研究者詳細 - 黒柳　秀人

論文 - 黒柳　秀人

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UNC5B is an isoform-dependent target for ectodomain shedding.

Kotaro Sugimoto, Eichi Watabe, Mio Takuma, Kaname Nagahara, Toshinori Sawano, Mihoko Kajita, Junichi Takagi, Hidehito Kuroyanagi, Kyoko Shirakabe

The Journal of Biochemistry ( Oxford University Press ) mvaf043 2025年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Cytoplasmic mutant RBM20 causes arrhythmogenicity in murine atria.

Kensuke Ihara, Satoshi Iwamiya, Masaki Ikuta, Yurie Soejima, Yuichi Hiraoka, Atsushi Nakano, Susumu Minamisawa, Tetsushi Furukawa, Hidehito Kuroyanagi, Tetsuo Sasano

Journal of Molecular and Cellular Cardiology ( Elsevier Ltd. ) 205 ( August 2025 ) 1 - 12 2025年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Profiling of RBM20-Regulated CaMKIIδ Splice Variants Across the Heart, Skeletal Muscle, and Olfactory Bulbs.

Yui Maeda, Yuri Yamasu, Hidehito Kuroyanagi

Genes to Cells ( John Wiley & Sons, Inc. ) 30 ( 3 ) e70021 2025年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20K21385, JP23K18221, JP22H04925

Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ), encoded by the Camk2d gene, plays key regulatory roles in various Ca2+-regulated cellular processes. Extensive alternative splicing of the Camk2d gene generates multiple CaMKIIδ splice variants that exhibit differential roles. Despite significant advances in understanding the functions of CaMKIIδ, the full repertoire of Camk2d splice variants in a variety of tissues and their distinct roles in physiological and pathological contexts remain incompletely characterized due to the complex nature of multiple alternative splicing events. Here, we conducted long-read amplicon sequencing to investigate the murine Camk2d splice variants in the heart, skeletal muscle, and olfactory bulbs and show that Camk2d mRNAs in the heart and skeletal muscle have shorter 3'UTRs. Our results in this study suggest that a key regulator of Camk2d splicing, RNA-binding motif protein 20 (RBM20), whose gain-of-function mutations cause dilated cardiomyopathy, is crucial for the expression of heart-specific splice variants. Olfactory bulbs specifically express novel splice variants that utilize a mutually exclusive exon 6B and/or an alternative polyadenylation site in a novel exon 17.5 in an RBM20-inependent manner. The tissue-specific repertoire of CaMKIIδ splice variants and their aberrant expression in disease model animals will help in understanding their roles in physiological and pathological contexts.

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Alternative splicing of a single exon causes a major impact on the affinity of Caenorhabditis elegans tropomyosin isoforms for actin filaments.

Shoichiro Ono, Eichi Watabe, Keita Morisaki, Kanako Ono, Hidehito Kuroyanagi

Frontiers in Cell and Developmental Biology ( Frontiers Media S.A. ) 11 1208913 2023年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20H04839, JP20H03181

Tropomyosin is generally known as an actin-binding protein that regulates actomyosin interaction and actin filament stability. In metazoans, multiple tropomyosin isoforms are expressed, and some of them are involved in generating subpopulations of actin cytoskeleton in an isoform-specific manner. However, functions of many tropomyosin isoforms remain unknown. Here, we report identification of a novel alternative exon in the #ICaenorhabditis elegans#IR tropomyosin gene and characterization of the effects of alternative splicing on the properties of tropomyosin isoforms. Previous studies have reported six tropomyosin isoforms encoded by the #IC. elegans lev-11#IR tropomyosin gene. We identified a seventh isoform, LEV-11U, that contained a novel alternative exon, exon 7c (E7c). LEV-11U is a low-molecular-weight tropomyosin isoform that differs from LEV-11T only at the exon 7-encoded region. #IIn silico#IR analyses indicated that the E7c-encoded peptide sequence was unfavorable for coiled-coil formation and distinct from other tropomyosin isoforms in the pattern of electrostatic surface potentials. #IIn vitro#IR, LEV-11U bound poorly to actin filaments, whereas LEV-11T bound to actin filaments in a saturable manner. When these isoforms were transgenically expressed in the #IC. elegans#IR striated muscle, LEV-11U was present in the diffuse cytoplasm with tendency to form aggregates, whereas LEV-11T co-localized with sarcomeric actin filaments. Worms with a mutation in E7c showed reduced motility and brood size, suggesting that this exon is important for the optimal health. These results indicate that alternative splicing of a single exon can produce biochemically diverged tropomyosin isoforms and suggest that a tropomyosin isoform with poor actin affinity has a novel biological function.

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Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.

Hirotatsu Imai, Daisuke Utsumi, Hidetsugu Torihara, Kenzo Takahashi, Hidehito Kuroyanagi, Akio Yamashita

Nucleic Acids Research ( Oxford University Press ) 51 ( 14 ) e76 2023年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20H04839, JP20H03181

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

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Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

Jue Ju, Tomohiko Aoyama, Yuka Yashiro, Seisuke Yamashita, Hidehito Kuroyanagi, Kozo Tomita

Nucleic Acids Research ( Oxford University Press ) 51 ( 5 ) 2434 - 2446 2023年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20H03181, JP17H05596, JP20H04839

In #ICaenorhabditis elegans#IR, the N#U6#UR-methyladenosine (m#U6#URA) modification by METT10, at the 3'-splice sites in #IS#IR-adenosyl-L-methionine (SAM) synthetase (#Isams#IR) precursor mRNA (pre-mRNA), inhibits #Isams#IR pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of #IC. elegans#IR METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m#U6#URA modification in the 3'-UTR hairpins of methionine adenosyltransferase (#IMAT2A#IR) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that #IC. elegans#IR METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of #Isams#IR pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. #IC. elegans#IR METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of #IC. elegans#IR METT10 facilitates the m#U6#URA modification of the 3'-splice sites of #Isams#IR pre-mRNAs. These results suggest the well-conserved mechanisms for the m#U6#URA modification of substrate RNAs between #IHomo sapiens#IR and #IC. elegans#IR, despite their different regulation mechanisms for SAM homeostasis.

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ナンセンスコドン介在的mRNA分解（NMD）と共役した選択的スプライシングによる遺伝子発現の制御

黒柳秀人

生化学 ( 公益社団法人日本生化学会 ) 94 ( 6 ) 868 - 874 2022年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

筆者らは，線虫のナンセンスコドン介在的mRNA分解（NMD）欠損変異株を利用して，全長mRNAの直接シーケンシング解析により，NMDの基質となるスプライスバリアントの網羅的探索を行い，259遺伝子の289バリアントを同定した．このうち，S-アデノシル-L-メチオニン（SAM）合成酵素をコードするsams遺伝子群では，SAMの濃度に応じて3′スプライス部位のAGジヌクレオチドのアデニン塩基がN6-メチルアデノシン（m6A）修飾を受けることで発現量が間接的にフィードバック制御されるという新奇の制御機構の発見に至った．本稿では，直接RNAシーケンシング解析を利用したmRNA全長配列解析やsams遺伝子の発現量の選択的スプライシングによる制御の意義などについて概説する．

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I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy.

