九州大学 研究者情報
論文一覧
中村 崇裕(なかむら たかひろ) データ更新日:2022.06.14

教授 /  農学研究院 生命機能科学部門 生命機能分子化学


原著論文
1. Kurataka Otsuka, Akihito Mamiya, Mineko Konishi, Mamoru Nozaki, Atsuko Kinoshita, Hiroaki Tamaki, Masaki Arita, Masato Saito, Kayoko Yamamoto, Takushi Hachiya, Ko Noguchi, Takashi Ueda, Yusuke Yagi, Takehito Kobayashi, Takahiro Nakamura, Yasushi Sato, Takashi Hirayama, Munetaka Sugiyama, Temperature-dependent fasciation mutants provide a link between mitochondrial RNA processing and lateral root morphogenesis, eLife, 10.7554/eLife.61611, 10, e61611, 2021.01.
2. Takehito Kobayashi, Yusuke Yagi, Takahiro Nakamura, Comprehensive Prediction of Target RNA Editing Sites for PLS-Class PPR Proteins in Arabidopsis thaliana, Plant and Cell Physiology, 10.1093/pcp/pcy251, 60, 4, 862-874, 2019.04, [URL], 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..
3. Kawabe Yoshinori, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05, [URL], 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..
4. Takayoshi Imai, Yusuke Yagi, Takahiro Nakamura, Recent progress toward RNA manipulation with engineered pentatricopeptide repeat proteins, Applied RNA Bioscience, 10.1007/978-981-10-8372-3_10, 151-160, 2018.04, [URL], 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..
5. Yoshinori Kawabe, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems, J. Biosci. Bioeng., 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05.
6. Kobayashi T, Yagi Y, Nakamura T, Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells., Methods Mol Biol. , 10.1007/978-1-4939-4931-1_11, 1469, 147-155, 2016.08.
7. Kazama T, Itabashi E, Fujii S, Nakamura T, Toriyama K, Mitochondrial ORF79 levels determine the timing of pollen abortion in cytoplasmic male sterile, The Plant Journal, doi: 10.1111/tpj.13135, 85, 6, 707-716, 2016.03.
8. Tomohiko Kazama, Etsuko Itabashi, Shinya Fujii, Takahiro Nakamura, Kinya Toriyama, Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice, Plant Journal, 10.1111/tpj.13135, 85, 6, 707-716, 2016.03, [URL], 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..
9. Yagi Y, Shirakawa M, Nakamura T, The challenges faced by EditForce Inc., to go beyond genome editing, Nature, Sponsor feature, 2015.12, 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..
10. Sakuma T, Takenaga M, Kawabe Y, Nakamura T, Kamihira M, Yamamoto T, Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids., Int J Mol Sci., 10.3390/ijms161023849, 16, 10, 23849-23866, 2015.10.
11. Kenji Okuda, Harumi Shoki, Miho Arai, Toshiharu Shikanai, Ian Small, Takahiro Nakamura, Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing, PLANT JOURNAL, 10.1111/tpj.12687, 80, 5, 870-882, 2014.12, 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..
12. Takayoshi Imai, Takahiro Nakamura, Maeda, Taku, Xuzhu Gao, Yoshimitsu Kakuta, Takashi Nakashima, makoto kimura, Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at T psi C loop in precursor tRNAs, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2014.07.030, 450, 4, 1541-1546, 2014.08.
13. Yusuke Yagi, Takahiro Nakamura, Ian Small, The potential for manipulating RNA with pentatricopeptide repeat proteins, PLANT JOURNAL, 10.1111/tpj.12377, 78, 5, 772-782, 2014.06, 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..
14. Tomohiko Kazama, Yusuke Yagi, Kinya Toriyama, Takahiro Nakamura, Heterogeneity of the 5 '-end in plant mRNA may be involved in mitochondrial translation, FRONTIERS IN PLANT SCIENCE, 10.3389/fpls.2013.00517, 4, 517, 2013.12.
15. Mizuho Ichinose, Chieko Sugita, Yusuke Yagi, Takahiro Nakamura, Mamoru Sugita, Two DYW Subclass PPR Proteins are Involved in RNA Editing of ccmFc and atp9 Transcripts in the Moss Physcomitrella patens: First Complete Set of PPR Editing Factors in Plant Mitochondria, PLANT AND CELL PHYSIOLOGY, 10.1093/pcp/pct132, 54, 11, 1907-1916, 2013.11.
