the Emb27 expression level in the embryo corresponds with the phenotypic expression of the emb27 mutants. In the W22 genetic background, an Emb27 transcription level higher than 6% of the wild-type level renders normal embryogenesis, whereas lower than that arrests embryogenesis. Mutation of Emb27 reduces the level of plastid 16S rRNA and the accumulation of the plastid-encoded proteins. As a secondary effect, splicing of several plastid introns was impaired in emb27-1 and 2 other plastid translation-defective mutants, emb15 and emb16, suggesting that plastome-encoded factors are required for the splicing of these introns, such as Maturase K (MatK). Our results indicate that EMB27 is essential for plastid protein translation, embryogenesis, and seedling development in maize and reveal an expression threshold of Emb27 for maize embryogenesis.
Assuntos
Regulação da Expressão Gênica de Plantas , Mutação , Proteínas de Plantas , Plastídeos , Plântula , Sementes , Zea mays , Zea mays/genética , Zea mays/embriologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutação/genética , Plastídeos/genética , Plastídeos/metabolismo , Fenótipo , Splicing de RNA/genética , Íntrons/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Identifying the PPR-E+-NUWA-DYW2 editosome improves our understanding of the C-to-U RNA editing in plant organelles. However, the mechanism of RNA editing remains to be elucidated. Here, we report that GLUTAMINE-RICH PROTEIN23 (GRP23), a previously identified nuclear transcription regulator, plays an essential role in mitochondrial RNA editing through interacting with MORF (multiple organellar RNA-editing factor) proteins and atypical DYW-type pentatricopeptide repeat (PPR) proteins. GRP23 is targeted to mitochondria, plastids, and nuclei. Analysis of the grp23 mutants rescued by embryo-specific complementation shows decreased editing efficiency at 352 sites in mitochondria and 6 sites in plastids, with a predominant specificity for sites edited by the PPR-E and PPR-DYW proteins. GRP23 interacts with atypical PPR-DYW proteins (MEF8, MEF8S, DYW2, and DYW4) and MORF proteins (MORF1 and MORF8), whereas the four PPR-DYWs interact with the two MORFs. These interactions may increase the stability of the GRP23-MORF-atypical PPR-DYW complex. Furthermore, analysis of mef8Nâ³64aamef8s double mutants shows that MEF8/MEF8S are required for the editing of the PPR-E protein-targeted sites in mitochondria. GRP23 could enhance the interaction between PPR-E and MEF8/MEF8S and form a homodimer or heterodimer with NUWA. Genetic complementation analysis shows that the C-terminal domains of GRP23 and NUWA possess a similar function, probably in the interaction with the MORFs. NUWA also interacts with atypical PPR-DYWs in yeast. Both GRP23 and NUWA interact with the atypical PPR-DYWs, suggesting that the PPR-E proteins recruit MEF8/MEF8S, whereas the PPR-E+ proteins specifically recruit DYW2 as the trans deaminase, and then GRP23, NUWA, and MORFs facilitate and/or stabilize the E or E+-type editosome formation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Edição de RNA , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Edição de RNA/genética , RNA Mitocondrial/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA-protein complexes in an adenosine triphosphate-dependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize (Zea mays) DEAD-box RNA helicase 48 (ZmRH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis, and seed development. Loss of ZmRH48 function severely arrested embryogenesis and endosperm development, leading to defective kernel formation. ZmRH48 is targeted to mitochondria, where its deficiency dramatically reduced the splicing efficiency of five cis-introns (nad5 intron 1; nad7 introns 1, 2, and 3; and ccmFc intron 1) and one trans-intron (nad2 intron 2), leading to lower levels of mitochondrial complexes I and III. ZmRH48 interacts with two unique pentatricopeptide repeat (PPR) proteins, PPR-SMR1 and SPR2, which are required for the splicing of over half of all mitochondrial introns. PPR-SMR1 interacts with SPR2, and both proteins interact with P-type PPR proteins and Zm-mCSF1 to facilitate intron splicing. These results suggest that ZmRH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.
Assuntos
Proteínas de Plantas , Zea mays , Humanos , Zea mays/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Íntrons/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Regulação da Expressão Gênica de Plantas , Sementes/metabolismo , Mitocôndrias/metabolismo , RNA/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismoRESUMO
C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins).
