Language：English   Publishing type：Research paper (scientific journal)  

EditForce Inc., established in May 2015, provides an alternative option to genome editing and a novel tool for versatile RNA editing at the genomic scale, called ‘transcriptome editing’, based on pentatricopeptide repeat (PPR) protein engineering technologies. Our core technologies have been invented at Kyushu University, Japan. The company is located in Fukuoka city in Kyushu, a southwestern island of Japan. EditForce is an innovative company in the post-genomic era. Our mission is to provide novel DNA/RNA operating tools to understand and modify various living entities and to translate our PPR technologies in various biological industries, including the pharmaceutical and agricultural industries.

Language：English   Publishing type：Research paper (scientific journal)  

In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. Target cytidines are specifically recognized by pentatricopeptide repeat (PPR) proteins of the PLS subfamily, which have additional C-terminal E or E-DYW motifs. Recent in silico analysis proposed a model for site recognition by PLS-subfamily PPR proteins: one-PPR motif to one-nucleotide correspondence with the C-terminal last S motif aligning to the nucleotide at position -4 with respect to the editing site. Here we show quantitative biochemical data on site recognition by four PLS-subfamily proteins: CRR28 and OTP85 are DYW-class members while CRR21 and OTP80 are E-class members. The minimal RNA segments required for high affinity binding by these PPR proteins were experimentally determined. The results were generally consistent with the in silico based model. However, we clarified that several PPR motifs, including the C-terminal L2 and S motifs of CRR21 and OTP80, are dispensable for the RNA binding, suggesting distinct contributions of each PPR motif to site recognition. We also demonstrate that the DYW motif interacts with the target C and its 5′ proximal region (-3 to 0), whereas the E motif is not involved in binding.

DOI： 10.1111/tpj.12687

Language：English   Publishing type：Research paper (scientific journal)  

The pentatricopeptide repeat (PPR) protein family, particularly prevalent in plants, includes many sequence-specific RNA binding proteins involved in all aspects of organelle RNA metabolism including RNA stability, processing, RNA 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 2-3 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 can be routinely used by the scientific community.

DOI： 10.1111/tpj.12377

Language：English  

DOI： 10.3389/fpls.2013.00517

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA-binding proteins that are commonly found in plants. Organelle transcript processing and stability are mediated by PPR proteins in a gene-specific manner through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. However, the sequence-specific RNA recognition mechanism of the PPR protein remains largely unknown. Here, we show the principle underlying RNA recognition for PPR proteins involved in RNA editing. The distance between the PPR-RNA alignment and the editable C was shown to be conserved. Amino acid variation at 3 particular positions within the motif determined recognition of a specific RNA in a programmable manner, with a 1-motif to 1-nucleotide correspondence, with no gap sequence. Data from the decoded nucleotide frequencies for these 3 amino acids were used to assign accurate interacting sites to several PPR proteins for RNA editing and to predict the target site for an uncharacterized PPR protein.

DOI： 10.1371/journal.pone.0057286

Language：English   Publishing type：Research paper (scientific journal)  

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.

Language：English   Publishing type：Research paper (scientific journal)  

The phytohormone abscisic acid (ABA) plays pivotal roles in the regulation of developmental and environmental responses in plants. Identification of cytoplasmic ABA receptors enabled the elucidation of the main ABA signaling pathway, connecting ABA perception to either nuclear events or the action of several transporters. However, the physiological functions of ABA in cellular processes largely remain unknown. To obtain greater insight into the ABA response, we performed genetic screening to isolate ABA-related mutants of Arabidopsis and isolated several novel ABA-hypersensitive mutants. We further characterized one of those mutants—ahg11. Map-based cloning showed that AHG11 encodes a PPR type protein, which has potential roles in RNA editing. An AHG11-GFP fusion protein indicated that AHG11 mainly localized to the mitochondria. Consistent with this observation, the nad4 transcript, which normally undergoes RNA editing, lacks a single RNA editing event conferring a conversion of an amino acid residue in ahg11 mutants. The geminating ahg11 seeds have higher levels of reactive oxygen species responsive genes. Presumably partial impairment of mitochondrial function caused by an amino acid conversion in one of the Complex I components induces redox imbalance, which in turn confers abnormal response to the plant hormone.

