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 RNA-stabilization factors in chloroplasts of vascular plants

2018 ◽  
Vol 62 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Nikolay Manavski ◽  
Lisa-Marie Schmid ◽  
Jörg Meurer
Keyword(s):  
Stress Responses ◽  
Vascular Plants ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Degradation ◽  
Decay Rates ◽  
Transcriptional Level ◽  
Chloroplast Gene Expression ◽  
Chloroplast Rna

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.

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 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 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.

scholarly journals Chloroplast cold-resistance is mediated by the acidic domain of the RNA binding protein CP31A

2019 ◽  
Author(s):  
Ayako Okuzaki ◽  
Marie-Kristin Lehniger ◽  
Jose M Muino ◽  
Benjamin Lenzen ◽  
Thilo Rühe ◽  
...  
Keyword(s):  
Cold Resistance ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Acidic Domain ◽  
Cold Sensitive ◽  
Chloroplast Rna ◽  
Rna Accumulation

AbstractChloroplast RNA metabolism is characterized by long-lived mRNAs that undergo a multitude of post-transcriptional processing events. Chloroplast RNA accumulation responds to environmental cues, foremost light and temperature. A large number of nuclear-encoded RNA-binding proteins (RBPs) are required for chloroplast RNA metabolism, but we do not yet know how chloroplast RBPs convert abiotic signals into gene expression changes. Previous studies showed that the chloroplast ribonucleoprotein 31A (CP31A) is required for the stabilization of multiple chloroplast mRNAs in the cold, and that the phosphorylation of CP31A at various residues within its N-terminal acidic domain (AD) can alter its affinity for RNA in vitro. Loss of CP31A leads to cold sensitive plants that exhibit bleached tissue at the center of the vegetative rosette. Here, by applying RIP-Seq, we demonstrated that CP31A shows increased affinity for a large number of chloroplast RNAs in vivo in the cold. Among the main targets of CP31A were RNAs encoding subunits of the NDH complex and loss of CP31A lead to reduced accumulation of ndh transcripts. Deletion analyses revealed that cold-dependent RNA binding and cold resistance of chloroplast development both depend on the AD of CP31A. Together, our analysis established the AD of CP31A as a key mediator of cold acclimation of the chloroplast transcriptome.One sentence summaryCold exposure induces increased RNA association of the RRM protein CP31A, which mediates cold-resistance of Arabidopsis thaliana via its acidic domain

scholarly journals Searching for a Match: Structure, Function and Application of Sequence-Specific RNA-Binding Proteins

2019 ◽  
Vol 60 (9) ◽  
pp. 1927-1938 ◽  
Author(s):  
Lauren K Dedow ◽  
Julia Bailey-Serres
Keyword(s):  
Gene Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Specificity ◽  
Pentatricopeptide Repeat ◽  
Rna Sequences ◽  
Regulatory Processes ◽  
Ppr Proteins ◽  
Modular Structures

Abstract Plants encode over 1800 RNA-binding proteins (RBPs) that modulate a myriad of steps in gene regulation from chromatin organization to translation, yet only a small number of these proteins and their target transcripts have been functionally characterized. Two classes of eukaryotic RBPs, pentatricopeptide repeat (PPR) and pumilio/fem-3 binding factors (PUF), recognize and bind to specific sequential RNA sequences through protein–RNA interactions. These modular proteins possess helical structural units containing key residues with high affinity for specific nucleotides, whose sequential order determines binding to a specific target RNA sequence. PPR proteins are nucleus-encoded, but largely regulate post-transcriptional gene regulation within plastids and mitochondria, including splicing, translation and RNA editing. Plant PUFs are involved in gene regulatory processes within the cell nucleus and cytoplasm. The modular structures of PPRs and PUFs that determine sequence specificity has facilitated identification of their RNA targets and biological functions. The protein-based RNA-targeting of PPRs and PUFs contrasts to the prokaryotic cluster regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that target RNAs in prokaryotes. Together the PPR, PUF and CRISPR-Cas systems provide varied opportunities for RNA-targeted engineering applications.

Two Novel PLS-Class Pentatricopeptide Repeat Proteins Are Involved in the Group II Intron Splicing of Mitochondrial Transcripts in the Moss Physcomitrella patens

2020 ◽  
Vol 61 (10) ◽  
pp. 1687-1698
Author(s):  
Mizuho Ichinose ◽  
Airi Ishimaru ◽  
Chieko Sugita ◽  
Kensaku Nakajima ◽  
Yasuhiro Kawaguchi ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Physcomitrella Patens ◽  
Rna Binding Proteins ◽  
Group Ii Intron ◽  
Pentatricopeptide Repeat ◽  
Mobility Shift ◽  
Intron 1 ◽  
Ppr Proteins ◽  
Group Ii

Abstract 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 a certain set of PLS-class PPR proteins may influence the splicing efficiency of mitochondrial group II introns.

scholarly journals PPR20 Is Required for the cis-Splicing of Mitochondrial nad2 Intron 3 and Seed Development in Maize

2019 ◽  
Vol 61 (2) ◽  
pp. 370-380 ◽  
Author(s):  
Yan-Zhuo Yang ◽  
Shuo Ding ◽  
Yong Wang ◽  
Hong-Chun Wang ◽  
Xin-Yuan Liu ◽  
...  
Keyword(s):  
Seed Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Endosperm Development ◽  
Binding Activity ◽  
Pentatricopeptide Repeat ◽  
Ppr Protein ◽  
Exact Function ◽  
Ppr Proteins ◽  
P Type

Abstract 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.

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