Assuntos
Proteínas de Plantas/metabolismo , Edição de RNA , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Zea mays/fisiologia , Complexo I de Transporte de Elétrons/metabolismo , Desenvolvimento Embrionário/genética , Endosperma/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação com Perda de Função , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The self-splicing of group II introns during RNA processing depends on their catalytic structure and is influenced by numerous factors that promote the formation of that structure through direct binding. Here we report that C-to-U editing at a specific position in two nad7 introns is essential to splicing, which also implies that the catalytic activity of non-functional group II introns could be restored by editing. We characterized a maize (Zea mays) mutant, dek46, with a defective kernel phenotype; Dek46 encodes a pentatricopeptide repeat DYW protein exclusively localized in mitochondria. Analyses of the coding regions of mitochondrial transcripts did not uncover differences in RNA editing between dek46 mutant and wild-type maize, but showed that splicing of nad7 introns 3 and 4 is severely reduced in the mutant. Furthermore, editing at nucleotide 22 of domain 5 (D5-C22) of both introns is abolished in dek46. We constructed chimeric introns by swapping D5 of P.li.LSUI2 with D5 of nad7 intron 3. In vitro splicing assays indicated that the chimeric intron containing D5-U22 can be self-spliced, but the one containing D5-C22 cannot. These results indicate that DEK46 functions in the C-to-U editing of D5-C22 of both introns, and the U base at this position is critical to intron splicing.
Assuntos
Íntrons , Mitocôndrias/metabolismo , Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Splicing de RNA , RNA de Plantas/genética , RNA de Plantas/metabolismo , Sementes/metabolismo , Zea mays/metabolismoRESUMO
Pentatricopeptide repeat (PPR) proteins are involved in the C-to-U RNA editing of organellar transcripts. The maize genome contains over 600 PPR proteins and few have been found to function in the C-to-U RNA editing in chloroplasts. Here, we report the function of ZmPPR26 in the C-to-U RNA editing and chloroplast biogenesis in maize. ZmPPR26 encodes a DYW-type PPR protein targeted to chloroplasts. The zmppr26 mutant exhibits albino seedling-lethal phenotype. Loss of function of ZmPPR26 abolishes the editing at atpA-1148 site, and decreases the editing at ndhF-62, rpl20-308, rpl2-2, rpoC2-2774, petB-668, rps8-182, and ndhA-50 sites. Overexpression of ZmPPR26 in zmppr26 restores the editing efficiency and rescues the albino seedling-lethal phenotype. Abolished editing at atpA-1148 causes a Leu to Ser change at AtpA-383 that leads to a reduction in the abundance of chloroplast ATP synthase in zmppr26. The accumulation of photosynthetic complexes are also markedly reduced in zmppr26, providing an explanation for the albino seedling-lethal phenotype. These results indicate that ZmPPR26 is required for the editing at atpA-1148 and is important for editing at the other seven sites in maize chloroplasts. The editing at atpA-1148 is critical for AtpA function, assembly of ATP synthase complex, and chloroplast biogenesis in maize.
Assuntos
Edição de RNA , Zea mays , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
Pentatricopeptide repeat (PPR) proteins play an important role in post-transcriptional regulation of mitochondrial gene expression. Functions of many PPR proteins and their roles in plant growth and development remain unknown. Through characterization of an empty pericarp32 (emp32) mutant, we identified the function of Emp32 in mitochondrial intron splicing and seed development in maize. The loss-of-function mutant emp32 shows embryo lethality with severely arrested embryo and endosperm development, and over-expression of Emp32 rescues the embryo-lethality. EMP32 is a P-type PPR protein targeted to mitochondria. Loss of function in Emp32 dramatically decreases the splicing efficiency of nad7 intron 2, while complementation of Emp32 restores the splicing efficiency. Although nad7 intron 2 is partially spliced in the wild type, over-expression of Emp32 does not increase the splicing efficiency. The splicing deficiency of nad7 intron 2 blocks the assembly of mitochondrial complex I and dramatically reduces its activity, which may explain the embryo-lethality in emp32. In addition to the one copy of nad7 in the maize mitochondrial genome, we identified one to six copies of nad7 in the nuclear genomes in different maize inbred lines. These copies appear not to be expressed. Together, our results revealed that the P-type PPR protein EMP32 is required for the cis-splicing of nad7 intron 2 and seed development in maize.