DOI： 10.1093/jxb/ers188

Language：English   Publishing type：Research paper (scientific journal)  

葉緑体とミトコンドリアの遺伝子発現は核にコードされる遺伝子によって転写後のRNAのレベルで大きく制御されている。最近の研究で、植物で特有に大きなファミリーを形成するPPR蛋白質が配列特異的なRNA結合蛋白質として、前述のオルガネラRNA代謝に重要な役割を担うことが明らかになってきた。PPR蛋白質中の複数のPPRモチーフ(35アミノ酸）の繰り返しがRNA結合に働くと推測されているが、その詳細は明らかでない。我々はここに、PPR蛋白質のRNA結合の分子基盤を示した。まず、Pfamにおけるモチーフの定義がPPRモチーフのRNA結合ユニットとしての働きを正しく示すことを明らかにした。また、2個のPPRモチーフからなる一連の組換え蛋白質を用いた生化学的、計算科学的な解析から、5個のアミノ酸（1、4、8、12、ii(-2））がPPRモチーフのRNA結合表面を形成することを明らかにした。SELEX法などにより、配列特異的なRNA結合を解析したところ、PPRとRNAの相互作用の親和性、配列特異性に関わることをいくつかの特徴的なアミノ酸を見いだした。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1038/s41598-023-50776-z

Language：English   Publisher：Scientific Reports  

Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.

DOI： 10.1038/s41598-023-50776-z

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1016/j.ggedit.2022.100021

Language：English   Publisher：Communications Biology  

Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5′ but not apparent 3′ preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.

DOI： 10.1038/s42003-022-03927-3

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.7554/eLife.61611

Language：English   Publishing type：Research paper (scientific journal)  

Hundreds of RNA editing events, that is conversion of cytidines (Cs) to uridines (Us), have been observed in the mitochondrial and plastid transcriptome in vascular plants. Defects of C-to-U RNA editing affect a wide variety of physiological processes. These editing sites are recognized by pentatricopeptide repeat (PPR) superfamily proteins. PPR proteins are sequence-specific RNA binding proteins that participate in multiple aspects of organellar RNA metabolism. They are categorized into P and PLS subclasses, where PLS-class proteins are largely identified as RNA editing PPRs. Elucidating the principle involved in PPR-RNA recognition, the so-called PPR code, has enhanced our understanding of the recognition of RNA editing sites, thereby enabling prediction of target RNA editing sites for uncharacterized PLS-class proteins. Computational PPR-RNA prediction in RNA editing can be applied to the study of PPR-deficient plants that are genetically isolated from physiological abnormalities. However, the use of PPR-RNA prediction in RNA editing is still restricted due to ambiguous procedures and prediction reliability. Here, we refined the PPR code dataset, and the reliability of the computational prediction was quantitatively evaluated using known RNA editing PPRs. With this knowledge, a computational analysis was conducted in the 'PPR-to-editing site' and 'editing site-to-PPR' directions, against 199 PLS-class proteins and 499 organelle RNA editing sites in Arabidopsis thaliana. We propose 52 plausible PPR-RNA pairs for uncharacterized proteins and editing sites. The presented data will facilitate the study of organellar RNA editing involved in diverse physiological processes in A. thaliana.

DOI： 10.1093/pcp/pcy251

Language：English   Publishing type：Research paper (scientific journal)  

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.

DOI： 10.1016/j.jbiosc.2017.12.003

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2017.12.003

Language：English  

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are widely distributed in plants. They contain 2 to 30 repeating units of ~35-amino acid PPR motifs. They are known to play important roles in RNA processing, RNA editing, and translational regulation. Recent studies on the RNA recognition mode of PPR proteins revealed that one PPR motif interacts with one nucleotide. In addition, it was revealed that amino acids at three specific positions in a single motif serve to specify its binding base. Thus, mutation of these amino acids can cause a modification of the binding specificity of PPR motifs. Indeed, the engineered PPR motifs fused with various effector domains are shown to bind to and manipulate RNAs in a controlled manner. In this review, we summarize the recent progress in structural studies on PPR motifs. We focus on their RNA recognition mode and discuss the potentials of PPR as novel, versatile tools for RNA manipulation.