Takuma Yamamoto, Rie Sano, Aya Miura, Mai Imasaka, Yoshiro Naito, Minori Nishiguchi, Kensuke Ihara, Naruhito Otani, Yoshihiko Kominato, Masaki Ohmuraya, Hidehito Kuroyanagi & Hajime Nishio

Journal of Molecular Medicine ( Springer Nature ) 100 ( 12 ) 1741 - 1754 2022年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20K07659, JP20K21385

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model.

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Periostin Exon-21 Antibody Neutralization of Triple-Negative Breast Cancer Cell-Derived Periostin Regulates Tumor-Associated Macrophage Polarization and Angiogenesis.

Tatsuya Fujikawa, Fumihiro Sanada, Yoshiaki Taniyama, Kana Shibata, Naruto Katsuragi, Nobutaka Koibuchi, Kaori Akazawa, Yuko Kanemoto, Hidehito Kuroyanagi, Kenzo Shimazu, Hiromi Rakugi and Ryuichi Morishita

Cancers ( MDPI ) 13 ( 20 ) 5072 - 5072 2021年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20K07659, JP20H03181 and JP20K21385

Periostin (Pn) is involved in multiple processes of cancer progression. Previously, we reported that Pn expression is correlated with mesenchymal tumor markers and poor prognosis in triple-negative breast cancer (TNBC). In the TNBC xenograft model, chemotherapy increased expression of a Pn alternative splicing variant (ASV) with exon 21, and administration of the neutralizing antibody against Pn with exon 21 (Pn-21 Ab) overcame chemoresistance with a reduction in the mesenchymal cancer cell fraction. In the present study, the role of Pn ASV with exon 21 in TNBC progression has been addressed. We first established a stable cell line carrying a fluorescence-based splicing reporter. Pn-positive TNBC has higher expression of genes related to tumor-associated macrophage (TAM) recruitment and ECM-receptor interaction than Pn-negative cells. In a xenograft model, only Pn-positive cells initiated tumor formation, and the Pn-21 Ab suppressed tumor cell growth, accompanied by decreased M2 TAM polarization and the number of tumor vessels. These data suggest that cancer cell-derived Pn ASV educates TAMs and regulates angiogenesis, which in turn establishes a microenvironmental niche that is supportive of TNBC.

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Loss of Caenorhabditis elegans homologue of human MOB4 compromises life span, health life span and thermotolerance.

Momotaj Jahan, Hiroaki Iwasa, Hidehito Kuroyanagi, Yutaka Hata

Genes to Cells ( The Molecular Biology Society of Japan/John Wiley & Sons Australia, Ltd ) 26 ( 10 ) 798 - 806 2021年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Mob1/phocein family proteins are conserved from yeast to mammals. Human has four MOB genes, MOB1, 2, 3 and 4. Human MOB1 protein, which is a component of the Hippo pathway, is involved in the inhibition of yes-associated protein (YAP1) through large tumor suppressor (LATS) kinases and plays a tumor suppressive role. In contrast, MOB4 activates YAP1. Caernorhabditis elegans (C. elegans) also has four MOB genes. Moreover, C. elegans has homologues of YAP1 (Ce_YAP-1) and LATS kinases (WTS-1). Nevertheless, our previous study revealed that the Hippo pathway is not conserved in C. elegans and that heat shock activates Ce_YAP-1. We also reported that Ce_YAP-1 is involved in the regulation of life span, healthy lifespan and thermotolerance. In this study, we raised a question whether and how C. elegans homologue of MOB4 (Ce_MOB-4) is involved in the regulation of Ce_YAP-1. Ce_MOB-4 is ubiquitously expressed in adult worms. This expression pattern is similar to that of Ce_YAP-1. mob-4 loss-of-function mutants show short life span, short health life span and compromise thermotolerance. However, heat shock activates Ce_YAP-1 in mob-4 mutant. In conclusion, the role of MOB4 in the activation of YAP1 is not conserved in C. elegans.

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m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis.

Eichi Watabe, Marina Togo-Ohno, Yuma Ishigami, Shotaro Wani, Keiko Hirota, Mariko Kimura-Asami, Sharmin Hasan, Satomi Takei, Akiyoshi Fukamizu, Yutaka Suzuki, Tsutomu Suzuki, Hidehito Kuroyanagi

The EMBO Journal ( EMBO Press ) 40 ( 14 ) e106434 2021年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20H03181, JP18H05272, JP20H04839, JP17K07746, JP17H01519, JP23116004, JP16H06279, JP26113003, JP20112004, JP24657116, JP25118506, JP15H01467, JP26291003, JP15H01350, JP15KK0252, JP17H05596, JP17H03633, 1D102, JPMJER2002

Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in vitro. Direct RNA sequencing coupled with machine learning confirms m6A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6A modification at the 3'SS of the sams genes.

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Author Correction: Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs

Jay W. Schneider, Saji Oommen, Muhammad Y. Qureshi, Sean C. Goetsch, David R. Pease, Rhianna S. Sundsbak, Wei Guo, Mingming Sun, Han Sun, Hidehito Kuroyanagi, Dennis A. Webster, Alexander W. Coutts, Kimberly A. Holst, Brooks S. Edwards, Nikolas Newville, Matthew A. Hathcock, Tamene Melkamu, Francesca Briganti, Wu Wei, Maria G. Romanelli, Scott C. Fahrenkrug, Doug E. Frantz, Timothy M. Olson, Lars M. Steinmetz, Daniel F. Carlson, Timothy J. Nelson & Wanek Program Preclinical Pipeline

Nature Medicine ( Springer Nature Limited ) 27 ( 7 ) 1309 2021年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Nature Medicine ( Springer Nature Limited ) 26 ( 11 ) 1788 - 1800 2020年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

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A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice.

Kensuke Ihara, Tetsuo Sasano, Yuichi Hiraoka, Marina Togo-Ohno, Yurie Soejima, Motoji Sawabe, Megumi Tsuchiya, Hidesato Ogawa, Tetsushi Furukawa & Hidehito Kuroyanagi

Scientific Reports ( Springer Nature ) 10 ( 1 ) 17894 - 17894 2020年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20K21385

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.

