Phosphoiylation -dependent regulation of alternative splicing

选择性剪接的磷酸化依赖性调控

基本信息

批准号：
14380326
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：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380326/
关键词：
alternative splicing Phosphorylation SR proteins SRPK Clk mRNA processing protein kinase inhibitor Clk 特異的阻害剤

项目摘要

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The selection of splice site can be altered by numerous extracellular stimuli, including growth factors, cytokines, hormones, depolarization, osmotic shock, and UVC irradiation, through synthesis, phosphorylation, and a change in localization of serine/arginine-rich (SR) proteins. SR proteins are a family of essential factors required for constitutive splicing of pre-mRNA and play an important role in modulating alternative splicing. They are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs (RRMs) at the amino terminus and an RS domain at the carboxy terminus.Phosphorylation state of SR proteins appears to influence their activities in general and alternative splicing. To date, several kinases have been reported to phosphorylate SR proteins, … More including SRPK-familykinases (SRPK1 and SRPK2), Clk/Sty family kinases, which consisted of four members (Clkl/Sty and Clk2-4), DNA topoisomerase I, p34cdc2 kinase and hPRP4, Addition of purified SRPK1 to permeabilized cells, or overexpression of SRPK1, 2 or one of CIk family members in transfected cells result in an apparent disassembly of the nuclear speckles.The kinase inhibitor DRB (5,6-dichloro-1-β-D-ribo-furanosylbenzimidazole), which was previously shown to inhibit casein kinase II, inhibited Clk2 and induced the redistribution of SR proteins. Recently, through extensive screening of a chemical library, a specific inhibitor of Clkl/Sty and Clk4 was found and named as TG003. Clk 1/Sty-dependent splicing and disassembly of nuclear speckles were suppressed by TG003. Moreover, administration of TG003 rescued the embryonic defects induced by excessive Clk/Sty activity in Xen opus. As the importance of alternative splicing has been illustrated by the increasing number of human diseases attributed to missplicing events, TGOO3 will be applicable for the therapeutic manipulation of.abnormal splicing induced by activated Clk/Sty.Considering the therapeutic application of inhibitors of SR protein kinases, prevention of virus is one of hopeful fields, because splicing is a crucial step for virus multiplication. In the case of HIV, 8 acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat and rev mRNAs, and the usage of these splicing sites are regulated by SR proteins and hnRNPs. In addition, Akusjarvi group reported that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. The anti-virus effects of inhibitors of SR protein kinases are now under investigation in our laboratory. Less

剪接位点使用的调节提供了控制基因表达和蛋白质组多样性产生的通用机制，在许多生物过程中发挥着重要作用。剪接位点的选择可以通过许多细胞外刺激来改变，包括生长因子、细胞因子、通过合成、磷酸化和富含丝氨酸/精氨酸 (SR) 蛋白的定位变化，激素、去极化、渗透压休克和 UVC 照射是 SR 蛋白所需的一系列重要因素。它们在真核生物中高度保守，其特点是在氨基末端有一个或两个 RNA 识别基序 (RRM)，在羧基末端有一个 RS 结构域。 SR 蛋白的磷酸化状态似乎会影响其一般剪接和选择性剪接的活性。迄今为止，据报道有几种激酶可以磷酸化 SR 蛋白，……更多包括。 SRPK 家族激酶（SRPK1 和 SRPK2）、由四个成员（Clk1/Sty 和 Clk2-4）开始的 Clk/Sty 家族激酶、DNA 拓扑异构酶 I、p34cdc2 激酶和 hPRP4、向透化细胞中添加纯化的 SRPK1，或过表达转染细胞中的 SRPK1、2 或 CIk 家族成员之一会导致激酶抑制剂DRB（5,6-二氯-1-β-D-核糖基苯并咪唑）先前被证明可以抑制酪蛋白激酶II，最近通过广泛筛选，可以抑制Clk2并诱导SR蛋白的重新分布。在化学文库中，发现了 Clk1/Sty 和 Clk4 的特异性抑制剂，并将其命名为 Clk 1/Sty 依赖性剪接和分解。 TG003 抑制了核斑点的发生，此外，TG003 的施用挽救了 Xen opus 中由过度的 Clk/Sty 活性引起的胚胎缺陷。 TGOO3将适用于由激活的Clk/Sty诱导的异常剪接的治疗操作。考虑到SR蛋白激酶抑制剂的治疗应用，预防病毒是其中之一充满希望的领域，因为剪接是病毒增殖的关键步骤，就 HIV 而言，8 个受体位点竞争性地产生 vif、vpu、vpr、nef、env、tat 和 rev mRNA，以及这些的用途。剪接位点受 SR 蛋白和 hnRNP 调节。此外，Akusjarvi 小组报道，从晚期腺病毒感染的细胞中纯化的 SR 蛋白作为剪接增强子或被失活。通过病毒诱导的去磷酸化来剪接阻遏蛋白 SR 蛋白激酶抑制剂的抗病毒作用目前正在我们的实验室中进行研究。