DOI： 10.1007/978-981-10-8372-3_10

Language：English   Publishing type：Research paper (scientific journal)  

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear-encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)-type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD-CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS-associated gene in LD-CMS rice, similar to its role in BT-CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT-CMS rice. We also show that RF2 promotes degradation of atp6-orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT-CMS rice. The amount of ORF79 protein in LD-CMS rice was one-twentieth of the amount in BT-CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD-CMS and BT-CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD-CMS and BT-CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD-CMS and BT-CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.

DOI： 10.1111/tpj.13135

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms161023849

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2014.07.030

Language：English  

DOI： 10.1093/pcp/pct132

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.4161/rna.24908

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1093/pcp/pcs069

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1093/nar/gkr1084

Language：English   Publishing type：Research paper (scientific journal)  

原色素体（plastid）から葉緑体（chloroplast）への分化の過程で、色素体の遺伝子発現システムは、光合成を可能とする葉緑体のそれに大きく変化する。イネのv1（virescent1）変異体では、低温状態で葉緑体分化が損なわれる。我々は本論文で、v1遺伝子を同定し、この遺伝子が葉緑体局在のNUS1蛋白質をコードすることを明らかにした。NUS1のC末端側は、原核生物でリボソームRNAの転写に働く転写集結抑制因子と良く似た構造を有す。また、NUS1は葉緑体成熟途中で特異的に発現していた。RNA免疫沈降実験、およびゲルシフト法により、NUS1は16S rRNAの上流領域を含むいくつかの葉緑体RNAと結合することを明らかにした。NUS1変異体では、低温下での葉緑体形成途中で、葉緑体中のrRNAの成熟および蓄積が損なわれており、その結果、葉緑体遺伝子の転写および翻訳の広範囲にわたる異常が観察された。以上の結果は、NUS1が低温ストレス状態での原色素体から葉緑体への分化において、その遺伝子発現系の構築に重要な役割を果たすことを示唆している。

DOI： 10.1111/j.1365-313X.2011.04755.x

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1105/tpc.108.064667

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.febslet.2008.11.017

Language：English   Publishing type：Research paper (scientific journal)  

In BT-type cytoplasmic male sterile rice (Oryza sativa L.) with Chinsurah Boro II cytoplasm, cytoplasmic male sterility (CMS) is caused by an accumulation of the cytotoxic peptide ORF79. The ORF79 protein is expressed from a dicistronic gene atp6-orf79, which exists in addition to the normal atp6 gene in the BT-type mitochondrial genome. The CMS is restored by a PPR (pentatricopeptide-repeat) gene, Rf1, via RNA processing. However, it has not yet been elucidated how the accumulation of ORF79 is reduced by the action of the Rf1 protein. Here, we report that the level of processed orf79 transcripts in the restorer line was reduced to 50% of the unprocessed atp6-orf79 transcripts in the CMS line. Ninety percent of the processed orf79 transcripts, which remained after degradation, were not associated with the ribosome for translation. Our data suggests that the processing of atp6-orf79 transcripts diminishes the expression of orf79 by the translational reduction and degradation of the processed orf79 transcripts.

DOI： 10.1111/j.1365-313X.2008.03529.x

Language：English   Publishing type：Research paper (scientific journal)  

Small, regulatory, non-coding RNA (ncRNA) is involved in various cell functions in both prokaryotes and eukaryotes. However, information on ncRNA in cyanobacteria is still scarce. We studied ncRNA genes by computational screening to compare the intergenic regions of the Synechococcus elongatus PCC 6301 genome with the genomes of three freshwater cyanobacteria. We identified an ncRNA gene in S. elongatus, which has been previously described as yfr1 in marine cyanobacteria. The S. elongatus yfr1 gene is 65 nucleotides long and is positioned between guaB and trxA. We found a high conservation of the yfr1 gene in most cyanobacterial lineages. A yfr1-deficient mutant showed reduced growth under various stress conditions, e.g. oxidative stress and high salt stress conditions, and showed unusual accumulation of sbtA mRNA. A gel shift assay demonstrated interaction of the Yfr1 RNA with sbtA mRNA in vitro. This suggests that the sbtA transcript is a target RNA for the Yfr1 RNA.

