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Ian Small - One of the best experts on this subject based on the ideXlab platform.

  • the Pentatricopeptide Repeat protein emb2654 is essential for trans splicing of a chloroplast small ribosomal subunit transcript
    Plant Physiology, 2017
    Co-Authors: Nader Aryamanesh, Ian Small, Hannes Ruwe, Lilian M V P Sanglard, Leila Eshraghi, John D Bussell, Katharine A Howell, Catherine Colas Des Francssmall
    Abstract:

    We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class Pentatricopeptide Repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA "footprint" located on the 5' half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.

  • the solution structure of the Pentatricopeptide Repeat protein ppr10 upon binding atph rna
    Nucleic Acids Research, 2015
    Co-Authors: Benjamin S Gully, Ian Small, Alice Barkan, Kate Shearston, Will A Stanley, Nathan Cowieson, Charles S Bond
    Abstract:

    The Pentatricopeptide Repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts ZEA MAYS: PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, ATPH: and PSAJ, HAS BEEN DEMONSTRATED TO FOLLOW: a recognition code where the identity of two amino acids per Repeat determines the base-specificity. A recently solved ZmPPR10: PSAJ: complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein-RNA contacts than inferred PREVIOUSLY: Here we describe the solution structure of the ZmPPR10: ATPH: complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA-protein interface.

  • the design and structural characterization of a synthetic Pentatricopeptide Repeat protein
    Acta Crystallographica Section D-biological Crystallography, 2015
    Co-Authors: Benjamin S Gully, Kunal R Shah, Mihwa Lee, Kate Shearston, Nicole M Smith, Agata Sadowska, Amanda J Blythe, Kalia Bernathlevin, Will A Stanley, Ian Small
    Abstract:

    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 the de novo design 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 A 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.

  • the Pentatricopeptide Repeat proteins tang2 and organelle transcript processing439 are involved in the splicing of the multipartite nad5 transcript encoding a subunit of mitochondrial complex i
    Plant Physiology, 2014
    Co-Authors: Catherine Colas Des Francssmall, Sandra K Tanz, Andeol Falcon De Longevialle, Elizabeth C Lowe, Caroline Smith, Michael W Bevan, Ian Small
    Abstract:

    Pentatricopeptide Repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two Pentatricopeptide Repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far.

  • the potential for manipulating rna with Pentatricopeptide Repeat proteins
    Plant Journal, 2014
    Co-Authors: Yusuke Yagi, T Nakamura, Ian Small
    Abstract:

    The Pentatricopeptide Repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence-specific RNA-binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2-30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA-binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR-based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.

Alice Barkan - One of the best experts on this subject based on the ideXlab platform.

  • in vivo stabilization of endogenous chloroplast rnas by customized artificial Pentatricopeptide Repeat proteins
    Nucleic Acids Research, 2021
    Co-Authors: Nikolay Manavski, Alice Barkan, Margarita Rojas, Sebastien Mathieu, Louisvalentin Meteignier, Andreas Brachmann, Kamel Hammani
    Abstract:

    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.

  • in vivo stabilization of endogenous chloroplast rnas by customized artificial Pentatricopeptide Repeat proteins
    bioRxiv, 2020
    Co-Authors: Nikolay Manavski, Alice Barkan, Margarita Rojas, Louisvalentin Meteignier, Andreas Brachmann, Kamel Hammani
    Abstract:

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

  • the arabidopsis Pentatricopeptide Repeat protein lpe1 and its maize ortholog are required for translation of the chloroplast psbj rna
    Plant Journal, 2019
    Co-Authors: Rosalind Williamscarrier, Margarita Rojas, Susan Belcher, Carolyn Brewster, Prakitchai Chotewutmontri, Sonja Ljungdahl, Alice Barkan
    Abstract:

    The expression of chloroplast genes relies on a host of nucleus-encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the Pentatricopeptide Repeat protein LPE1, reported to stimulate translation of the chloroplast psbA mRNA in Arabidopsis. Mutation of the maize LPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplast psbJ open reading frame (ORF) but has no effect on psbA expression. To reflect this function, we named the maize LPE1 ortholog Translation of psbJ 1 (TPJ1). Arabidopsis lpe1 mutants likewise exhibit a loss of psbJ translation, and have, in addition, a decrease in psbN translation. We detected a small decrease in ribosome occupancy on the psbA mRNA in Arabidopsis lpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 and hcf173) in conjunction with in vitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of the psbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 in psbN translation contributes to the PSII deficiency in lpe1 mutants.

