The P-class pentatricopeptide repeat protein binds to the 5’ untranslated and translated regions of chloroplast psbI-ycf12 transcript and is involved in mRNA stability
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PROVIDER: PRJNA497789 | ENA |
REPOSITORIES: ENA
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Project description:Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among them, pentatricopeptide repeat (PPR) proteins are known to be a key player in posttranscriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited a reduced growth of the protonemata and lower photosynthetic activity. Immuno-blot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poorly formation of the PSII super-complexes in the KO mutants. To access whether PpPPR_21 is involved in the chloroplast gene expression, chloroplast genome-wide microarray analysis and northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII, did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels of WT and the KO mutants. A complemented PpPPR_21 KO moss transformed with the cognate full-length PpPPR_21 cDNA rescued the psbI transcript accumulation level. RNA binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5’-untraslated and translated region of the psbI mRNA. The present study suggests that PpPPR_21 may be essential for accumulation of a stable psbI-ycf12 mRNA.
2019-03-26 | GSE121554 | GEO
Project description:Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5′ capping and 3′ polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3′-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3′-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.
2021-04-07 | GSE139514 | GEO
Project description:The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.
| S-EPMC6899631 | biostudies-literature
Project description:In Trypanosoma brucei, most mitochondrial mRNAs undergo U-insertion/deletion editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: Pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for ribosome recruitment. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes exonucleolytic degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point prevents translation of incompletely edited mRNAs. Our findings also implicate the RNA editing substrate binding complex (RESC) in mediating the interaction between the 5′-end bound pyrophosphohydrolase MERS1 and 3′-end associated KPAF4 to enable mRNA circularization. This event is critical for transcript stability during the editing process.
2019-04-02 | PXD012008 | Pride
Project description:BackgroundEven though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis ( http://ppr.plantenergy.uwa.edu.au ).ResultsThe homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants.ConclusionsThese results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.
| S-EPMC6555926 | biostudies-literature
Project description:The pentatricopeptide repeat (PPR) proteins form one of the largest families in higher plants and are believed to be involved in the posttranscriptional processes of gene expression in plant organelles. It has been shown by using a genetic approach focusing on NAD(P)H dehydrogenase (NDH) activity that a PPR protein CRR4 is essential for a specific RNA editing event in chloroplasts. Here, we discovered Arabidopsis crr21 mutants that are specifically impaired in the RNA editing of the site 2 of ndhD (ndhD-2), which encodes a subunit of the NDH complex. The CRR21 gene encodes a member of the PPR protein family. The RNA editing of ndhD-2 converts the Ser-128 of NdhD to leucine. In crr21, the activity of the NDH complex is specifically impaired, suggesting that the Ser128Leu change has important consequences for the function of the NDH complex. Both CRR21 and CRR4 belong to the E+ subgroup in the PLS subfamily that is characterized by the presence of a conserved C-terminal region (the E/E+ domain). This E/E+ domain is highly conserved and exchangeable between CRR21 and CRR4, although it is not essential for the RNA binding. Our results suggest that the E/E+ domain has a common function in RNA editing rather than of recognizing specific RNA sequences.
| S-EPMC1876591 | biostudies-literature
Project description:BACKGROUND:The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown. RESULTS:In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton. CONCLUSIONS:Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.
| S-EPMC6693126 | biostudies-literature
Project description:Pentatricopeptide repeat (PPR) proteins are RNA binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5' region of nad5 exon 4 and the bulged-A region in domain VI of atp9 group II intron 1 while the recombinant PpPPR_9 bound to the translated region of ORF622 in cox1 intron 3. These results suggest that PLS-class PPR proteins may influence the splicing efficiency of mitochondrial group II introns.
2020-08-01 | GSE145050 | GEO
Project description:Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5' end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5' UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPR proteins can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.
| S-EPMC8191804 | biostudies-literature
Project description:Extensive studies have been carried out on chloroplast gene expression and chloroplast development; however, the regulatory mechanism is still largely unknown. Here, we characterized Pigment-Defective Mutant4 (PDM4), a P-type PPR protein localized in chloroplast. The pdm4 mutant showed seedling-lethal and albino phenotype under heterotrophic growth conditions. Transmission electron microscopic analysis revealed that thylakoid structure was totally disrupted in pdm4 mutant and eventually led to the breakdown of chloroplasts. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in pdm4 mutant. Besides, transcript profile analysis detected that, in pdm4 mutant, the expression of plastid-encoded RNA polymerase-dependent genes was markedly affected, and deviant chloroplast rRNA processing was also observed. In addition, we found that PDM4 functions in the splicing of group II introns and may also be involved in the assembly of the 50S ribosomal particle. Our results demonstrate that PDM4 plays an important role in chloroplast gene expression and chloroplast development in Arabidopsis.
| S-EPMC7432182 | biostudies-literature