DOI： 10.1093/pcp/pcm098

Language：English   Publishing type：Research paper (scientific journal)  

In higher plants, RNA editing is a post-transcriptional process that converts C to U in organelle mRNAs. We have previously shown that an Arabidopsis thaliana crr4 mutant is defective with respect to RNA editing for creating the translational initial codon of the plastid ndhD gene (the ndhD-1 site). CRR4 contains 11 pentatricopeptide repeat motifs but does not contain any domains that are likely to be involved in the editing activity. The green fluorescent protein fused to the putative transit peptide of CRR4 targeted the plastid. The recombinant CRR4 expressed in Escherichia coli specifically bound to the 25 nucleotides of the upstream and the 10 nucleotides of the downstream sequences surrounding the editing site of ndhD-1. The target C nucleotide of this editing is not essential for the binding of CRR4. Taken together with the genetic evidence, we conclude that the pentatricopeptide repeat protein CRR4 is a sequence-specific RNA-binding protein that acts as a site recognition factor in plastid RNA editing.

DOI： 10.1074/jbc.M608184200

Language：English   Publishing type：Research paper (scientific journal)  

The moss Physcomitrella patens is a newly established model plant that is widely used for the characterization of gene function by targeted gene knockout or over-expression. The target gene disruption occurs in both the nuclear and chloroplast genomes. We applied DNA microarray technology to the P. patens plastid genome for large-scale analysis of transcripts. A microarray was constructed containing 108 DNA fragments to detect all annotated plastid genes. We analyzed the transcript profile in a knockout transformant for the arginine tRNA gene, trnR-CCG, and confirmed previous results that rbcL and psal transcripts accumulate in similar levels to wild-type moss, and accD transcript level is higher than those of wild-type moss. Additionally, the plastid DNA microarray revealed that most plastid genes were expressed at similar levels in wild-type and transformant mosses. This indicates that tmR-CCG is not essential for the expression of plastid genes.

DOI： 10.1055/s-2005-865620

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1042/BST0320571

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The nonphotosynthetic mutant of Arabidopsis hcf152 is impaired in the processing of the chloroplast polycistronic transcript, psbB-psbT-psbH-petB-petD, resulting in non-production of the essential photosynthetic cytochrome b₆f complex. The nucleus-encoded HCF152 gene was identified to encode a pentatricopeptide repeat (PPR) protein composed primarily of 12 PPR motifs, similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. To understand the molecular mechanism of how HCF152 modulates chloroplast gene expression, the molecular and biochemical properties should be revealed. To this end, HCF152 and several truncated versions were produced in bacteria and analyzed for RNA-binding and protein-protein interaction. It was found that two HCF152 polypeptides bind to form a homodimer, and that this binding is impaired by a single amino acid substitute near the carboxyl terminus, replacing leucine with proline. Recombinant HCF152 bound with higher affinity RNA molecules, resembling the petB exon-intron junctions, as well as several other molecules. The highest affinity was found to RNA composed of the poly(A) sequence. When truncated proteins composed of different numbers of PPR motifs were analyzed for RNA-binding, it was found that two PPR motifs were required for RNA-binding, but had very low affinity. The affinity to RNA increased significantly when proteins composed of more PPR motifs were analyzed, displaying the highest affinity with the full-length protein composed of 12 PPR motifs. Together, our data characterized the nuclear-encoded HCF152 to be a chloroplast RNA-binding protein that may be involved in the processing or stabilization of the petB transcript by binding to the exonintron junctions.

DOI： 10.1046/j.1432-1033.2003.03796.x

Language：English   Publishing type：Research paper (scientific journal)  

The plastid genome of higher plants includes about 120 genes. We adopted genomic array technologies to the tobacco plastid genome. A microarray was constructed, consisting of 220 DNA fragments that cover the whole genome sequence. Each DNA fragment corresponds to a single known gene or an intergenic region. We evaluated reliability of this microarray by comparing the plastid RNA level in light- or dark-grown tobacco seedlings. The transcripts encoding photosynthetic subunits increased significantly in light-grown tissues as expected. Furthermore, we found unexpected signals in several intergenic regions, suggesting the existence of novel transcripts in tobacco plastids.

