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 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 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.

scholarly journals Cofactor-Independent RNA Editing by a Synthetic S-Type PPR Protein

2021 ◽  
Author(s):  
Kalia Bernath-Levin ◽  
Jason Schmidberger ◽  
Suvi Honkanen ◽  
Bernard Gutmann ◽  
Yueming Kelly Sun ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Processing ◽  
Tandem Repeats ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Screening Method ◽  
Pentatricopeptide Repeat ◽  
Wide Range ◽  
Ppr Proteins ◽  
P Type

ABSTRACT Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors, and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method we show that synthetic S-type PPR proteins are easy to design, bind with high affinity and specificity, and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

scholarly journals Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants

2016 ◽  
Vol 85 (4) ◽  
pp. 532-547 ◽  
Author(s):  
Shifeng Cheng ◽  
Bernard Gutmann ◽  
Xiao Zhong ◽  
Yongtao Ye ◽  
Mark F. Fisher ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Land Plants ◽  
Pentatricopeptide Repeat ◽  
Structural Motifs ◽  
Repeat Proteins ◽  
Pentatricopeptide Repeat Proteins

scholarly journals The design and structural characterization of a synthetic pentatricopeptide repeat protein

2015 ◽  
Vol 71 (2) ◽  
pp. 196-208 ◽  
Author(s):  
Benjamin S. Gully ◽  
Kunal R. Shah ◽  
Mihwa Lee ◽  
Kate Shearston ◽  
Nicole M. Smith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Consensus Sequence ◽  
Pentatricopeptide Repeat ◽  
Ppr Protein ◽  
Ppr Proteins

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to thede novodesign of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

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.

Chloroplast RNA-binding and pentatricopeptide repeat proteins

2004 ◽  
Vol 32 (4) ◽  
pp. 571-574 ◽  
Author(s):  
T. Nakamura ◽  
G. Schuster ◽  
M. Sugiura ◽  
M. Sugita
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Transcriptional Level ◽  
Pentatricopeptide Repeat ◽  
Higher Plant ◽  
Chloroplast Gene Expression ◽  
Ppr Proteins ◽  
Chloroplast Rna ◽  
Recognition Motifs

Chloroplast gene expression is mainly regulated at the post-transcriptional level by numerous nuclear-encoded RNA-binding protein factors. In the present study, we focus on two RNA-binding proteins: cpRNP (chloroplast ribonucleoprotein) and PPR (pentatricopeptide repeat) protein. These are suggested to be major contributors to chloroplast RNA metabolism. Tobacco cpRNPs are composed of five different proteins containing two RNA-recognition motifs and an acidic N-terminal domain. The cpRNPs are abundant proteins and form heterogeneous complexes with most ribosome-free mRNAs and the precursors of tRNAs in the stroma. The complexes could function as platforms for various RNA-processing events in chloroplasts. It has been demonstrated that cpRNPs contribute to RNA stabilization, 3′-end formation and editing. The PPR proteins occur as a superfamily only in the higher plant species. They are predicted to be involved in RNA/DNA metabolism in chloroplasts or mitochondria. Nuclear-encoded HCF152 is a chloroplast-localized protein that usually has 12 PPR motifs. The null mutant of Arabidopsis, hcf152, is impaired in the 5′-end processing and splicing of petB transcripts. HCF152 binds the petB exon–intron junctions with high affinity. The number of PPR motifs controls its affinity and specificity for RNA. It has been suggested that each of the highly variable PPR proteins is a gene-specific regulator of plant organellar RNA metabolism.

scholarly journals The Analysis of the Editing Defects in the dyw2 Mutant Provides New Clues for the Prediction of RNA Targets of Arabidopsis E+-Class PPR Proteins

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 280 ◽  
Author(s):  
Bastien Malbert ◽  
Matthias Burger ◽  
Mauricio Lopez-Obando ◽  
Kevin Baudry ◽  
Alexandra Launay-Avon ◽  
...  
Keyword(s):  
Mitochondrial Genes ◽  
Pentatricopeptide Repeat ◽  
Repeat Proteins ◽  
Rna Targets ◽  
Ppr Proteins ◽  
Almost All ◽  
Pentatricopeptide Repeat Proteins

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

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