  • ribonucleoprotein capture by in vivo expression of a designer Pentatricopeptide Repeat protein in arabidopsis
    The Plant Cell, 2019
    Co-Authors: James J Mcdermott, Rosalind Williamscarrier, Kenneth P Watkins, Alice Barkan
    Abstract:

    Pentatricopeptide Repeat (PPR) proteins bind RNA via a mechanism that facilitates the customization of sequence specificity. However, natural PPR proteins have irregular features that limit the degree to which their specificity can be predicted and customized. We demonstrate here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein characterization. We show by RNA coimmunoprecipitation sequencing (RIP-seq) that artificial PPR proteins designed to bind the Arabidopsis (Arabidopsis thaliana) chloroplast psbA mRNA bind with high specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass spectrometry showed the psbA translational activator HCF173 and two RNA binding proteins of unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and CP33C binding sites placed them in different locations on psbA mRNA. These results demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other applications that rely on the ability to target a protein to a specified RNA sequence.

  • unexpected functional versatility of the Pentatricopeptide Repeat proteins pgr3 ppr5 and ppr10
    Nucleic Acids Research, 2018
    Co-Authors: Margarita Rojas, Christian Schmitzlinneweber, Rafael G Miranda, Hannes Ruwe, Reimo Zoschke, Nora Hase, Alice Barkan
    Abstract:

    Pentatricopeptide Repeat (PPR) proteins are a large family of helical Repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3′-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3′-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.

Toshiharu Shikanai - One of the best experts on this subject based on the ideXlab platform.

  • the Pentatricopeptide Repeat protein pgr3 is required for the translation of petl and ndhg by binding their 5 utrs
    Plant and Cell Physiology, 2021
    Co-Authors: Haruka Higashi, Mizuki Takenaka, Yoshinobu Kato, Tomoya Fujita, Shintaro Iwasaki, Masayuki Nakamura, Yoshiki Nishimura, Toshiharu Shikanai
    Abstract:

    PGR3 is a P-class Pentatricopeptide Repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5' UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes' access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

  • The Restorer-of-fertility-like 2 Pentatricopeptide Repeat protein and RNase P are required for the processing of mitochondrial orf291 RNA in Arabidopsis.
    Plant Journal, 2016
    Co-Authors: Sota Fujii, Philippe Giege, Takamasa Suzuki, Tetsuya Higashiyam, Nobuya Koizuka, Toshiharu Shikanai
    Abstract:

    Eukaryotes harbor mitochondria obtained via ancient symbiosis events. The successful evolution of energy production in mitochondria has been dependent on the control of mitochondrial gene expression by the nucleus. In flowering plants, the nuclear-encoded Pentatricopeptide Repeat (PPR) superfamily proteins are widely involved in mitochondrial RNA metabolism. Here, we show that an Arabidopsis nuclear-encoded RNA-binding protein, Restorer-of-fertility-like PPR protein 2 (RFL2), is required for RNA degradation of the mitochondrial orf291 transcript via endonucleolytic cleavage of the transcript in the middle of its reading frame. Both in vivo and in vitro, this RNA cleavage requires the activity of mitochondrial proteinaceous RNase P, which is possibly recruited to the site by RFL2. The site of RNase P cleavage likely forms a tRNA-like structure in the orf291 transcript. This study presents an example of functional collaboration between a PPR protein and an endonuclease in RNA cleavage. Furthermore, we show that the RFL2-binding region within the orf291 gene is hypervariable in the family Brassicaceae, possibly correlated with the rapid evolution of the RNA-recognition interfaces of the RFL proteins.

  • mutagenesis of individual Pentatricopeptide Repeat motifs affects rna binding activity and reveals functional partitioning of arabidopsis proton gradient regulation3
    The Plant Cell, 2013
    Co-Authors: Sota Fujii, Nozomi Sato, Toshiharu Shikanai
    Abstract:

    Pentatricopeptide Repeat (PPR) proteins bind RNA and act in multiple eukaryotic processes, including RNA editing, RNA stability, and translation. Here, we investigated the mechanism underlying the functional versatility of Arabidopsis thaliana PROTON GRADIENT REGULATION3 (PGR3), a chloroplast protein harboring 27 PPR motifs. Previous studies suggested that PGR3 acts in (1) stabilization of photosynthetic electron transport L (petL) operon RNA, (2) translation of petL, and (3) translation of ndhA. We showed here that replacement of the 4th amino acid of the 12th PPR with nonpolar or charged amino acids abolished functions (1) and (2) but not (3) of PGR3 by compromising the function of this specific PPR. This discovery enabled us to knock out the RNA binding ability of individual PPR motifs. Consequently, we showed that the 16 N-terminal PPRs were sufficient for function (1) via sequence-specific RNA binding, whereas the 11 C-terminal motifs were essential for functions (2) and (3) by activating translation. We also clarified that the 14th amino acid of the 12th PPR should be positively charged to make the PPR functionally active. Our finding opens up the possibility of selectively manipulating the functions of PPR proteins.