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mRNA Editing, Processing and Quality Control in Caenorhabditis elegans

Joshua A Arribere, Hidehito Kuroyanagi, Heather A Hundley

Genetics ( Genetics Society of America ) 215 ( 3 ) 531 - 568 2020年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

関連する研究費コード: JP20112004, JP25118506, JP26291003, JP15H01350, JP15H01467, JP15KK0252, JP17H03633, JP17H05596, JP20H03181, JP221S0002 and JP16H06279 (PAGS)

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Alternative splicing of the Caenorhabditis elegans lev-11 tropomyosin gene is regulated in a tissue-specific manner.

Watabe E, Ono S, Kuroyanagi H

Cytoskeleton (Hoboken, N.J.) 75 ( 10 ) 427 - 436 2018年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Tropomyosin isoforms contribute to generation of functionally divergent actin filaments. In the nematode Caenorhabditis elegans, multiple isoforms are produced from lev‐11, the single tropomyosin gene, by combination of two separate promoters and alternative pre‐mRNA splicing. In this study, we report that alternative splicing of lev‐11 is regulated in a tissue‐specific manner so that a particular tropomyosin isoform is expressed in each tissue. Reverse‐transcription polymerase chain reaction analysis of lev‐11 mRNAs confirms five previously reported isoforms (LEV‐11A, LEV‐11C, LEV‐11D, LEV‐11E and LEV‐11O) and identifies a new sixth isoform LEV‐11T. Using transgenic alternative‐splicing reporter minigenes, we find distinct patterns of preferential exon selections in the pharynx, body wall muscles, intestine and neurons. The body wall muscles preferentially process splicing to produce high‐molecular‐weight isoforms, LEV‐11A, LEV‐11D and LEV‐11O. The pharynx specifically processes splicing to express a low‐molecular‐weight isoform LEV‐11E, whereas the intestine and neurons process splicing to express another low‐molecular‐weight isoform LEV‐11C. The splicing pattern of LEV‐11T was not predominant in any of these tissues, suggesting that this is a minor isoform. Our results suggest that regulation of alternative splicing is an important mechanism to express proper tropomyosin isoforms in particular tissue and/or cell types in C. elegans.

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UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.

Iwasa H, Sarkar A, Shimizu T, Sawada T, Hossain S, Xu X, Maruyama J, Arimoto-Matsuzaki K, Withanage K, Nakagawa K, Kurihara H, Kuroyanagi H, Hata Y

Cancer science 109 ( 9 ) 2767 - 2780 2018年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

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Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization.

Murayama R, Kimura-Asami M, Togo-Ohno M, Yamasaki-Kato Y, Naruse TK, Yamamoto T, Hayashi T, Ai T, Spoonamore KG, Kovacs RJ, Vatta M, Iizuka M, Saito M, Wani S, Hiraoka Y, Kimura A, Kuroyanagi H

Scientific reports ( Nature Publishing Group ) 8 ( 1 ) 8970 - 8970 2018年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study.

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Tropomyosin isoforms differentially affect muscle contractility in the head and body regions of the nematode Caenorhabditis elegans.

Barnes DE, Watabe E, Ono K, Kwak E, Kuroyanagi H, Ono S

Molecular biology of the cell ( American Society for Cell Biology ) 29 ( 9 ) 1075 - 1088 2018年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Tropomyosin, one of the major actin filament–binding proteins, regulates actin–myosin interaction and actin-filament stability. Multicellular organisms express a number of tropomyosin isoforms, but understanding of isoform-specific tropomyosin functions is incomplete. The nematode Caenorhabditis elegans has a single tropomyosin gene, lev-11, which has been reported to express four isoforms by using two separate promoters and alternative splicing. Here, we report a fifth tropomyosin isoform, LEV-11O, which is produced by alternative splicing that includes a newly identified seventh exon, exon 7a. By visualizing specific splicing events in vivo, we find that exon 7a is predominantly selected in a subset of the body wall muscles in the head, while exon 7b, which is the alternative to exon 7a, is utilized in the rest of the body. Point mutations in exon 7a and exon 7b cause resistance to levamisole-induced muscle contraction specifically in the head and the main body, respectively. Overex-pression of LEV-11O, but not LEV-11A, in the main body results in weak levamisole resistance. These results demonstrate that specific tropomyosin isoforms are expressed in the head and body regions of the muscles and contribute differentially to the regulation of muscle contractility.

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Alternative Splicing Regulator RBM20 and Cardiomyopathy.

Watanabe T, Kimura A, Kuroyanagi H

Frontiers in molecular biosciences 5 ( NOV ) 105 2018年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

RBM20 is a vertebrate-specific RNA-binding protein with two zinc finger (ZnF) domains, one RNA-recognition motif (RRM)-type RNA-binding domain and an arginine/serine (RS)-rich region. RBM20 has initially been identified as one of dilated cardiomyopathy (DCM)-linked genes. RBM20 is a regulator of heart-specific alternative splicing and Rbm20ΔRRM mice lacking the RRM domain are defective in the splicing regulation. The Rbm20ΔRRM mice, however, do not exhibit a characteristic DCM-like phenotype such as dilatation of left ventricles or systolic dysfunction. Considering that most of the RBM20 mutations identified in familial DCM cases were heterozygous missense mutations in an arginine-serine-arginine-serine-proline (RSRSP) stretch whose phosphorylation is crucial for nuclear localization of RBM20, characterization of a knock-in animal model is awaited. One of the major targets for RBM20 is the TTN gene, which is comprised of the largest number of exons in mammals. Alternative splicing of the TTN gene is exceptionally complicated and RBM20 represses >160 of its consecutive exons, yet detailed mechanisms for such extraordinary regulation are to be elucidated. The TTN gene encodes the largest known protein titin, a multi-functional sarcomeric structural protein specific to striated muscles. As titin is the most important factor for passive tension of cardiomyocytes, extensive heart-specific and developmentally regulated alternative splicing of the TTN pre-mRNA by RBM20 plays a critical role in passive stiffness and diastolic function of the heart. In disease models with diastolic dysfunctions, the phenotypes were rescued by increasing titin compliance through manipulation of the Ttn pre-mRNA splicing, raising RBM20 as a potential therapeutic target.

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An emerging model organism Caenorhabditis elegans for alternative pre-mRNA processing in vivo.