DOI： 10.1093/pcp/pcg101

Language：English   Publishing type：Research paper (scientific journal)  

The psbB-psbT-psbH-petB-petD operon of higher plant chloroplasts is a heterogeneously composed transcriptional unit that undergoes complex RNA processing events until the mature oligocistronic RNAs are formed. To identify the nucleus-encoded factors required for the processing and expression of psbB-psbT-psbH-petB-petD transcripts, we performed mutational analysis using Arabidopsis. The allelic nuclear mutants hcf152-1 and hcf152-2 were identified that are affected specifically in the accumulation of the plastidial cytochrome b₆f complex. In both mutants, reduced amounts of spliced petB RNAs (encoding the cytochrome b₆ subunit) were detected, thus explaining the observed protein deficiencies. Additionally, mutant hcf152-1 is affected in the accumulation of transcripts cleaved between the genes psbH and petB. As a result of a close T-DNA insertion, the HCF152 gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF152 encodes a pentatricopeptide repeat (PPR) protein with 12 putative PPR motifs that is located inside the chloroplast. The protein shows a significant structural, but not primary, sequence similarity to the maize protein CRP1, which is involved in the processing and translation of the chloroplast petD and petA RNAs. In addition, we found that HCF152 is an RNA binding protein that binds certain areas of the petB transcript. The protein possibly exists in the chloroplast as a homodimer and is not associated with other proteins to form a high molecular mass complex.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Post-transcriptional RNA processing is an important step in the regulation of chloroplast gene expression, and a number of chloroplast ribonucleoproteins (cpRNPs) are likely to be involved in this process. The major tobacco cpRNPs are composed of five species: cp28, cp29A, cp29B, cp31, and cp33 and these are divided into three groups (I, II, and III). By immunoprecipitation, gel filtration, and Western blot analysis, we demonstrated that these cpRNPs are abundant stromal proteins that exist as complexes with ribosome-free mRNAs. Many ribosome-free psbA mRNAs coprecipitate with cpRNPs, indicating that the majority of stromal psbA mRNAs are associated with cpRNPs. In addition, an in vitro mRNA degradation assay indicated that exogenous psbA mRNA is more rapidly degraded in cpRNP-depleted extracts than in nondepleted extracts. When the depleted extract was reconstituted with recombinant cpRNPs, the psbA mRNA in the extract was protected from degradation to a similar extent as the psbA mRNA in the nondepleted extract. Moreover, restoration of the stabilizing activity varied following addition of individual group-specific cpRNPs alone or in combination. When the five cpRNPs were supplemented in the depleted extract, full activity was restored. We propose that these cpRNPs act as stabilizing factors for nonribosome-bound mRNAs in the stroma.

DOI： 10.1074/jbc.M008817200

Language：English   Publishing type：Research paper (scientific journal)  

This study demonstrates that quantitative RT-PCR can be used to measure transient expression of genes introduced into plant cells by particle bombardment. An expression construct for β-glucuronidase was introduced into tobacco seedlings by particle bombardment followed by real-time quantitative RT-PCR of β-glucuronidase mRNA using a fluorogenic TaqMan probe. β-glucuronidase mRNA expression peaked within 2 h after gene transfer. β-glucuronidase protein activity was maximally elevated in plant cells 8 h after gene transfer and remained elevated for up to 50 h. This method is very sensitive, quantitating the target GUS transcript in 10 pg total RNA.

DOI： 10.1007/BF02824017

Language：English   Publishing type：Research paper (scientific journal)  

Tobacco chloroplasts possess five conserved ribonucleoproteins (cpRNPs). To elucidate the function of cpRNPs we analyzed their localization and target nucleic acid molecules in chloroplasts. Immunoprecipitation of the stromal extract and Northern analysis revealed that cpRNPs are associated in vivo with not only various species of chloroplast mRNAs but also intron-containing precursor (pre-) tRNAs. This observation strongly suggests that cpRNPs are involved in RNA processing, including mRNA stability and pre-tRNA splicing.

