Manipulation of aberrant splicing.

异常剪接的操纵。

• 批准号: 16390074
• 负责人: HAGIWARA Masatoshi
• 金额: $ 9.6万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390074/
• 关键词: alternative splicing; mRNA; Clk; CLASP; virus; SRPK; SRPIN340; 抗ウイルス薬; インシュリン; Clk1; 4; TG003; SR蛋白質; Sty; 蛋白リン酸化酵素阻害剤

Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes. This surprising finding underscores the importance of alternative splicing, through which a single gene can generate structurally and functionally distinct protein isoforms. Based on genome wide analysis, 75% of human genes are thought to encode at least two alternatively spliced isoforms. The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis.A benzothiazole compound TG003, a kinase inhibitor that targets Clk1 and Clk4, suppressed dissociation of nuclear speckles, altered the splicing patterns, and rescued the embryonic defects induced by excessive Clk activity. The emerging inhibitors of the signal transduction pathways regulating pre-mRNA alternative sp … More licing may open the way to therapies against diseases caused by miss-splicing. We recently found that Clk is involved in the insulin-dependent alternative splising.We identified a novel serine/arginine (SR)-rich-related protein as a binding partner of Clk4 mutant lacking kinase activity and designated it CLASP (Clk4-associating SR-related protein). CLASP is a binding partner of Clk4 and may be involved in the regulation of the activity of Clk kinase family. CLASP mRNA was highly expressed in brain, and both CLASP and Clk4 mRNAs were expressed in the hippocampus, the cerebellum, and the olfactory bulb. Two forms of CLASP were produced by a frameshift following alternative splicing. The staining of a short form of CLASP (CLASP-S) showed a nucleoplasmic pattern, while the long form of CLASP (CLASP-L) was localized as nuclear dots. Overexpression of CLASPL promoted exon EB inclusion from CR-1 and CR-2 pre-mRNA of Clk1. We are currently studying the role of CLASP on the survival of neuronal cells.We have developed a transgenic alternative splicing reporter system that visualizes expression profiles of mutually exclusive alternative exons of a nematode C.elegans at a single cell level in vivo. We isolated mutant worms defective in the tissue-specific expression of the reporter and identified a novel evolutionarily conserved trans-acting factor, ASD-1 (alternative-splicing-defective-1), which also regulated tissue-specific alternative splicing of an endogenous gene. These results indicate that the reporter worm system can be used to analyze expression profiles of the alternative splicing isoforms, and to identify trans-acting factors and cis-acting elements. Furthermore, our results also demonstrated that regulation factors and elements of the tissue-specific alternative splicing events are conserved in vertebrates and nematodes.Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. We originally developed SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. SRPIN340 suppressed propagation of HIV, Sindbis virus, SARS virus, and cytomegalovirus, suggesting that they may require SRPK-dependent SR protein phosphorylation for their multiplication. Less

最近的整个基因组序列分析表明，使用数量有限的基因实现了高度的蛋白质组学复杂性。这个令人惊讶的发现强调了替代剪接的重要性，单个基因可以在结构和功能上不同的蛋白质同工型中产生。基于基因组广泛的分析，人们认为75％的人基因至少编码了两个剪接的同工型。剪接场所使用的调节提供了一种用于控制基因表达和产生蛋白质组多样性的多功能机制，在许多生物学过程中扮演着重要的作用，例如胚胎发育，细胞生长和凋亡。苯甲唑化合物TG003，一种针对CLK1的splk1和clkk4的模式，抑制了核分化的核能，并抑制了核分化，并抑制了核分化，并抑制了核分化的效果。过量CLK活性引起的胚胎缺陷。信号转导途径的新兴抑制剂调节前MRNA替代性SP…更多的许可可能为通过错过而导致的疾病疗法开辟了道路。我们最近发现，CLK参与了胰岛素依赖性替代分裂。我们确定了一种新型的丝氨酸/精氨酸（SR）与富含激酶的结合伴侣，它是缺乏激酶活性的Clk4突变体的结合伴侣，并指定了IT CLASP（CLK4-CORAPOCIATIC SR相关的SR相关蛋白）。扣子是CLK4的结合伙伴，可能参与了clk激酶家族活性的调节。扣子mRNA在大脑中高度表达，cLASP和CLK4 mRNA在海马，小脑和嗅球中表达。替代剪接后，通过框架进行了两种形式的扣子。短形式的扣子（clasp-s）的染色显示出核浆模式，而CLASPL的长形扣（clasp-l）过表达促进了从Crk1的Cr-1和Cr-2 Pre-MRNA促进外显子EB纳入。我们目前正在研究扣子在神经元细胞存活中的作用。我们已经开发了一个转基因替代剪接报告基因系统，该系统可视化线虫的相互排斥替代外显子的表达谱。我们分离了报告基因的组织特异性表达中有缺陷的突变虫，并鉴定出一种新型的进化构成的反式作用因子ASD-1（替代性 - 替代缺陷1），该因子还调节了内源基因的组织特异性替代剪接。这些结果表明，报告基因蠕虫系统可用于分析替代剪接同工型的表达谱，并鉴定跨作用因子和顺式作用元件。此外，我们的结果还表明，组织特异性替代剪接事件的调节因子和元素在脊椎动物和线虫中保守。尽管病毒基因组通常很小，但它编码了一系列广泛的蛋白质。该病毒利用宿主-RNA处理机制提供了表达这种蛋白质组学多样性所需的替代剪接能力。丝氨酸 - 精氨酸（SR）蛋白和激活它们的激酶对于这种替代剪接机制至关重要。我们最初开发了SR蛋白磷酸化抑制剂340（SRPIN340），该抑制剂优先抑制SRPK1和SRPK2，并下调SRP75。 SRPIN340抑制了HIV，Sindbis病毒，SARS病毒和巨细胞病毒的传播，这表明它们可能需要SRPK依赖性的SR蛋白磷酸化来进行繁殖。较少的

项目成果

A spliceosomal intron-binding protein, IBP160, links position-dependent assembly of intorn-encoded box C/C snoRNP to pre-mRNA splicing.

剪接体内含子结合蛋白 IBP160 将 intorn 编码的盒 C/C snoRNP 的位置依赖性组装与前 mRNA 剪接连接起来。

• 发表时间: 2006
• 期刊: Mol. Cell 23(5)
• 作者: 三輪 正直;金居 正幸;内田 真啓;花井修次;Fukuhara T;Hirose T
• 通讯作者: Hirose T

A novel histone-exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A/H2B.

一种新型组蛋白交换因子，蛋白磷酸酶 2Cγ，介导 H2A/H2B 的交换和去磷酸化。

• 发表时间: 2006
• 期刊: Journal of Cell Biology 175
• 作者: Kline;S.;Cheeseman;I.M.;Hori;T.;Fukagawa;T.;Desai;A.;Masahiro Okada;Hiroshi Kimura
• 通讯作者: Hiroshi Kimura

A transgenic reporter reveals cell-type-specific expression profiles and regulation mechanisms of alternatively-spliced exons in vivo.

转基因报告基因揭示了细胞类型特异性表达谱和体内可变剪接外显子的调节机制。

• 发表时间: 2006
• 期刊: Nature Methods 3
• 作者: Kuroyanagi;H
• 通讯作者: H