Assuntos
NADH Desidrogenase/genética , Proteínas de Plantas/fisiologia , Splicing de RNA/genética , Zea mays , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Íntrons/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Pentatricopeptide repeat (PPR) proteins are helical repeat RNA-binding proteins that function in RNA processing by conferring sequence-specific RNA-binding activity. Owing to the lethality of PPR mutants, functions of many PPR proteins remain obscure. In this study, we report the function of PPR20 in intron splicing in mitochondria and its role in maize seed development. PPR20 is a P-type PPR protein targeted to mitochondria. The ppr20 mutants display slow embryo and endosperm development. Null mutation of PPR20 severely reduces the cis-splicing of mitochondrial nad2 intron 3, resulting in reduction in the assembly and activity of mitochondrial complex I. The ppr20-35 allele with a Mu insertion in the N-terminal region shows a much weaker phenotype. Molecular analyses revealed that the mutant produces a truncated transcript, coding for PPR20ΔN120 lacking the N-terminal 120 amino acids. Subcellular localization revealed that PPR20ΔN120:GFP is able to target to mitochondria as well, suggesting the sequence diversity of the mitochondrial targeting peptides. Another mutant zm_mterf15 was also found to be impaired in the splicing of mitochondrial nad2 intron 3. Further analyses are required to identify the exact function of PPR20 and Zm_mTERF15 in the splicing of nad2 intron 3.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Íntrons/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Splicing de RNA , Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Alelos , Complexo I de Transporte de Elétrons/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , Mutação , Fenótipo , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Ligação a RNA , Sementes/citologia , Sementes/genética , Zea mays/genéticaRESUMO
In land plants, cytidine-to-uridine (C-to-U) editing of organellar transcripts is an important post-transcriptional process, which is considered to remediate DNA genetic mutations to restore the coding of functional proteins. Pentatricopeptide repeat (PPR) proteins have key roles in C-to-U editing. Owing to its large number, however, the biological functions of many PPR proteins remain to be identified. Through characterizing a small kernel4 (smk4) mutant, here we report the function of Smk4 and its role in maize growth and development. Null mutation of Smk4 slows plant growth and development, causing small plants, delayed flowering time, and small kernels. Cloning revealed that Smk4 encodes a new E-subclass PPR protein, and localization indicated that SMK4 is exclusively localized in mitochondria. Loss of Smk4 function abolishes C-to-U editing at position 1489 of the cytochrome c oxidase1 (cox1) transcript, causing an amino acid change from serine to proline at 497 in Cox1. Cox1 is a core component of mitochondrial complex IV. Indeed, complex IV activity is reduced in the smk4, along with drastically elevated expression of alternative oxidases (AOX). These results indicate that SMK4 functions in the C-to-U editing of cox1-1489, and this editing is crucial for mitochondrial complex IV activity, plant growth, and kernel development in maize.
Assuntos
Mitocôndrias/metabolismo , Edição de RNA , Sementes/embriologia , Sementes/genética , Zea mays/embriologia , Zea mays/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Respiração Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de AminoácidosRESUMO
Splicing of plant organellar group II introns is under accurate nuclear control that employs many nucleus-encoded protein cofactors from various families. For mitochondrial introns, only a few splicing factors have been characterized because disruption of their functions often causes embryo lethality. Here, we report the function of Empty Pericarp8 (Emp8) in the splicing of three group II introns in mitochondria, complex I biogenesis, and seed development in maize. Emp8 encodes a P subfamily pentatricopeptide repeat protein that localizes in mitochondria. The loss-of-function mutants of Emp8 are embryo lethal, showing severely arrested embryo and endosperm development in maize. The respiration rate in the emp8 mutants is reduced with substantially enhanced expression of alternative oxidases. Transcript analysis indicated that the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 are abolished, and the cis-splicing of nad2 intron 1 is severely impaired in the emp8 mutants. These defects consequently lead to the disassembly of mitochondrial complex I and a dramatic reduction in its activity. Together, these results suggest that Emp8 is required for the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 and nad2 intron 1, which is essential to mitochondrial complex I assembly and hence to embryogenesis and endosperm development in maize.