Wani S, Kuroyanagi H

Wiley interdisciplinary reviews. RNA ( WILEY ) 8 ( 6 ) e1428 2017年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

A nematode Caenorhabditis elegans is an intron-rich organism and up to 25% of its pre-mRNAs are estimated to be alternatively processed. Its compact genomic organization enables construction of fluorescence splicing reporters with intact genomic sequences and visualization of alternative processing patterns of interest in the transparent living animals with single-cell resolution. Genetic analysis with the reporter worms facilitated identification of trans-acting factors and cis-acting elements, which are highly conserved in mammals. Analysis of unspliced and partially spliced pre-mRNAsin vivo raised models for alternative splicing regulation relying on specific order of intron excision. RNA-seq analysis of splicing factor mutants and CLIP-seq analysis of the factors allow global search for target genes in the whole animal. An mRNA surveillance system is not essential for its viability or fertility, allowing analysis of unproductively spliced noncoding mRNAs. These features offer C. elegans as an ideal model organism for elucidating alternative pre-mRNA processing mechanisms in vivo. Examples of isoform-specific functions of alternatively processed genes are summarized. (C) 2017 Wiley Periodicals, Inc.

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Evolutionarily conserved autoregulation of alternative pre-mRNA splicing by ribosomal protein L10a.

Takei S, Togo-Ohno M, Suzuki Y, Kuroyanagi H

Nucleic acids research ( OXFORD UNIV PRESS ) 44 ( 12 ) 5585 - 5596 2016年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Alternative splicing of pre-mRNAs can regulate expression of protein-coding genes by generating unproductive mRNAs rapidly degraded by nonsense-mediated mRNA decay (NMD). Many of the genes directly regulated by alternative splicing coupled with NMD (AS-NMD) are related to RNA metabolism, but the repertoire of genes regulated by AS-NMD in vivo is to be determined. Here, we analyzed transcriptome data of wild-type and NMD-defective mutant strains of the nematode worm Caenorhabditis elegans and demonstrate that eight of the 82 cytoplasmic ribosomal protein (rp) genes generate unproductively spliced mRNAs. Knockdown of any of the eight rp genes exerted a dynamic and compensatory effect on alternative splicing of its own transcript and inverse effects on that of the other rp genes. A large subunit protein L10a, termed RPL-1 in nematodes, directly and specifically binds to an evolutionarily conserved 39-nt stretch termed L10ARE between the two alternative 5' splice sites in its own pre-mRNA to switch the splice site choice. Furthermore, L10ARE-mediated splicing autoregulation of the L10a-coding gene is conserved in vertebrates. These results indicate that L10a is an evolutionarily conserved splicing regulator and that homeostasis of a subset of the rp genes are regulated at the level of pre-mRNA splicing in vivo.

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Splicing factors control C. elegans behavioural learning in a single neuron by producing DAF-2c receptor.

Tomioka M, Naito Y, Kuroyanagi H, Iino Y

Nature communications ( NATURE PUBLISHING GROUP ) 7 11645 - 11645 2016年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Alternative splicing generates protein diversity essential for neuronal properties. However, the precise mechanisms underlying this process and its relevance to physiological and behavioural functions are poorly understood. To address these issues, we focused on a cassette exon of the Caenorhabditis elegans insulin receptor gene daf-2, whose proper variant expression in the taste receptor neuron ASER is critical for taste-avoidance learning. We show that inclusion of daf-2 exon 11.5 is restricted to specific neuron types, including ASER, and is controlled by a combinatorial action of evolutionarily conserved alternative splicing factors, RBFOX, CELF and PTB families of proteins. Mutations of these factors cause a learning defect, and this defect is relieved by DAF-2c (exon 11.5+) isoform expression only in a single neuron ASER. Our results provide evidence that alternative splicing regulation of a single critical gene in a single critical neuron is essential for learning ability in an organism.

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RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing

Kanako Kuwasako, Mari Takahashi, Satoru Unzai, Kengo Tsuda, Seiko Yoshikawa, Fahu He, Naohiro Kobayashi, Peter Güntert, Mikako Shirouzu, Takuhiro Ito, Akiko Tanaka, Shigeyuki Yokoyama, Masatoshi Hagiwara, Hidehito Kuroyanagi, Yutaka Muto

Nature Structural and Molecular Biology ( Nature Publishing Group ) 21 ( 9 ) 778 - 786 2014年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.

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Comprehensive analysis of mutually exclusive alternative splicing in C. elegans.

Hidehito Kuroyanagi, Satomi Takei, Yutaka Suzuki

Worm 3 e28459 - e28459 2014年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Mutually exclusive selection of one exon in a cluster of exons is a rare form of alternative pre-mRNA splicing, yet suggests strict regulation. However, the repertoires of regulation mechanisms for the mutually exclusive (ME) splicing in vivo are still unknown. Here, we experimentally explore putative ME exons in C. elegans to demonstrate that 29 ME exon clusters in 27 genes are actually selected in a mutually exclusive manner. Twenty-two of the clusters consist of homologous ME exons. Five clusters have too short intervening introns to be excised between the ME exons. Fidelity of ME splicing relies at least in part on nonsense-mediated mRNA decay for 14 clusters. These results thus characterize all the repertoires of ME splicing in this organism.

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選択的スプライシング可視化システム

木村まり子, 黒柳秀人

Medical Science Digest ( (株)ニュー・サイエンス社 ) 39 ( 11 ) 502 - 503 2013年10月

掲載種別: 研究論文（その他学術会議資料等）

選択的スプライシングレポーター系は、任意の目的遺伝子のメッセンジャーRNA前駆体の選択的スプライシングパターンを、生細胞または生体内で複数の蛍光タンパク質の発現により検出する可視化システムである。選択的スプライシング制御に影響を与える化合物のスクリーニングやスプライシング制御因子の同定、シスエレメントの探索など、本可視化システムの応用範囲は多岐に渡る。本稿では選択的スプライシングレポーターの実例と活用例を紹介する。(著者抄録)

Switch-like regulation of tissue-specific alternative pre-mRNA processing patterns revealed by customized fluorescence reporters.

Hidehito Kuroyanagi

Worm 2 ( 3 ) e23834 - e23834 2013年07月

掲載種別: 研究論文（その他学術会議資料等）

Alternative processing of precursor mRNAs (pre-mRNAs), including alternative transcription start sites, alternative splicing and alternative polyadenylation, is the major source of protein diversity and plays crucial roles in development, differentiation and diseases in higher eukaryotes. It is estimated from microarray analyses and deep sequencing of mRNAs from synchronized worms that up to 25% of protein-coding genes in Caenorhabditis elegans undergo alternative pre-mRNA processing and that many of them are subject to developmental regulation. Recent progress in visualizing the alternative pre-mRNA processing patterns in living worms with custom-designed fluorescence reporters has enabled genetic analyses of the regulatory mechanisms for alternative processing events of interest in vivo. Expression of the tissue-specific isoforms of actin depolymerising factor (ADF)/cofilin, UNC-60A and UNC-60B, is regulated by a combination of alternative splicing and alternative polyadenylation of pre-mRNA from a single gene unc-60. We recently found that muscle-specific splicing regulators ASD-2 and SUP-12 cooperatively switch the pre-mRNA processing patterns of the unc-60 gene in body wall muscles. Here I summarize the bichromatic fluorescence reporter system utilized for visualizing the tissue-specific alternative processing patterns of the unc-60 pre-mRNA. I also discuss the model for the coordinated regulation of the UNC-60B-type pre-mRNA processing in body wall muscles by ASD-2 and SUP-12.

