Manipulation of aberrant splicing.

最近的全基因组序列分析表明，蛋白质组的高度复杂性是由有限数量的基因实现的。这一令人惊讶的发现强调了选择性剪接的重要性，通过这种剪接，单个基因可以产生结构和功能上不同的蛋白质亚型。基于全基因组分析，75%的人类基因被认为编码至少两种可供选择的剪接异构体。剪接位点使用的调控为控制基因表达和蛋白质组多样性的产生提供了一种广泛的机制，在许多生物学过程中发挥着重要作用，如胚胎发育、细胞生长和细胞凋亡。针对CLK1和CLK4的激酶抑制剂苯并噻唑化合物TG003可以抑制核斑点的解离，改变剪接模式，并挽救由于CLK活性过高而导致的胚胎缺陷。新出现的调节前信使核糖核酸替代SP…的信号转导通路的抑制物更多的舔可能会为治疗由错配引起的疾病开辟道路。我们最近发现CLK参与了胰岛素依赖的选择性剪接。我们发现了一个新的富含丝氨酸/精氨酸(SR)的蛋白作为缺乏激酶活性的Clk4突变体的结合伙伴，并将其命名为CLASP(Clk4-Associating SR-Related Protein)。CLASP是CLK4的结合伙伴，可能参与了CLK激酶家族活性的调节。CLASP基因在脑中高表达，在海马区、小脑和嗅球中均有表达。两种形式的卡环是通过交替拼接后的框架移位产生的。短型卡环(卡环-S)染色呈核质图案，而长型卡环(卡环-L)则定位于核点。CLASPL的过表达促进了CLK1的CR-1和CR-2前mRNA的EB外显子包涵体。我们目前正在研究CLAP在神经细胞存活中的作用。我们开发了一种转基因选择性剪接报告系统，可以在体内的单细胞水平上可视化线虫相互排斥的选择性外显子的表达谱。我们分离了报告基因组织特异性表达缺陷的突变虫子，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内源基因的选择性剪接。这些结果表明，报告蠕虫系统可以用来分析选择性剪接异构体的表达谱，并识别反式作用因子和顺式作用元件。此外，我们的结果还表明，组织特异性选择性剪接事件的调节因子和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，但它编码一系列广泛的蛋白质。该病毒利用宿主RNA处理机制来提供表达这种蛋白质组多样性所需的选择性剪接能力。富含丝氨酸的精氨酸(SR)蛋白和激活它们的激酶是这种选择性剪接机制的核心。我们最初开发了SR蛋白磷酸化抑制物340(SRPIN340)，它优先抑制SRPK1和SRPK2，并下调SRp75。SRPIN340抑制HIV、Sindbis病毒、SARS病毒和巨细胞病毒的增殖，提示它们可能需要依赖SRPK的SR蛋白磷酸化才能增殖。较少

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