RESUMO
Plant mitochondrial genes contain cis- and trans-group II introns that must be spliced before translation. The mechanism by which these introns are spliced is not well understood. Several families of proteins have been implicated in the intron splicing, of which the pentatricopeptide repeat (PPR) proteins are proposed to confer the substrate binding specificity. However, very few PPRs are characterized. Here, we report the function of a P-type PPR protein, EMP12, and its role in seed development. EMP12 is targeted to mitochondria. Loss-of-function mutation in Emp12 severely arrests embryo and endosperm development, causing embryo lethality. The trans-splicing of mitochondrial nad2 intron 2 and cis-splicing of nad2 intron 4 are abolished, whereas the cis-splicing of nad2 intron 1 is reduced in emp12 mutants. As a result, complex I assembly is disrupted, and its activity is strongly reduced in the mutants. The expression of the alternative oxidase and several components of other mitochondrial complexes is increased, possibly in response to the defective complex I. These results suggest that Emp12 is required for the trans-splicing of nad2 intron 2 and cis-splicing of nad2 introns 1 and 4, and is important to complex I biogenesis, and embryogenesis and endosperm development in maize.
Assuntos
Proteínas Mitocondriais/genética , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Zea mays/genética , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Íntrons , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/metabolismo , Splicing de RNA , Sementes/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
Vitamin B6, an essential cofactor for a range of biochemical reactions and a potent antioxidant, plays important roles in plant growth, development, and stress tolerance. Vitamin B6 deficiency causes embryo lethality in Arabidopsis (Arabidopsis thaliana), but the specific role of vitamin B6 biosynthesis in endosperm development has not been fully addressed, especially in monocot crops, where endosperm constitutes the major portion of the grain. Through molecular characterization of a small kernel2 (smk2) mutant in maize, we reveal that vitamin B6 has differential effects on embryogenesis and endosperm development in maize. The B6 vitamer pyridoxal 5'-phosphate (PLP) is drastically reduced in both the smk2 embryo and the endosperm. However, whereas embryogenesis of the smk2 mutant is arrested at the transition stage, endosperm formation is nearly normal. Cloning reveals that Smk2 encodes the glutaminase subunit of the PLP synthase complex involved in vitamin B6 biosynthesis de novo. Smk2 partially complements the Arabidopsis vitamin B6-deficient mutant pdx2.1 and Saccharomyces cerevisiae pyridoxine auxotrophic mutant MML21. Smk2 is constitutively expressed in the maize plant, including developing embryos. Analysis of B6 vitamers indicates that the endosperm accumulates a large amount of pyridoxamine 5'-phosphate (PMP). These results indicate that vitamin B6 is essential to embryogenesis but has a reduced role in endosperm development in maize. The vitamin B6 required for seed development is synthesized in the seed, and the endosperm accumulates PMP probably as a storage form of vitamin B6.
Assuntos
Glutaminase/metabolismo , Mutação/genética , Sementes/embriologia , Vitamina B 6/biossíntese , Zea mays/embriologia , Zea mays/enzimologia , Sequência de Aminoácidos , Arabidopsis/genética , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Citosol/metabolismo , Endosperma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glutaminase/química , Fenótipo , Plantas Geneticamente Modificadas , Subunidades Proteicas/metabolismo , Piridoxina/metabolismo , Saccharomyces cerevisiae/metabolismo , Sementes/genética , Zea mays/genéticaRESUMO
Pentatricopeptide repeat (PPR) proteins comprise a large family of sequence-specific RNA binding proteins in land plants. Because of its large family size and frequent embryo lethality in the mutants, molecular functions and physiological roles of many PPR proteins are unknown. Through characterization of an empty pericarp9 (emp9) mutant in maize (Zea mays), we defined the functions of EMP9 in mitochondrial RNA editing, respiratory complex formation and seed development. Mu insertions in different regions of Emp9 facilitated dissection of the domain functions of the EMP9. Through genetic and functional analyses of multiple alleles, we showed that deletions of two N-terminal PPR motifs and partial E+ domain do not eliminate the editing function of EMP9. Emp9 encodes an E+ subclass PPR protein that is localized in mitochondria. Loss of EMP9 function abolishes the C-to-U editing of ccmB-43 and rps4-335 sites in mitochondria. The loss of editing at ccmB-43 and rps4-335 affects the maturation of cytochrome c and impairs the biogenesis of mitochondrial respiratory complexes, particularly complex III. This work extends our understanding of PPR-E+ protein in editing function and seed development, and provides insights into the molecular function of mitochondrial CcmB protein in higher plants.