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Yes-associated protein homolog, YAP-1, is involved in the thermotolerance and aging in the nematode Caenorhabditis elegans

Hiroaki Iwasa, Sainawaer Maimaiti, Hidehito Kuroyanagi, Shodai Kawano, Kazutoshi Inami, Shikshya Timalsina, Mitsunobu Ikeda, Kentaro Nakagawa, Yutaka Hata

Experimental Cell Research ( Academic Press Inc. ) 319 ( 7 ) 931 - 945 2013年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The mammalian Hippo pathway comprises mammalian Ste20-like kinases (MST1/2) and large tumor suppressor kinases (LATS1/2). LATS1/2, which are activated by MST1/2, phosphorylate a transcriptional co-activator, yes-associated protein (YAP), and induce the recruitment of YAP by 14-3-3 to cytoplasm, so that the TEAD-dependent gene transcriptions are turned off. Although the core components of the Hippo pathway are well conserved in metazoans, it has been discussed that Caenorhabditis elegans lacks YAP ortholog, we found that F13E6.4 gene encodes a protein that shows sequence similarities to YAP in the N-terminal TEAD-binding domain and in the WW domain. We designated this gene as yap-1. YAP-1 is widely expressed in various cells such as epithelial cells, muscles, hypodermal cells, gonadal sheath cells, spermatheca, and hypodermal cells. YAP-1 is distributed in cytoplasm and nuclei. wts-1 (LATS ortholog) and ftt-2 (14-3-3 ortholog) knockdowns cause nuclear accumulation of YAP-1, supporting that the subcellular localization of YAP-1 is regulated in a similar way as that of YAP. Heat shock also causes the nuclear accumulation of YAP-1 but after heat shock, YAP-1 translocates to cytoplasm. Knockdowns of DAF-21 (HSP90 ortholog) and HSF-1block the nuclear export of YAP-1 during this recovery. YAP-1 overexpression is beneficial for thermotolerance, whereas YAP-1 hyperactivity induced by wts-1 and ftt-2 knockdowns is deleterious on thermal response and yap-1 deficiency promotes health aging. In short, YAP-1 partially shares basal characters with mammalian YAP and plays a role in thermal stress response and healthy aging. © 2013 Elsevier Inc.

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Position-dependent and neuron-specific splicing regulation by the CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans

Hidehito Kuroyanagi, Yohei Watanabe, Yutaka Suzuki, Masatoshi Hagiwara

Nucleic Acids Research 41 ( 7 ) 4015 - 4025 2013年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

A large fraction of protein-coding genes in metazoans undergo alternative pre-mRNA splicing in tissue-or cell-type-specific manners. Recent genome-wide approaches have identified many putative-binding sites for some of tissue-specific trans-acting splicing regulators. However, the mechanisms of splicing regulation in vivo remain largely unknown. To elucidate the modes of splicing regulation by the neuron-specific CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans, we performed deep sequencing of poly(A) + RNAs from the unc-75(+)-and unc-75-mutant worms and identified more than 20 cassette and mutually exclusive exons repressed or activated by UNC-75. Motif searches revealed that (G/U)UGUUGUG stretches are enriched in the upstream and downstream introns of the UNC-75-repressed and-activated exons, respectively. Recombinant UNC-75 protein specifically binds to RNA fragments carrying the (G/U)UGUUGUG stretches in vitro. Bi-chromatic fluorescence alternative splicing reporters revealed that the UNC-75-target exons are regulated in tissue-specific and (G/U)UGUUGUG element-dependent manners in vivo. The unc-75 mutation affected the splicing reporter expression specifically in the nervous system. These results indicate that UNC-75 regulates alternative splicing of its target exons in neuron-specific and position-dependent manners through the (G/U)UGUUGUG elements in C. elegans. This study thus reveals the repertoire of target events for the CELF family in the living organism. © 2013 The Author(s).

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CELF Family RNA-Binding Protein UNC-75 Regulates Two Sets of Mutually Exclusive Exons of the unc-32 Gene in Neuron-Specific Manners in Caenorhabditis elegans

Hidehito Kuroyanagi, Yohei Watanabe, Masatoshi Hagiwara

PLoS Genetics 9 ( 2 ) e1003337 - e1003337 2013年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene. © 2013 Kuroyanagi et al.

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Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1

Hiroaki Iwasa, Hidehito Kuroyanagi, Sainawaer Maimaiti, Mitsunobu Ikeda, Kentaro Nakagawa, Yutaka Hata

EXPERIMENTAL CELL RESEARCH ( ELSEVIER INC ) 319 ( 3 ) 1 - 11 2013年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling. (c) 2012 Elsevier Inc. All rights reserved.

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Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans

Genta Ohno, Kanako Ono, Marina Togo, Yohei Watanabe, Shoichiro Ono, Masatoshi Hagiwara, Hidehito Kuroyanagi

PLoS Genetics 8 ( 10 ) e1002991 - e1002991 2012年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript. © 2012 Ohno et al.

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Dual role of the carboxyl-terminal region of pig liver l-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity*

Kumiko Hirai, Hidehito Kuroyanagi, Yoshitaka Tatebayashi, Yoshitaka Hayashi, Kanako Hirabayashi-Takahashi, Kuniaki Saito, Seiich Haga, Tomihiko Uemura, Susumu Izumi

JOURNAL OF BIOCHEMISTRY ( OXFORD UNIV PRESS ) 148 ( 6 ) 639 - 650 2010年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMO delta C50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (delta C20, delta C30 and delta C50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

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Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans

Hidehito Kuroyanagi, Genta Ohno, Hiroaki Sakane, Hiroyuki Maruoka, Masatoshi Hagiwara

Nature Protocols 5 ( 9 ) 1495 - 1517 2010年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Transgenic multicolor fluorescence reporters enable the visualization of alternative splicing patterns at a single-cell resolution in living organisms and facilitate further genetic analyses to identify cis-elements and trans-acting factors involved in splicing regulation. In this paper, we describe a method of generating fluorescence alternative splicing reporters for the nematode Caenorhabditis elegans. We describe strategies for designing minigene reporters and methods for constructing them DNA fragments ('modules', such as promoter/3′ cassettes, a genomic fragment of interest and a fluorescent protein cassette) that exist in separate vectors are assembled using site-directed recombination. We also describe strategies and methods for mutant screening and single-nucleotide polymorphism mapping using fluorescence reporters. This is the first detailed description of the design and construction of fluorescence alternative splicing reporters for C. elegans and their use in subsequent genetic analyses. It takes 2-4 months to construct minigenes and generate extrachromosomal lines for visualizing spatiotemporal distribution of alternative splicing events in vivo. Identification of regulators by integration of transgenes, mutant screening and mapping of the responsible genes takes a further 6-12 months. The fluorescence-reporter construction described here can also be applied to the vertebrate cell culture system. © 2010 Nature America, Inc. All rights reserved.