16. Yusuke Yagi, Makoto Tachikawa, Hisayo Noguchi, Soichiro Satoh, Junichi Obokata, Takahiro Nakamura, Pentatricopeptide repeat proteins involved in plant organellar RNA editing, RNA BIOLOGY, 10, 9, 1419-1425, 2013.09.
17. Yusuke Yagi, Shimpei Hayashi, Keiko Kobayashi, Takashi Hirayama, Takahiro Nakamura, Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants., PLoS ONE, 10.1371/journal.pone.0057286, 8, 3, e57286, 2013.03, 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..
18. Yusuke Yagi, Makoto Tachikawa, Hisayo Noguchi, Soichirou Satoh, Junichi Obokata, Takahiro Nakamura, Pentatricopeptide repeat proteins involved in plant organellar RNA editing, RNA biology, 10.4161/rna.24908, 10, 9, 1236-1242, 2013.01, [URL], 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..
19. Takahiro Nakamura, Yusuke Yagi, Keiko Kobayashi, Mechanistic insight into pentatricopeptide repeat proteins as sequence-specific RNA-binding proteins for organellar RNAs in plants, Plant and Cell Physiology, 10.1093/pcp/pcs069, 53, 7, 1171-1179, 2012.07, [URL], 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..
20. Takahiro Nakamura, Yusuke Yagi, Keiko Kobayashi, Mechanistic Insight into Pentatricopeptide Repeat Proteins as Sequence-Specific RNA-Binding Proteins for Organellar RNAs in Plants, Plant Cell Physiol, 53, 7, 1171-1179, 2012.06, 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..
21. Maki Murayama, Shimpei Hayashi1, Noriyuki Nishimura1, Mayumi Ishide, Keiko Kobayashi, Yusuke Yagi, Tadao Asami, Takahiro Nakamura, Kazuo Shinozaki, Takashi Hirayama, Isolation of Arabidopsis ahg11, a weak ABA hypersensitive mutant defective in nad4 RNA editing , J. Exp. Bot., 10.1093/jxb/ers188, 2012.06, 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. .
22. Kobayashi K, Kawabata M, Hisano K, Kazama T, Matsuoka K, Sugita M, Nakamura T, Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein, Nucleic Acids Res., 40, 2712-2723, 2012.03, 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。.
23. Keiko Kobayashi, Masuyo Kawabata, Keizo Hisano, Tomohiko Kazama, Ken Matsuoka, Mamoru Sugita, Takahiro Nakamura, Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein, Nucleic Acids Research, 10.1093/nar/gkr1084, 40, 6, 2712-2723, 2012.03, [URL], 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..
24. Kusumi K, Sakata C, Nakamura T, Kawasaki S, Yoshimura A, Iba K, A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions, Plant J., 10.1111/j.1365-313X.2011.04755.x, 68, 1039-1050, 2011.12, 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。.
25. Okuda K, Chateigner-Boutin AL, Nakamura T, Delannoy E, Sugita M, Myouga F, Motohashi R, Shinozaki K, Small I, Shikanai T, Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts, Plant Cell, 21, 146-156, 2009.05.
26. Kenji Okuda, Anne Laure Chateigner-Boutin, Takahiro Nakamura, Etienne Delannoy, Mamoru Sugita, Fumiyoshi Myouga, Reiko Motohashi, Kazuo Shinozaki, Ian Small, Toshiharu Shikanai, Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts, Plant Cell, 10.1105/tpc.108.064667, 21, 1, 146-156, 2009.01, [URL], 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..
27. Nakamura T, Sugita M, A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endo-ribonuclease activity, FEBS Lett., 582, 163-168, 2008.12.
28. Takahiro Nakamura, Mamoru Sugita, A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endoribonuclease activity, FEBS Letters, 10.1016/j.febslet.2008.11.017, 582, 30, 4163-4168, 2008.12, [URL], 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..
29. Kazama T, Nakamura T, Watanabe M, Sugita M, Toriyama K, Suppression mechanism of mitochondrial ORF79 accumulation by Rf1 protein in BT-type cytoplasmic male sterile rice, Plant J., 55, 37661-7, 2008.08.
30. Tomohiko Kazama, Takahiro Nakamura, Masao Watanabe, Mamoru Sugita, Kinya Toriyama, Suppression mechanism of mitochondrial ORF79 accumulation by Rf1 protein in BT-type cytoplasmic male sterile rice, Plant Journal, 10.1111/j.1365-313X.2008.03529.x, 55, 4, 619-628, 2008.08, [URL], 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..
