scholarly journals Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein

2010 ◽  
Vol 108 (1) ◽  
pp. 415-420 ◽  
Author(s):  
Jana Prikryl ◽  
Margarita Rojas ◽  
Gadi Schuster ◽  
Alice Barkan
Keyword(s):  
Binding Site ◽  
Large Family ◽  
Pentatricopeptide Repeat ◽  
Repeat Proteins ◽  
Rna Remodeling ◽  
Ppr Proteins ◽  
Maize Protein ◽  
Rna Stabilization ◽  
The Stability

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI–atpH intergenic region (i) blocks both 5′→3′ and 3′→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5′-terminus in conjunction with a generic 5′→3′ exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10’s ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins.

scholarly journals Mitochondrial Pentatricopeptide Repeat Protein, EMB2794, Plays a Pivotal Role in NADH Dehydrogenase Subunit nad2 mRNA Maturation in Arabidopsis thaliana

2020 ◽  
Vol 61 (6) ◽  
pp. 1080-1094
Author(s):  
Fernanda Marchetti ◽  
Maximiliano Cainzos ◽  
Sofía Shevtsov ◽  
Juan Pablo Córdoba ◽  
Laure Dora Sultan ◽  
...  
Keyword(s):  
Nadh Dehydrogenase ◽  
Developmental Defect ◽  
Pentatricopeptide Repeat ◽  
Mrna Maturation ◽  
Ppr Proteins ◽  
In Trans ◽  
Ppr Genes ◽  
The Stability

Abstract 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.

scholarly journals In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

2020 ◽  
Author(s):  
Nikolay Manavski ◽  
Louis-Valentin Meteignier ◽  
Margarita Rojas ◽  
Andreas Brachmann ◽  
Alice Barkan ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Specificity ◽  
Pentatricopeptide Repeat ◽  
Functional Capabilities ◽  
Ppr Protein ◽  
Repeat Proteins ◽  
Ppr Proteins ◽  
Physical Barriers

ABSTRACTPentatricopeptide 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 PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

scholarly journals Identification of Pentatricopeptide Repeat Proteins in the Model OrganismDictyostelium discoideum

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Sam Manna ◽  
Jessica Brewster ◽  
Christian Barth
Keyword(s):  
Dictyostelium Discoideum ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Gene Products ◽  
Antisense Inhibition ◽  
Pentatricopeptide Repeat ◽  
Repeat Proteins ◽  
Ppr Proteins ◽  
Pentatricopeptide Repeat Proteins ◽  
Encoding Genes

Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protistDictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

scholarly journals Pentatricopeptide Repeat Proteins in Trypanosoma brucei Function in Mitochondrial Ribosomes

2007 ◽  
Vol 27 (19) ◽  
pp. 6876-6888 ◽  
Author(s):  
Mascha Pusnik ◽  
Ian Small ◽  
Laurie K. Read ◽  
Thomas Fabbro ◽  
André Schneider
Keyword(s):  
Trypanosoma Brucei ◽  
Large Subunit ◽  
Direct Interaction ◽  
Pentatricopeptide Repeat ◽  
Amino Acid Motif ◽  
Mitochondrial Ribosomes ◽  
Repeat Proteins ◽  
Ppr Proteins ◽  
Pentatricopeptide Repeat Proteins ◽  
Large Subunit Rrna

ABSTRACT The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A comparative analysis shows that seven out of eight selected PPR proteins are mitochondrially localized and essential for oxidative phosphorylation. Six of these are required for the stabilization of mitochondrial rRNAs and, like ribosomes, are associated with the mitochondrial membranes. Furthermore, one of the PPR proteins copurifies with the large subunit rRNA. Finally, ablation of all of the PPR proteins that were tested induces degradation of the other PPR proteins, indicating that they function in concert. Our results show that a significant number of trypanosomal PPR proteins are individually essential for the maintenance and/or biogenesis of mitochondrial rRNAs.