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Fox-1 family of RNA-binding proteins

Hidehito Kuroyanagi

CELLULAR AND MOLECULAR LIFE SCIENCES ( SPRINGER BASEL AG ) 66 ( 24 ) 3895 - 3907 2009年12月 [ 査読有り ]

掲載種別: 研究論文（その他学術会議資料等）

The Fox-1 family of RNA-binding proteins are evolutionarily conserved regulators of tissue-specific alternative splicing in metazoans. The Fox-1 family specifically recognizes the (U)GCAUG stretch in regulated exons or in flanking introns, and either promotes or represses target exons. Recent unbiased bioinformatics analyses of alternatively spliced exons and comparison of various vertebrate genomes identified the (U)GCAUG stretch as a highly conserved and widely distributed element enriched in intronic regions surrounding exons with altered inclusion in muscle, heart, and brain, consistent with specific expression of Fox-1 and Fox-2 in these tissues. Global identification of Fox-2 target RNAs in living cells revealed that many of the Fox-2 target genes themselves encode splicing regulators. Further systematic elucidation of target genes of the Fox-1 family and other splicing regulators in various tissues will lead to a comprehensive understanding of splicing regulatory networks.

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STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo

Genta Ohno, Masatoshi Hagiwara, Hidehito Kuroyanagi

GENES & DEVELOPMENT ( COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT ) 22 ( 3 ) 360 - 374 2008年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.

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The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo

Hidehito Kuroyanagi, Genta Ohno, Shohel Mitani, Masatoshi Hagiwara

MOLECULAR AND CELLULAR BIOLOGY ( AMER SOC MICROBIOLOGY ) 27 ( 24 ) 8612 - 8621 2007年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the fibroblast growth factor receptor gene, egl-15, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-15 alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-15 RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-15(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.

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The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans

Keiko Gengyo-Ando, Hidehito Kuroyanagi, Tetsuo Kobayashi, Motohide Murate, Kazushi Fujimoto, Shigeo Okabe, Shohei Mitani

EMBO REPORTS ( NATURE PUBLISHING GROUP ) 8 ( 2 ) 152 - 157 2007年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Rab5, a small guanosine triphosphatase, is known to regulate the tethering and docking reaction leading to SNARE (soluble NSF attachment protein receptors)-mediated fusion between endosomes. However, it is uncertain how the signal of the activated Rab5 protein is transduced by its downstream effectors during endosome fusion. Here, we show that the Sec1/Munc18 gene vps-45 is essential for not only viability and development but also receptor-mediated and fluid-phase endocytosis pathways in Caenorhabditis elegans. We found that VPS-45 interacts with a Rab5 effector, Rabenosyn-5 (RABS-5), and the mutants of both vps-45 and rabs-5 show similar endocytic phenotypes. In the macrophage-like cells of vps-45 and rabs-5 mutants, aberrantly small endosomes were accumulated, and the endosome fusion stimulated by the mutant RAB-5 Q780 is suppressed by these mutations. Our results indicate that VPS-45 is a key molecule that functions downstream from RAB-5, cooperating with RABS-5, to regulate the dynamics of the endocytic system in multicellular organisms.

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Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

Hidehito Kuroyanagi, Tetsuo Kobayashi, Shohei Mitani, Masatoshi Hagiwara

NATURE METHODS ( NATURE PUBLISHING GROUP ) 3 ( 11 ) 909 - 915 2006年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Alternative splicing of pre-mRNAs allows multicellar organisms to create a huge diversity of proteomes from a finite number of genes. But extensive studies in vitro or in cultured cells have not fully explained the regulation mechanisms of tissue-specific or developmentally regulated alternative splicing in living organisms. Here we report a transgenic reporter system that allows visualization of expression profiles of mutually exclusive exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A and 5B showed tissue-specific profiles, and we isolated mutants defective in the tissue specificity. We identified alternative-splicing defective-1 (asd-1), encoding a new RNA-binding protein of the evolutionarily conserved Fox-1 family, as a regulator of the egl-15 reporter. Furthermore, an asd-1; fox-1 double mutant was defective in the expression of endogenous egl-15 (5A) and phenocopied egl-15 (5A) mutant. This transgenic reporter system can be a powerful experimental tool for the comprehensive study of expression profiles and regulation mechanisms of alternative splicing in metazoans.

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Impaired cell cycle control of neuronal precursor cells in the neocortical primordium of presenilin-1-deficient mice

S Yuasa, M Nakajima, H Aizawa, N Sahara, KI Koizumi, T Sakai, M Usami, SI Kobayashi, H Kuroyanagi, H Mori, H Koseki, T Shirasawa

JOURNAL OF NEUROSCIENCE RESEARCH ( WILEY-LISS ) 70 ( 3 ) 501 - 513 2002年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Recent studies have implicated presenilin-1 (PS-1) in the processing of the amyloid precursor protein and Notch-1. We show that PS-1 has biological effects on differentiation and cell cycle control of neuronal precursor cells in vivo using PS-1-deficient mice. The expression of Class III beta-tubulin was upregulated throughout the neocortical primordia of PS-1-deficient El 4 embryos, especially on the ventricular surface. The increased speed of migration of the immature neurons from the ventricular zone outward in the PS-1-deficient neocortical primordia was indicated by an in vivo bromodeoxyuridine (BrdU)-labeling assay and a Dil-labeling assay in slice culture. Furthermore, we investigated the cell cycle of neuronal precursor cells in the neocortical ventricular zone using an in vivo cumulative BrdU-Iabeling assay. The length of the cell cycle in the neocortical precursor cells of wild-type mice was 11.4-hr whereas that of the PS-1-deficient mice was 15.4 hr. Among all phases of the cell cycle, S-phase exhibited the most prominent change in length, increasing from 2.4 hr in the wild-type mice to 7.4 hr in the mutant mice. The distribution of beta-catenin was specifically affected in the ventricular zone of the PS-1-deficient mice. These findings suggest that PS-1 is involved in the differentiation and the cell cycle control of neuronal precursor cells in the ventricular proliferating zone of the neocortical primordium. (C) 2002 Wiley-Liss, Inc.

