Clk Kinases and Splicing Regulation

项目摘要/摘要： 虽然剪接位点的适当选择促进了基因组多样性，但适应性生长和 在发展中，拼接错误会对功能产生巨大的有害影响，现在 被认为是许多人类疾病的根本原因。事实上，拼接错误与 患有肌营养不良症、阿尔茨海默病、帕金森氏症、精神障碍、共济失调和 癌症使得控制剪接位点选择的因素的研究对人类健康至关重要。 剪接发生在剪接体上，剪接体是由几个RNA和 大量的蛋白质。对正常的基因剪接至关重要的是正确选择5‘-3’剪接位点， 发生在剪接体发育早期的事件，其特异性由 必需的剪接因子家族，称为SR蛋白。SR蛋白的磷酸化状态 直接影响它们的亚细胞定位和剪接活动，但我们对这些 获得不同的形式充其量也是不完整的。蛋白激酶CLK家族的磷酸化 SR蛋白，并将它们动员到活跃的基因剪接部位。CLK不同于许多经典的 蛋白激酶，因为它们缺少底物结合的对接凹槽，而是含有无序的 我们展示的N-末端延伸连接到SR蛋白上。在本提案中，我们将探讨 N-末端在CLK1动员和核内SR蛋白识别中的作用 广泛的体内和体外实验。我们将研究CLK依赖的影响 SR蛋白构象的磷酸化、亚细胞定位以及与关键蛋白的相互作用 剪接体中剪接位选择的介体。这项提议的更大目标是定义CLK 在生物和生化水平上发挥作用，以便我们能够更好地了解 与拼接错误有关的人类疾病。

Project Summary/Abstract: While the proper selection of splice sites drives genomic diversity, adaptive growth and development, errors in splicing can have enormous detrimental effects on function and is now recognized as the underlying cause for many human diseases. Indeed, splicing errors are associated with muscular dystrophy, Alzheimer's disease, Parkinsonism, psychiatric disorders, ataxias and cancers making the study of factors that control splice-site selection vitally important for human health. Splicing occurs at the spliceosome, a macromolecular complex composed of several RNAs and numerous proteins. Critical to normal gene splicing is the proper selection of the 5'-3' splice sites, events that occur early in the development of the spliceosome and whose specificity is guided by an essential family of splicing factors known as SR proteins. The phosphorylation states of SR proteins directly impact their subcellular localization and splicing activities but our understanding of how these different forms are attained is, at best, incomplete. The CLK family of protein kinases phosphorylates SR proteins and mobilizes them to sites of active gene splicing. The CLKs differ from many classic protein kinases in that they lack a docking groove for substrate binding but instead contain a disordered N-terminal extension that we showed attaches to the SR protein. In this proposal we will explore the role of the N-terminus for the mobilization of CLK1 and recognition of SR proteins in the nucleus using a wide array of in vivo and in vitro experiments. We will study the effects of CLK-dependent phosphorylation on SR protein conformation, subcellular localization, and interactions with critical mediators of splice-site selection in the spliceosome. The larger goal of this proposal is to define CLK function at both biological and biochemical levels so that we can better understand the mechanisms of human diseases associated with errors in splicing.