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

最近的全基因组序列分析表明，通过有限数量的基因实现了高度的蛋白质组复杂性，这一令人惊讶的发现强调了选择性剪接的重要性，通过该剪接，单个基因可以基于全基因组产生结构和功能不同的蛋白质亚型。分析表明，75%的人类基因被认为编码至少两种选择性剪接异构体，剪接位点使用的调节为控制基因表达和蛋白质组多样性的产生提供了一种通用机制，在许多生物学中发挥着重要作用。苯并噻唑化合物 TG003 是一种针对 Clk1 和 Clk4 的激酶抑制剂，可抑制核斑点的解离，改变剪接模式，并挽救由过度的 Clk 活性引起的胚胎缺陷。调节前体 mRNA 替代 sp 的信号转导途径的抑制剂可能为治疗错误剪接引起的疾病开辟道路。我们鉴定了一种新的富含丝氨酸/精氨酸 (SR) 的相关蛋白作为缺乏激酶活性的 Clk4 突变体的结合伴侣，并将其命名为 CLASP（Clk4 相关 CLASP）。 CLASP mRNA 是 Clk4 的结合伴侣，可能参与 Clk 激酶家族活性的调节，并且 CLASP 和 Clk4 mRNA 在脑中均表达。海马、小脑和嗅球通过选择性剪接后的移码产生两种形式的 CLASP (CLASP-S) 的染色显示核质模式，而长形式的 CLASP (CLASP-) 的染色显示核质模式。 L) 定位为核点，CLASPL 的过表达促进了 Clk1 的 CR-1 和 CR-2 前 mRNA 的外显子 EB 包含。我们目前正在研究 CLASP 对神经元细胞存活的作用。我们开发了一种转基因选择性剪接报告系统，可以在体内单细胞水平可视化线虫相互排斥的选择性外显子的表达谱。线虫报告基因的组织特异性表达有缺陷，并鉴定出一种新的进化保守的反式作用因子 ASD-1（选择性剪接缺陷-1），它也调节组织特异性这些表明报告蠕虫系统可用于分析选择性剪接异构体的表达谱，并鉴定反式作用因子和顺式作用元件。此外，我们的结果还证明了调节因子。组织特异性选择性剪接事件的元件在脊椎动物和线虫中是保守的。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。宿主 RNA 加工机制提供表达这种蛋白质组多样性 (SR) 所需的选择性剪接能力，而激活它们的激酶是这种选择性剪接机制的核心。抑制剂 340 (SRPIN340)，优先抑制 SRPK1 和 SRPK2 并下调 SRp75，抑制 SRPIN340 的增殖。 HIV、辛德比斯病毒、SARS 病毒和巨细胞病毒，表明它们的增殖可能需要 SRPK 依赖性 SR 蛋白磷酸化。

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