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Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

D Nakai, S Yuasa, M Takahashi, T Shimizu, S Asaumi, K Isono, T Takao, Y Suzuki, H Kuroyanagi, K Hirokawa, H Koseki, T Shirsawa

Biochem. Biophys. Res. Commun. 289 ( 2 ) 463 - 471 2001年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to t

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Mouse coq7/clk-1 orthologue rescued slowed rhythmic behavior and extended life span of clk-1 longevity mutant in Caenorhabditis elegans.

M Takahashi, S Asaumi, S Honda, Y Suzuki, D Nakai, H Kuroyanagi, T Shimizu, Y Honda, T Shirasawa

Biochem. Biophys. Res. Commun. 286 ( 3 ) 534 - 540 2001年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The coq7/clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a regulatory role in biological rhythm and longevity. The mouse COQ7 is homologous to Saccharomyces cerevisiae COQ7/CAT5 that is required for the biosynthesis of coenzyme Q (ubiquinone), an essential messenger in mitochondrial respiration. In the present study, we characterized the expression and processing of mouse COQ7. We found that COQ7 is highly expressed in tissues with high energy demand such as heart, muscle, liver, and kidney in mice. Biochemical analysis revealed that COQ7 is targeted to mitochondria where it is processed to mature form. Transgenic expression of mouse coq7 completely rescued the slowed rhythmic behaviors of clk-1 such as defecation. In life-span analysis, transgenic expression reverted the extended life span of clk-1 to the comparable level with wild-type control. These data strongly suggested that coq7 plays a pivotal role in the regulation of biological rhythms and the determination of life span in mammalian species.

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Aberrant synaptic transmission in the hippocampal CA3 region and cognitive deterioration in protein-repair enzyme-deficient mice

Y Ikegaya, M Yamada, T Fukuda, H Kuroyanagi, T Shirasawa, N Nishiyama

HIPPOCAMPUS ( WILEY-LISS ) 11 ( 3 ) 287 - 298 2001年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

L-aspartate is the amino-acid residue most susceptible to spontaneous isomerization. This denaturation causes an alteration in the biological activity of the protein and is regarded as an aging process of the protein. Protein L-isoaspartyl methyltransferase (PIMT) repairs this posttranslational modification and thus is implicated in retarding the aging process of proteins. PIMT is highly expressed in the brain, and its deficiency results in progressive epilepsy after 4 weeks of age, with a fatal seizure in mice. Here we report the pathophysiological role of this repair system in the hippocampal slice of PIMT-deficient mice. The hippocampal mossy fiber-CA3 synapses of PIMT-deficient mice showed hyperexcitation that was repressed by a gamma -aminobutyric acid (GABA)(A) receptor agonist muscimol. In addition, the mossy fiber-CA3 synapses failed to show long-term potentiation or paired-pulse facilitation. No abnormality, however, was observed in Schaffer collateral-CA1 synapses or in perforant path-dentate gyrus synapses. Electron microscopic study revealed aberrant distribution of synaptic vesicles in the mossy fiber terminals and vacuolar degeneration at the axon hillock of dentate granule cells in PIMT-deficient mice. Furthermore, the PIMT-deficient mice showed impaired spatial memory in Morris water maze test and exhibited fewer anxiety-related behaviors in the elevated-plus test. These results suggest that the mossy fiber-CA3 system is vulnerable to aspartate isomerization and that the PIMT-mediated repair system is essential for maintenance of normal functions of the hippocampus. Hippocampus 2001;11:287-298. (C) 2001 Wiley-Liss, Inc.

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SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development.

H Kuroyanagi, T Kimura, K Wada, N Hisamoto, K Matsumoto, M Hagiwara

Mech. Dev. 99 ( 1-2 ) 51 - 64 2000年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.

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A mouse serine/threonine kinase homologous to C-elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons

T Tomoda, RS Bhatt, H Kuroyanagi, T Shirasawa, ME Hatten

NEURON ( CELL PRESS ) 24 ( 4 ) 833 - 846 1999年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The formation of the cerebellar circuitry depends on the outgrowth of connections between the two principal classes of neurons, granule neurons and Purkinje neurons. To identify genes that function in axon outgrowth, we have isolated a mouse homolog of C. elegans UNC51, which is required for axon formation, and tested its function in cerebellar granule neurons. Murine Unc51.1 encodes a novel serine/threonine kinase and is expressed in granule cells in the cerebellar cortex. Retroviral infection of immature granule cells with a dominant negative Unc51.1 results in inhibition of neurite outgrowth in vitro and in vivo. Moreover, infected neurons fail to express TAG-1 or neuron-specific beta-tubulin, suggesting that development is arrested prior to this initial step of differentiation. Thus, Unc51.1 signals the program of gene expression leading to the formation of granule cell axons.

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Mouse ULK2, a novel member of the UNC-51-like protein kinases: unique features of functional domains

J Yan, H Kuroyanagi, T Tomemori, N Okazaki, K Asato, Y Matsuda, Y Suzuki, Y Ohshima, S Mitani, Y Masuho, T Shirasawa, M Muramatsu

ONCOGENE ( STOCKTON PRESS ) 18 ( 43 ) 5850 - 5859 1999年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identified mouse and human cDNAs encoding UNC-51-like kinase (ULK1), Here we report the identification and characterization of the second murine member of this kinase family, ULK2, Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of similar to 150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a species-specific manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.

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Orthologues of the Caenorhabditis elegans longevity gene clk-1 in mouse and human

S Asaumi, H Kuroyanagi, N Seki, T Shirasawa

GENOMICS ( ACADEMIC PRESS INC ) 58 ( 3 ) 293 - 301 1999年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al, 1997, Science 275: 980-983), The primary structure of CLK-1 showed a significant homology 60 Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes. In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene, Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively. Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C. elegans CLK-1, and 37% identity to S. cerevisiae Coq7p/Cat5p. Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes. Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle. Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene. Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3-p13.11. (C) 1999 Academic Press.

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Human ULK1, a novel serine/threonine kinase related to UNC-51 kinase of Caenorhabditis elegans: CDNA cloning, expression, and chromosomal assignment

H Kuroyanagi, J Yan, N Seki, Y Yamanouchi, Y Suzuki, T Takano, M Muramatsu, T Shirasawa

GENOMICS ( ACADEMIC PRESS INC ) 51 ( 1 ) 76 - 85 1998年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The unc-51 gene, isolated from mutants of Caenorhabditis elegans exhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura, et at, 1994, Genes Dev. 8: 2389-2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pi of 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p of Saccharomyces cerevisiae. Phylogenetic analysis of ULK1, UNC-51, and Apg1p suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that the ULK1 gene spans 30-40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system of C. elegans. Both FISH and RH mapping confirmed the regional localization of ULK1 to human chromosome 12q24.3. (C) 1998 Academic Press.