DOI： 10.1016/S0014-5793(99)01390-3

Language：English   Publishing type：Research paper (scientific journal)  

Using ssDNA-cellulose column chromatography, a 34 kDa ribonucleoprotein (p34) has been purified from a 0.4 M KCl crude extract of spinach chloroplasts as an effective phosphate acceptor for casein kinase II (CK-II) in vitro. Monomeric and oligomeric CK-IIs were copurified with p34 by the column chromatography and the kinases were separated from p34 by means of Mono Q column chromatography. It was found that (i) the purified p34 (pi 4.9) was phosphorylated specifically by CK-II in vitro; and (ii) similar polypeptides, such as p35 (pI 4.7) and p39 (pI 4.9) in maize and p33 (pI 4.7) in liverwort, were detected as ssDNA-binding chloroplast proteins phosphorylated by CK-II in vitro. The findings suggest that (i) RNPs that function as phosphate acceptors for CK-II exist commonly in chloroplasts among plant cells; and (ii) the physiological activity of RNPs is regulated by their specific phosphorylation by CK-II in chloroplasts.

Language：Japanese   Book type：Scholarly book

Language：Japanese  

Language：Japanese   Book type：Scholarly book

Language：Japanese   Book type：Scholarly book

Event date： 2023.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2021.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：東京   Country：Japan  

Event date： 2021.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：on line   Country：Japan  

Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：on line   Country：Japan  

Event date： 2020.2

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2019.10

Language：English   Presentation type：Oral presentation (general)  

Venue：BArcelona, Spain   Country：Spain  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：奈良   Country：Japan  

Event date： 2019.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：熊本   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.12

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.1

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2017.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2017.5 - 2018.5

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：東京   Country：Japan  

Event date： 2016.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2016.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2016.2

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Fukuoka   Country：Japan  

The pentatricopeptide repeat (PPR) protein family is a eukaryotic specific protein family that is specially expanded in plant (~500), whereas very few in other organisms (five in yeast and six in human). Most PPR proteins are gene-specific regulators of mitochondria and chloroplast gene expression, suggesting that PPR protein is evolved for control of endosymbiotic organelle genome. PPR proteins function as sequence-specific RNA/DNA binding proteins in various RNA/DNA metabolisms including RNA cleavage, splicing, RNA editing, and translation, through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. Recently, we have cracked the RNA/DNA recognition code: one PPR motif corresponds to one nucleotide, and amino acid variances at three particular positions confer the nucleotide specificity with programmable manner, and further above principles could be applied for both RNA- and DNA-binding PPR proteins. Our finding opened another possibility, the use of PPR motif on “genome editing” (DNA manipulation) and “transcriptome editing” (RNA manipulation). I would like to present current perspective about PPR protein family in endosymbiotic genome regulation, and our recent activities of PPR engineering for genome and transcriptome editing.

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

様々な生物のゲノム配列情報が明らかになり、ゲノム機能の解明などの従来の還元的な生物学に加えて、新しい生命システムの構築などの構成的な生物学が加速しつつある。このため、数十億塩基対から構成されるゲノム中の目的の１つの遺伝子、もしくはゲノムから転写される目的の１つのRNAを特異的に操作・改変する技術が求められている。最近、設計可能な人工ヌクレアーゼなどを用いた革新的なDNA操作技術であるゲノム編集が確立し、ゲノム中の特定の遺伝子の破壊や外来遺伝子の狙った位置への導入が可能になり、その利用が大きく期待されているが、その基本技術は全て海外で特許化されており、産業利用を視野に入れた独自の国産技術の確立が望まれる。一方、生物の複雑さや種の独自性に、多様な選択的スプライシングや非常に多くの蛋白質非コードRNAが大きく関わることが示唆されているが、ゲノム編集と対となるような汎用的なRNA操作技術は確立されていない。
我々は植物に多く含まれるPPRタンパク質を材料に、DNAおよびRNAそれぞれの操作技術の開発を行っている。PPRタンパク質は、35アミノ酸からなるPPRモチーフの連続で構成され、配列特異的なRNAまたはDNA結合蛋白質として働く。我々は、PPRタンパク質のDNA/RNA認識コードを解明し、特定の配列に結合する人工のDNA結合タンパク質およびRNA結合タンパク質、それぞれを設計するための基礎知的基盤を確立した。本講演では、既存のゲノム編集技術、およびPPRタンパク質を利用したRNA/DNA操作技術の開発の現状と可能性について紹介します。

Event date： 2015.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Tukuba   Country：Japan  