Assuntos
Mitocôndrias/metabolismo , Biogênese de Organelas , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Edição de RNA/genética , Sementes/genética , Zea mays/embriologia , Zea mays/genética , Alelos , Arabidopsis/genética , Sequência de Bases , Endosperma/embriologia , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Mutação com Perda de Função , Plantas Geneticamente Modificadas , Sementes/embriologiaRESUMO
OBJECTIVE: To evaluate the efficacy of nimotuzumab combined with palitaxel liposome and carboplatin (LP) regimen for treatment of advanced non-small cell lung cancer (NSCLC), and to observe the changes of tumor markers and toxicities in the treatment. METHODS Forty-one patients with advanced NSCLC were randomly divided into 2 groups: 21 patients in the observation group were treated with nimotuzumab (200 mg per week for 6 weeks), palitaxel liposome 160 mg/m2 and carboplatin (AUC = 6). 20 patients in the control group were given LP regimen. Each group completed two cycles of chemotherapy. The level of tumor markers (CEA, CYFR21-1 and NSE) and toxicities were checked at one week before and after the treatment. Thoracic CT examinations were taken before treatment and at the fourth week and eighth week after treatment. RESULTS: In the observation group, there were 2 cases of CR, 7 cases of PR, 9 cases of SD and 3 cases of PD. The objective response rate (RR) was 42. 9% in the observation group. In the control group, there were 1 case of CR, 6 cases of PR, 8 cases of SD and 5 cases of PD, with a RR of 35.0% in this group. There was no significant difference in the RR between the two groups (P = 0.751). The time to progression (TIP) was 6. 9 months in the observation group and 5. 7 months in the control group, with a significant difference (P = 0.027). The levels of NSE decreased significantly in both groups and showed a significant difference (P = 0.039). The levels of CEA and CYFRA21 in both groups were decreased after treatment, but did not show a significant difference before and after treatment, respectively. Except 3 cases had I-II skin toxicities on the faces in the observation group, there was no significant difference in toxicities between the two groups. CONCLUSION: Nimotuzmab combined with LP regimen shows a synergistic effect, can increase the efficacy and prolong TFP in advanced NSCLC patients. The toxicities are mild and tolerable.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carboplatina/administração & dosagem , Antígeno Carcinoembrionário/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Terapia Combinada , Exantema/induzido quimicamente , Feminino , Humanos , Queratina-19/metabolismo , Lipossomos/administração & dosagem , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/administração & dosagem , Fosfopiruvato Hidratase/metabolismo , Indução de RemissãoRESUMO
The conversion of cytidines to uridines (C-to-U) at specific sites in mitochondrial and plastid transcripts is a post-transcriptional processing event that is important to the expression of organellar genes. Pentatricopeptide repeat (PPR) proteins are involved in this process. In this study, we report the function of a previously uncharacterized PPR-DYW protein, Empty pericarp17 (EMP17), in the C-to-U editing and kernel development in maize. EMP17 is targeted to mitochondria. The loss-function of EMP17 arrests maize kernel development, abolishes the editing at ccmF C -799 and nad2-677 sites, and reduces the editing at ccmF C -906 and -966 sites. The absence of editing causes amino acid residue changes in CcmFC-267 (Ser to Pro) and Nad2-226 (Phe to Ser), respectively. As CcmFC functions in cytochrome c (Cytc) maturation, the amount of Cytc and Cytc 1 protein is drastically reduced in emp17, suggesting that the CcmFC-267 (Ser to Pro) change impairs the CcmFC function. As a result, the assembly of complex III is strikingly decreased in emp17. In contrast, the assembly of complex I appears less affected, suggesting that the Nad2-226 (Phe to Ser) change may have less impact on Nad2 function. Together, these results indicate that EMP17 is required for the C-to-U editing at several sites in mitochondrial transcripts, complex III biogenesis, and seed development in maize.