31. Takahiro Nakamura, Kumiko Naito, Naoto Yokota, Chieko Sugita, Mamoru Sugita, A cyanobacterial non-coding RNA, Yfr1, is required for growth under multiple stress conditions, Plant and Cell Physiology, 10.1093/pcp/pcm098, 48, 9, 1309-1318, 2007.09, [URL], 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..
32. Okuda K, Nakamura T, Sugita M, Shimizu T, Shikanai T, A pentatricopeptide repeat protein is a site recognition factor in chloroplast RNA editing, J. Biol. Chem., 281, 37661-7, 2006.12.
33. Kenji Okuda, Takahiro Nakamura, Mamoru Sugita, Toshiyuki Shimizu, Toshiharu Shikanai, A pentatricopeptide repeat protein is a site recognition factor in chloroplast RNA editing, Journal of Biological Chemistry, 10.1074/jbc.M608184200, 281, 49, 37661-37667, 2006.12, [URL], 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..
34. Takahiro Nakamura, C. Sugiura, Y. Kobayashi, M. Sugita, Transcript profiling in plastid arginine tRNA-CCG gene knockout moss
Construction of Physcomitrella patens plastid DNA microarray, Plant Biology, 10.1055/s-2005-865620, 7, 3, 258-265, 2005.05, [URL], 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..
35. Nakamura T, Schuster G, Sugiura M, Sugita M, Chloroplast RNA-binding and pentatricopeptide repeat proteins, Biochem. Soc. Trans., 32, 571-4, 2004.08.
36. Takahiro Nakamura, G. Schuster, M. Sugiura, M. Sugita, Chloroplast RNA-binding and pentatricopeptide repeat proteins, Biochemical Society Transactions, 10.1042/BST0320571, 32, 4, 571-574, 2004.08, [URL], 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..
37. Takahiro Nakamura, Karin Meierhoff, Peter Westhoff, Gadi Schuster, RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA, European Journal of Biochemistry, 10.1046/j.1432-1033.2003.03796.x, 270, 20, 4070-4081, 2003.10, [URL], 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..
38. Takahiro Nakamura, Yumiko Furuhashi, Keiko Hasegawa, Hiroshi Hashimoto, Kazufumi Watanabe, Junichi Obokata, Mamoru Sugita, Masahiro Sugiura, Array-based analysis on tobacco plastid transcripts
Preparation of a genomic microarray containing all genes and all intergenic regions, Plant and Cell Physiology, 10.1093/pcp/pcg101, 44, 8, 861-867, 2003.08, [URL], 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..
39. Karin Meierhoff, Susanne Felder, Takahiro Nakamura, Nicole Bechtold, Gadi Schuster, HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs, Plant Cell, 15, 6, 1480-1495, 2003.06, 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..
40. Takahiro Nakamura, Mamoru Sugita, Nuclear-encoded proteins involved in function of chloroplast RNAs, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 48, 15 Suppl, 2168-2175, 2003.
41. Takahiro Nakamura, Masaru Ohta, Masahiro Sugiura, Mamoru Sugita, Chloroplast ribonucleoproteins function as a stabilizing factor of ribosome-free mRNAs in the stroma, Journal of Biological Chemistry, 10.1074/jbc.M008817200, 276, 1, 147-152, 2001.01, [URL], 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..
42. Tetsuya Miyamoto, Takahiro Nakamura, Issei Nagao, Junichi Obokata, Quantitative Analysis of Transiently Expressed mRNA in Particle-Bombarded Tobacco Seedlings, Plant Molecular Biology Reporter, 10.1007/BF02824017, 18, 2, 101-107, 2000.01, [URL], 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..
43. Takahiro Nakamura, Masaru Ohta, Masahiro Sugiura, Mamoru Sugita, Chloroplast ribonucleoproteins are associated with both mRNAs and intron-containing precursor tRNAs, FEBS Letters, 10.1016/S0014-5793(99)01390-3, 460, 3, 437-441, 1999.11, [URL], 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..
44. Motoki Kanekatsu, Akiyoshi Ezumi, Takahiro Nakamura, Kenzo Ohtsuki, Chloroplast ribonucleoproteins (RNPs) as phosphate acceptors for casein kinase II
Purification by ssDNA-cellulose column chromatography, Plant and Cell Physiology, 36, 8, 1649-1656, 1995.12, 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..

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