Determination of complete genome sequences (DNA) and the transcriptome (RNA) for a wide variety of organisms have opened a new biological era for understanding of the complex genetic functions that define living entities. New technologies have recently emerged that enable targeted editing of genomes in diverse systems, by using designer nucleases with programmable, sequence-specific DNA binding modules of ZF finger and TALE protein, and a guide RNA-based CRISPR/CAS9 system. These advances are driving new approaches to many areas including agriculture, biotechnology and studies of genome structure and function. In contract, versatile, programmable RNA binding module that enables manipulation of a single specific RNA in the living cell, is not available to date.
The PPR (pentatricopeptide repeat) motif-containing protein, that organizes a large family in plants, is a sequence-specific RNA or DNA binding protein involving in multiple aspects of organelle RNA/DNA metabolisms including RNA stability, processing, RNA editing, and transcription. PPR proteins consist of a tandem array of PPR motifs (degenerated 35 amino acids) in variety repeat length (2-27 repeats).
Recently, we cracked the RNA/DNA recognition code: one PPR motif corresponds to one nucleotide, and amino acid variances at three particular positions confer the nucleotide specificity with programmable manner, and the code can be shared between RNA- and DNA-binding PPR proteins. Here we show our recent progress and future perspective of the PPR protein engineering for genome and transcriptome editing.

Event date： 2015.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Nara   Country：Japan  

Recently established genome editing technologies using ZF, TALE and CRISPR/Cas9 are driving new approaches to many areas of biotechnology and studies of genome structure and function. In contract, versatile, programmable RNA binding module that enables manipulation of a specific RNA in the living cell, is not available to date. The PPR (pentatricopeptide repeat) motif-containing protein, that organizes a large family in plants, is a sequence-specific RNA/DNA binding protein involving in multiple aspects of organelle gene expression. PPR proteins consist of a tandem array of PPR motifs (35 amino acids) in variety repeat length (2-27 repeats). Using Biochemical and informatics approaches, we successfully cracked the RNA/DNA recognition code: one PPR motif corresponds to one nucleotide, and amino acid variances at three particular positions confer the nucleotide specificity with programmable manner, and further above principles could be applied for both RNA- and DNA-binding PPR proteins. Our finding facilitates the use of PPR motif on “genome editing” (DNA manipulation) and “transcriptome editing” (RNA manipulation). We would like to show our recent progress and future perspective for the PPR protein engineering for genome and transcriptome editing.

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

近年、設計可能な人工ヌクレアーゼなどを用いて、ゲノム中の特定の遺伝子を破壊したり、外来遺伝子を狙った位置に導入する「ゲノム編集」技術が確立し、先天的な疾患に関連する遺伝子の改変などの応用展開が期待されている。しかし、ゲノム編集の基幹技術は全て海外で特許化されており、かつ特許権の帰属が複雑なため、国産のゲノム編集技術の確立が望まれる。
一方、ゲノムプロジェクトによって、多様な選択的スプライシングや膨大な量の蛋白質非コードRNAが発見され、ゲノム情報の発現におけるRNAの生物学的重要性が再認識された。RNA段階の制御と様々な後天的な疾患との関連が示唆されている。しかし、ゲノム編集と対となるようなRNA操作技術は確立されていない。
植物に多く含まれるPPR (pentatricopeptide repeat) 蛋白質は、35アミノ酸からなるPPRモチーフの連続で構成され、配列特異的なRNAまたはDNA結合蛋白質として働く。我々は、１つのPPRモチーフが１つの塩基に対応すること、結合塩基がモチーフ中の3箇所のアミノ酸によりコード化できること、DNAおよびRNA結合型PPRモチーフが同じ動作原理で核酸を認識すること、を見いだした。すなわち、PPRモチーフをモジュール化・集積することで、RNAとDNAの両方について、特定の配列に結合する蛋白質分子を設計することができる。本講演では、現存のゲノム編集技術、および純国産のDNA/RNA結合モジュールであるPPRモチーフについて、開発の現状と可能性について紹介します。