Pentatricopeptide repeat (PPR) proteins as sequence-specificity factors in post-transcriptional processes in organelles

2007 ◽  
Vol 35 (6) ◽  
pp. 1643-1647 ◽  
Author(s):  
E. Delannoy ◽  
W.A. Stanley ◽  
C.S. Bond ◽  
I.D. Small
Keyword(s):  
Molecular Mechanisms ◽  
Rna Binding ◽  
Higher Plants ◽  
Large Family ◽  
Current Data ◽  
Sequence Specificity ◽  
Pentatricopeptide Repeat ◽  
Ppr Proteins

PPR (pentatricopeptide repeat) genes form a large family particularly prevalent in higher plants and targeted to organelles. They are involved in many post-transcriptional processes such as splicing, editing, processing and translation. Current data suggest that PPR proteins are involved in targeting effectors to the correct sites on the correct transcripts but the molecular mechanisms for RNA binding and effector recruitment by PPR proteins are not understood yet.

scholarly journals Multiple Mechanisms Are Used for Growth Rate and Stringent Control of leuV Transcriptional Initiation inEscherichia coli

1999 ◽  
Vol 181 (18) ◽  
pp. 5771-5782 ◽  
Author(s):  
Dmitry K. Pokholok ◽  
Maria Redlak ◽  
Charles L. Turnbough ◽  
Sara Dylla ◽  
Walter M. Holmes
Keyword(s):  
Growth Rate ◽  
Binding Site ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Cellular Level ◽  
Structural Variants ◽  
Stringent Control ◽  
The Stability

ABSTRACT Expression of the Escherichia coli leuV operon, which contains three tRNA1 Leu genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3′,5′-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of theleuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those ofrrnBp 1. The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuVpromoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from theleuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp 1. Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

scholarly journals The nuclear-localized PPR protein OsNPPR1 is important for mitochondrial function and endosperm development in rice

2019 ◽  
Vol 70 (18) ◽  
pp. 4705-4720 ◽  
Author(s):  
Yuanyuan Hao ◽  
Yunlong Wang ◽  
Mingming Wu ◽  
Xiaopin Zhu ◽  
Xuan Teng ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Endosperm Development ◽  
Pentatricopeptide Repeat ◽  
Coding Region ◽  
Rice Mutant ◽  
Ppr Protein ◽  
Isolation And Characterization ◽  
Ppr Proteins

Abstract Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants. Recent studies revealed the functions of PPR proteins in organellar RNA metabolism and plant development, but the functions of most PPR proteins, especially PPRs localized in the nucleus, remain largely unknown. Here, we report the isolation and characterization of a rice mutant named floury and growth retardation1 (fgr1). fgr1 showed floury endosperm with loosely arranged starch grains, decreased starch and amylose contents, and retarded seedling growth. Map-based cloning showed that the mutant phenotype was caused by a single nucleotide substitution in the coding region of Os08g0290000. This gene encodes a nuclear-localized PPR protein, which we named OsNPPR1, that affected mitochondrial function. In vitro SELEX and RNA-EMSAs showed that OsNPPR1 was an RNA protein that bound to the CUCAC motif. Moreover, a number of retained intron (RI) events were detected in fgr1. Thus, OsNPPR1 was involved in regulation of mitochondrial development and/or functions that are important for endosperm development. Our results provide novel insights into coordinated interaction between nuclear-localized PPR proteins and mitochondrial function.

scholarly journals A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Santana Royan ◽  
Bernard Gutmann ◽  
Catherine Colas des Francs-Small ◽  
Suvi Honkanen ◽  
Jason Schmidberger ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Sequence ◽  
Pentatricopeptide Repeat ◽  
Binding Assays ◽  
E Coli ◽  
Ppr Protein ◽  
Ppr Proteins

AbstractMembers of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

scholarly journals GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean

2021 ◽  
Vol 12 ◽  
Author(s):  
Xingxing Feng ◽  
Suxin Yang ◽  
Yaohua Zhang ◽  
Cheng Zhiyuan ◽  
Kuanqiang Tang ◽  
...  
Keyword(s):  
Rna Editing ◽  
Pentatricopeptide Repeat ◽  
Mobility Shift ◽  
Single Nucleotide ◽  
Chlorophyll Contents ◽  
Complex Processes ◽  
Ppr Protein ◽  
Ppr Proteins ◽  
Mobility Shift Assay

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.

scholarly journals The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3′-UTRs of yeast mitochondrial mRNAs

2021 ◽  
Vol 118 (15) ◽  
pp. e2009329118
Author(s):  
Hauke S. Hillen ◽  
Dmitriy A. Markov ◽  
Ireneusz D. Wojtas ◽  
Katharina B. Hofmann ◽  
Michael Lidschreiber ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Rna Stability ◽  
Mitochondrial Rna ◽  
Pentatricopeptide Repeat ◽  
Sequence Element ◽  
Posttranscriptional Gene Regulation ◽  
The Family ◽  
Ppr Proteins

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.

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