RESUMO
In plants, splicing of organellar group II introns involves numerous nucleus-encoded trans-factors. But, how these trans-factors function and interact is not well understood. Here we report the function of a pentatricopeptide repeat (PPR) protein PPR14 and its physical relationship with other splicing factors in mitochondria. Null mutations of PPR14 severely arrest the embryo and endosperm development, causing an empty pericarp phenotype. PPR14 is required for the splicing of NADH dehydrogenase 2 (nad2) intron 3 and nad7 introns 1 and 2 in mitochondria. The absence of nad2 and nad7 transcripts leads to disruption of the mitochondrial complex I assembly and abolishes its NADH dehydrogenase activity. This is accompanied with increased levels of other mitochondrial complexes and elevated expression of the alternative oxidase proteins. As the function of PPR14 overlaps with PPR-SMR1 and the CRM-domain containing protein Zm-mCSF1, we tested their interactions. Protein-protein interaction analysis indicated that PPR14 interacts with PPR-SMR1 and Zm-mCSF1, suggesting that these three proteins may form a complex. As PPR proteins and CRM-domain containing proteins have many members in mitochondria and chloroplasts, we propose that organellar group II intron splicing is probably mediated by a dynamic complex that includes different PPR and CRM proteins in plants.
RESUMO
OBJECTIVE: To evaluate the efficacy of Endosteal(TM) (rh-endostatin, YH-16) combined with docetaxel and carboplatin (TP) regimen for the adjuvant treatment of non-small lung cancer (NSCLC) and its impact on circulating blood markers. METHODS: 36 patients with stage Ib-IIIa postoperative NSCLC, were randomly divided into the treatment group, Endosteal(TM) plus TP regimen, and the control group, TP regimen only, respectively. DFS and toxicities of patients were observed. The numbers of CEC and the levels of tumor marker CEA, NSE and CYFR21-1 were measured. RESULTS: The numbers of CEC and the levels of CEA, NSE and CYFR21-1 decreased after treatment. There were significant differences in CEC and NSE between treatment group and control group after four cycles of treatment, respectively (P = 0.016 and 0.013). Disease-free survival time (DFS) was longer in treatment group than control group but without significant difference. CEC was significantly increased in recurrent and metastasis cases and decreased after effective treatment. CONCLUSION: Endosteal(TM) combined with TP regimen seem to be superior to TP alone in some short term index for the treatment of postoperative NSCLC even though long-term survival is still anticipated. CEC, as a biomarker, may be useful in predicting the efficacy of the such synergistic treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Endostatinas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Taxoides/uso terapêutico , Adulto , Idoso , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Docetaxel , Endostatinas/administração & dosagem , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Taxoides/administração & dosagemRESUMO
The recently identified PPR-E+/NVWA/DYW2 RNA editing complex provides insights into the mechanism of RNA editing in higher plant organelles. However, whether the complex works together with the previously identified editing factors RIPs/MORFs is unclear. In this paper, we identified a maize Smk6 gene, which encodes a mitochondrion-targeted PPR-E+protein with E1 and E2 domains at the C terminus. Loss of Smk6 function affects the C-to-U editing at nad1-740, nad4L-110, nad7-739, and mttB-138,139 sites, impairs mitochondrial activity and blocks embryogenesis and endosperm development. Genetic and molecular analysis indicated that SMK6 is the maize ortholog of the Arabidopsis SLO2, which is a component of the PPR-E+/NVWA/DYW2 editing complex. However, yeast two-hybrid analyses did not detect any interaction between SMK6 and any of the mitochondrion-targeted RIPs/MORFs, suggesting that RIPs/MORFs may not be a component of PPR-E+/NVWA/DYW2 RNA editing complex. Further analyses are required to provide evidence that RIP/MORFs and SMK6 do not physically interact in vivo.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Edição de RNA , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Zea mays/metabolismoRESUMO
The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3.