  • the Pentatricopeptide Repeat protein otp87 is essential for rna editing of nad7 and atp1 transcripts in arabidopsis mitochondria
    Journal of Biological Chemistry, 2011
    Co-Authors: Kamel Hammani, Kenji Okuda, Toshiharu Shikanai, Catherine Colas Des Francssmall, Sandra K Tanz, Mizuki Takenaka, Axel Brennicke, Ian Small
    Abstract:

    In plant organelles, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in RNA of both mitochondria and plastids, altering the information encoded by the gene. The cytidine to be edited is determined by a cis-element surrounding the editing site that is specifically recognized and bound by a trans-acting factor. All the trans-acting editing factors identified so far in plant organelles are members of a large protein family, the Pentatricopeptide Repeat (PPR) proteins. We have identified the Organelle Transcript Processing 87 (OTP87) gene, which is required for RNA editing of the nad7-C24 and atp1-C1178 sites in Arabidopsis mitochondria. OTP87 encodes an E-subclass PPR protein with an unusually short E-domain. The recombinant protein expressed in Escherichia coli specifically binds to RNAs comprising 30 nucleotides upstream and 10 nucleotides downstream of the nad7-C24 and atp1-C1178 editing sites. The loss-of-function of OTP87 results in small plants with growth and developmental delays. In the otp87 mutant, the amount of assembled respiratory complex V (ATP synthase) is highly reduced compared with the wild type suggesting that the amino acid alteration in ATP1 caused by loss of editing at the atp1-C1178 site affects complex V assembly in mitochondria.

  • the Pentatricopeptide Repeat protein otp82 is required for rna editing of plastid ndhb and ndhg transcripts
    Plant Journal, 2009
    Co-Authors: Kenji Okuda, Fumiyoshi Myouga, Reiko Motohashi, Kamel Hammani, Ian Small, Kazuo Shinozaki, Sandra K Tanz, Lianwei Peng, Yoichiro Fukao, Toshiharu Shikanai
    Abstract:

    Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the Pentatricopeptide Repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

T Nakamura - One of the best experts on this subject based on the ideXlab platform.

  • the potential for manipulating rna with Pentatricopeptide Repeat proteins
    Plant Journal, 2014
    Co-Authors: Yusuke Yagi, T Nakamura, Ian Small
    Abstract:

    The Pentatricopeptide Repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence-specific RNA-binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2-30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA-binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR-based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.

  • Pentatricopeptide Repeat proteins involved in plant organellar rna editing
    RNA Biology, 2013
    Co-Authors: Yusuke Yagi, Makoto Tachikawa, Hisayo Noguchi, Soichirou Satoh, Junichi Obokata, T Nakamura
    Abstract:

    C-to-U RNA editing has been widely observed in organellar RNAs in terrestrial plants. Recent research has revealed the significance of a large, plant-specific family of Pentatricopeptide Repeat (PPR) proteins for RNA editing and other RNA processing events in plant mitochondria and chloroplasts. PPR protein is a sequence-specific RNA-binding protein that identifies specific C residues for editing. Discovery of the RNA recognition code for PPR motifs, including verification and prediction of the individual RNA editing site and its corresponding PPR protein, expanded our understanding of the molecular function of PPR proteins in plant organellar RNA editing. Using this knowledge and the co-expression database, we have identified two new PPR proteins that mediate chloroplast RNA editing. Further, computational target assignment using the PPR RNA recognition codes suggests a distinct, unknown mode-of-action, by which PPR proteins serve a function beyond site recognition in RNA editing.

  • mechanistic insight into Pentatricopeptide Repeat proteins as sequence specific rna binding proteins for organellar rnas in plants
    Plant and Cell Physiology, 2012
    Co-Authors: T Nakamura, Yusuke Yagi, Keiko Kobayashi
    Abstract:

    The Pentatricopeptide Repeat (PPR) protein family is highly expanded in terrestrial plants. Arabidopsis contains 450 PPR genes, which represents 2% of the total protein-coding genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of organellar genes. Most PPR proteins affect a single or small subset of gene(s), acting in a gene-specific manner. Studies over the last 10 years have revealed the significance of this protein family in coordinated gene expression in different compartments: the nucleus, chloroplast and mitochondrion. Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.

  • identification and characterization of the rna binding surface of the Pentatricopeptide Repeat protein
    Nucleic Acids Research, 2012
    Co-Authors: Keiko Kobayashi, Mamoru Sugita, Tomohiko Kazama, Masuyo Kawabata, Keizo Hisano, Ken Matsuoka, T Nakamura
    Abstract:

    The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of Pentatricopeptide Repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and ‘ii’(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.

