Clk Kinases and Splicing Regulation

项目概要/摘要： 虽然剪接位点的适当选择驱动基因组多样性，适应性生长和 发展，拼接错误可能对功能产生巨大的不利影响，现在 被认为是许多人类疾病的根本原因。事实上，剪接错误与 肌肉萎缩症、阿尔茨海默病、帕金森综合症、精神疾病、共济失调和 癌症使得研究控制剪接位点选择的因素对人类健康至关重要。 剪接发生在剪接体上，剪接体是由几种RNA组成的大分子复合物， 许多蛋白质。正常基因剪接的关键是正确选择5 '-3'剪接位点， 在剪接体发育早期发生的事件，其特异性由一个 一个重要的剪接因子家族，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，但我们对这些基因如何表达的理解， 不同的形式，充其量是不完整的。蛋白激酶的CLK家族磷酸化 SR蛋白并将它们移动到活性基因剪接位点。CLK不同于许多经典的 蛋白激酶，因为它们缺乏用于底物结合的对接槽，而是含有无序的 我们显示的N-末端延伸连接到SR蛋白。在本建议书中，我们将探讨 N-末端对细胞核中CLK 1的移动和SR蛋白的识别的作用， 大量的体内和体外实验。我们将研究依赖于CLK的 磷酸化对SR蛋白构象、亚细胞定位以及与关键蛋白的相互作用的影响。 剪接体中剪接位点选择的介体。这项建议的更大目标是界定时钟 在生物和生物化学水平上发挥作用，以便我们能够更好地了解 与剪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.