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Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51.

J Yan, H Kuroyanagi, A Kuroiwa, Y Matsuda, H Tokumitsu, T Tomoda, T Shirasawa, M Muramatsu

Biochem. Biophys. Res. Commun. 246 ( 1 ) 222 - 227 1998年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

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cDNA cloning, expression, subcellular localization, and chromosomal assignment of mammalian aurora homologues, aurora-related kinase (ARK) 1 and 2.

M Shindo, H Nakano, H Kuroyanagi, T Shirasawa, M Mihara, D J Gilbert, N A Jenkins, N G Copeland, H Yagita, K Okumura

Biochem. Biophys. Res. Commun. 244 ( 1 ) 285 - 292 1998年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Chromosomal segregation during mitosis as well as meiosis is considered to be regulated by multiple kinases, but the precise mechanism remains largely unknown. A mutation in Drosophila, designated aurora, was identified as a responsible gene for a chromosomal segregation defect and encodes a putative serine-threonine kinase. Here we have identified mammalian aurora homologues, designated aurora-related kinase (ARK) 1 and ARK2. Kinase domains of murine ARK1 and ARK2 showed 61 and 62% identity, respectively, to that of aurora at the amino acid levels, respectively. Cell cycle analysis revealed that the expression of ARK1 was correlated with G2/M phase, while ARK2 was expressed during S and G2/M phases. Immunofluorescence analysis demonstrated that ARK2 was mainly localized to the midbody, while ARK1 has been reported to be localized to the spindle pole during mitosis. Collectively, these results suggest that these two kinases may have distinct roles with different expression timing and subcellular localization during the cell cycle progression. Interspecific backcross mapping revealed that Ark1 is located in a distal region of mouse chromosome 2, while Ark2 is located in a central re

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Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy.

A Yamamoto, H Takagi, D Kitamura, H Tatsuoka, H Nakano, H Kawano, H Kuroyanagi, Y Yahagi, S Kobayashi, K Koizumi, T Sakai, K Saito, T Chiba, K Kawamura, K Suzuki, T Watanabe, H Mori, T Shirasawa

J. Neurosci. 18 ( 6 ) 2063 - 2074 1998年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. We generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biological role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 weeks of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartatate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 weeks of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. We conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.

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Induction of protein tyrosine phosphatase epsilon transcripts during NGF-induced neuronal differentiation of PC12D cells and during the development of the cerebellum.

Y Mukouyama, H Kuroyanagi, T Shirasawa, T Tomoda, D Saffen, M Oishi, T Watanabe

Brain Res. Mol. Brain Res. 50 ( 1-2 ) 230 - 236 1997年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

We have investigated a possible role played by protein tyrosine phosphatase epsilon (PTPepsilon), which was recently cloned and predominantly expressed in brain, in neural differentiation and function. During neuronal cell differentiation of PC12D cells triggered by NGF or FGF, PTPepsilon transcripts were transiently induced at a time between the appearance of transcripts for immediate-early genes and for neuronal cell-specific markers. PTPepsilon was the only PTPase whose transcripts were induced during PC12D cell differentiation among over two dozen PTPase transcripts so far examined. Moreover, in situ hybridization revealed that PTPepsilon transcripts were detected in the neural tube of day 12 postcoitum embryo, and in the nervous system including brain, spinal cord, and ganglions in a ubiquitous manner in late gestational stages. In 4-day-old neonatal mice, the transcripts were widely distributed in the central nervous system where the strongest expression was detected in the hippocampus, cerebral cortex, and olfactory bulb. Interestingly, in day 7 and 16 neonatal brains, the strongest PTPepsilon gene expression was localized in the granular cells of cerebellum, which might ind

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Glutamate dehydrogenase mRNA is immediately induced after phencyclidine treatment in the rat brain.

E Shimizu, H Shirasawa, K Kodama, H Kuroyanagi, T Shirasawa, T Sato, B Simizu

Schizophr. Res. 25 ( 3 ) 251 - 258 1997年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c-fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia.

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Identification of a receptor-type protein tyrosine phosphatase expressed in postmitotic maturing neurons: its structure and expression in the central nervous system.

M Tagawa, T Shirasawa, Y Yahagi, T Tomoda, H Kuroyanagi, S Fujimura, S Sakiyama, N Maruyama

Biochem. J. 321 ( Pt 3) 865 - 871 1997年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPphi expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hi

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Induction, localization, and purification of a novel sialidase, deaminoneuraminidase (KDNase), from Sphingobacterium multivorum.

S Nishino, H Kuroyanagi, T Terada, S Inoue, Y Inoue, F A Troy, K Kitajima

J. Biol. Chem. 271 ( 6 ) 2909 - 2913 1996年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNalpha2-->3Galbeta1-->3GalNAc alpha1-->3[KDNalpha2--> (8KDN alpha2-->)n-->6]GalNAcol, as a sole carbon source induced KDNase SM activity 15 40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to det

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Discovery of a new type of sialidase, "KDNase," which specifically hydrolyzes deaminoneuraminyl (3-deoxy-D-glycero-D-galacto-2-nonulosonic acid) but not N-acylneuraminyl linkages.

K Kitajima, H Kuroyanagi, S Inoue, J Ye, F A Troy, Y Inoue

J. Biol. Chem. 269 ( 34 ) 21415 - 21419 1994年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

The release of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN, deaminoneuraminic acid) residues from their alpha-ketosidic linkage is required to determine the structural and functional role of KDN-glycoconjugates in sources as disparate as trout egg polysialoglycoproteins and human cancers. We report for the first time the isolation and characterization of a novel type of sialidase (KDNase), which specifically hydrolyzes KDN ketosidic but not N-acylneuraminyl linkages. KDNase activity was assayed using 4-methylumbelliferyl KDN (4-MU-KDN). A KDNase-producing microorganism was identified as Sphingobacterium multivorum. The affinity-purified enzyme was designated KDNase SM to denote its origin and that it was free of N-acylneuraminidase, proteolytic, and other glycosidase activities. KDNase SM activity toward 4-MU-KDN was not inhibited by the N-acylneuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. KDNase SM released free KDN from naturally occurring substrates, including (KDN)GM3, KDN-glycoprotein, which bears a number of O-linked chains of KDN alpha 2-->3Gal beta 1-->3GalNAc alpha 1-->3 (KDN alpha 2-->(-->8KDN alpha 2-->)n-->6)GalNAc alpha 1-->, and the bian

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<黒柳 秀人>

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<黒柳　秀人>

論文 - 黒柳　秀人