  • a conserved dyw domain of the Pentatricopeptide Repeat protein possesses a novel endoribonuclease activity
    FEBS Letters, 2008
    Co-Authors: T Nakamura, Mamoru Sugita
    Abstract:

    Many plant Pentatricopeptide Repeat (PPR) proteins are known to contain a highly conserved C-terminal DYW domain whose function is unknown. Recently, the DYW domain has been proposed to play a role in RNA editing in plant organelles. To address this possibility, we prepared recombinant DYW proteins and tested their cytidine deaminase activity. However, we could not detect any activity in the assays we used. Instead, we found that the recombinant DYW domains possessed endoribonuclease activity and cleaved before adenosine residues in the RNA molecule. Some DYW-containing PPR proteins may catalyze site-specific cleavage of target RNA species.

Kenji Okuda - One of the best experts on this subject based on the ideXlab platform.

  • The E domains of Pentatricopeptide Repeat proteins from different organelles are not functionally equivalent for RNA editing
    The Plant journal : for cell and molecular biology, 2013
    Co-Authors: Anne-laure Chateigner-boutin, Kenji Okuda, Sota Fujii, Sandra K Tanz, Catherine Colas Des Francs-small, Ian Small
    Abstract:

    RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of Pentatricopeptide Repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the C terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue. Here, we describe two mutants, otp71 and otp72, disrupted in genes encoding mitochondrial E-type PPR proteins with single editing defects in ccmFN 2 and rpl16 transcripts, respectively. Comparisons between the E domains of these proteins and previously reported editing factors from chloroplasts suggested that there are characteristic differences in the proteins between the two organelles. To test this, we swapped E domains between two mitochondrial and two chloroplast editing factors. In all cases investigated, E domains from the same organelle (chloroplast or mitochondria) were found to be exchangeable; however, swapping the E domain between organelles led to non-functional editing factors. We conclude that the E domains of mitochondrial and plastid PPR proteins are not functionally equivalent, and therefore that an important component of the putative editing complexes in the two organelles may be different.

  • the Pentatricopeptide Repeat protein otp87 is essential for rna editing of nad7 and atp1 transcripts in arabidopsis mitochondria
    Journal of Biological Chemistry, 2011
    Co-Authors: Kamel Hammani, Kenji Okuda, Toshiharu Shikanai, Catherine Colas Des Francssmall, Sandra K Tanz, Mizuki Takenaka, Axel Brennicke, Ian Small
    Abstract:

    In plant organelles, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in RNA of both mitochondria and plastids, altering the information encoded by the gene. The cytidine to be edited is determined by a cis-element surrounding the editing site that is specifically recognized and bound by a trans-acting factor. All the trans-acting editing factors identified so far in plant organelles are members of a large protein family, the Pentatricopeptide Repeat (PPR) proteins. We have identified the Organelle Transcript Processing 87 (OTP87) gene, which is required for RNA editing of the nad7-C24 and atp1-C1178 sites in Arabidopsis mitochondria. OTP87 encodes an E-subclass PPR protein with an unusually short E-domain. The recombinant protein expressed in Escherichia coli specifically binds to RNAs comprising 30 nucleotides upstream and 10 nucleotides downstream of the nad7-C24 and atp1-C1178 editing sites. The loss-of-function of OTP87 results in small plants with growth and developmental delays. In the otp87 mutant, the amount of assembled respiratory complex V (ATP synthase) is highly reduced compared with the wild type suggesting that the amino acid alteration in ATP1 caused by loss of editing at the atp1-C1178 site affects complex V assembly in mitochondria.

  • the Pentatricopeptide Repeat protein otp82 is required for rna editing of plastid ndhb and ndhg transcripts
    Plant Journal, 2009
    Co-Authors: Kenji Okuda, Fumiyoshi Myouga, Reiko Motohashi, Kamel Hammani, Ian Small, Kazuo Shinozaki, Sandra K Tanz, Lianwei Peng, Yoichiro Fukao, Toshiharu Shikanai
    Abstract:

    Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the Pentatricopeptide Repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

  • Pentatricopeptide Repeat proteins with the dyw motif have distinct molecular functions in rna editing and rna cleavage in arabidopsis chloroplasts
    The Plant Cell, 2009
    Co-Authors: Kenji Okuda, Fumiyoshi Myouga, Reiko Motohashi, Etienne Delannoy, Anne Laure Chateignerboutin, Mamoru Sugita, Takahiro Nakamura, Kazuo Shinozaki, Ian Small
    Abstract:

    The plant-specific DYW subclass of Pentatricopeptide Repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.

  • conserved domain structure of Pentatricopeptide Repeat proteins involved in chloroplast rna editing
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Kenji Okuda, Fumiyoshi Myouga, Reiko Motohashi, Kazuo Shinozaki, Toshiharu Shikanai
    Abstract:

    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.