Event date： 2015.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

様々な生物のゲノム配列情報が明らかになり、ゲノム機能の解明などの従来の還元的な生物学に加えて、新しい生命システムの構築などの構成的な生物学が加速しつつある。このため、数十億塩基対から構成されるゲノム中の目的の１つの遺伝子、もしくはゲノムから転写される目的の１つのRNAを特異的に操作・改変する技術が求められている。最近、設計可能な人工ヌクレアーゼなどを用いた革新的なDNA操作技術であるゲノム編集が確立し、ゲノム中の特定の遺伝子の破壊や外来遺伝子の狙った位置への導入が可能になり、その利用が大きく期待されているが、その基本技術は全て海外で特許化されており、産業利用を視野に入れた独自の国産技術の確立が望まれる。一方、生物の複雑さや種の独自性に、多様な選択的スプライシングや非常に多くの蛋白質非コードRNAが大きく関わることが示唆されているが、ゲノム編集と対となるような汎用的なRNA操作技術は確立されていない。
我々は植物に多く含まれるPPRタンパク質を材料に、DNAおよびRNAそれぞれの操作技術の開発を行っている。PPRタンパク質は、35アミノ酸からなるPPRモチーフの連続で構成され、配列特異的なRNAまたはDNA結合蛋白質として働く。我々は、PPRタンパク質のDNA/RNA認識コードを解明し、特定の配列に結合する人工のDNA結合タンパク質およびRNA結合タンパク質、それぞれを設計するための基礎知的基盤を確立した。本シンポジウムでは、既存のゲノム編集技術、およびPPRタンパク質を利用したRNA/DNA操作技術の開発の現状と可能性について紹介します。

Event date： 2015.1

Language：English   Presentation type：Oral presentation (general)  

Venue：Ventura, CA   Country：United States  

Event date： 2014.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：倉敷   Country：Japan  

Event date： 2014.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：小倉   Country：Japan  

Event date： 2014.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2014.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2014.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：富山   Country：Japan  

Event date： 2013.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2013.1

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：岡山   Country：Japan  

Event date： 2013.1

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Fukuoka   Country：Japan  

Event date： 2012.10

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Sapporo   Country：Japan  

Event date： 2012.3

Presentation type：Symposium, workshop panel (public)  

Venue：京都産業大学   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：京都産業大学   Country：Japan  

Event date： 2012.1

Presentation type：Oral presentation (general)  

Venue：東京都千代田区、JST・東京本部別館   Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：三島   Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Language：English   Presentation type：Oral presentation (general)  

Venue：Kokura, Kita-kyushu   Country：Japan  

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：4

Type：Competition, symposium, etc. 

Number of participants：100

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：8

Type：Scientific advice/Review 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：3

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：6

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：10

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：7

Type：Scientific advice/Review 

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：100

Type：Scientific advice/Review 

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Other

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Seminar, workshop

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Seminar, workshop

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Lecture

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Lecture

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Lecture

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Other

PPR技術の医療、農業、工業への応用を目的としたベンチャー企業を設立した

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Seminar, workshop

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Lecture

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Other

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Lecture

久留米市に「ゲノム編集」実証ラボ開設

ゲノム編集産業化ネットワーク、編集技術の産業利用を後押し

国産化ゲノム編集技術、注目のPPRたんぱく質

Focalpointにて技術の紹介

大学発ベンチャー表彰2019の受賞企業特集

遺伝子編集技術をもつ企業として大学初ベンチャーが紹介

解禁・ゲノム編集（４）　いでよ国産技術

日本初の新規DNA/RNA操作技術の開発

ゲノム上書き、病をデリート（バイオ医療NEXT）

日本発ゲノム編集ツールPPRは原核生物にも存在

新バイオ技術始動

ゲノム編集 日本出遅れ。九州大学が新しいゲノム編集技術を開発。

九大発VC ３億円調達へ、ゲノム編集を商業利用へ

ゲノム編集　RNAに照準

第4世代のゲノム編集技術が招く新世界

九州大学発 独自のDNA、RNA操作技術

ゲノム編集

わが国初のゲノム編集ベンチャーの設立

我が国の第4世代のゲノム編集技術

ゲノム編集新世紀

遺伝子の編集技術の確立

九⼤と広⼤、DNA結合型PPRたんぱく質の特許を出願、ゲノム編集の国産技術開発へ

PPRたんぱく質、RNA・DNA認識コード解明