論文 - 黒柳　秀人

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）

　概要を見る

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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

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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）

　概要を見る

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）

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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）

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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）

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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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