Coordination of SR Protein Phosphorylation and RNA Splicing

项目摘要/摘要： 对人类和低等生物基因组的比较表明， 人类的复杂性不是通过基因数量的急剧增加实现的，而是通过 可选择的剪接事件将基因的不同部分连接在一起以产生不同的 蛋白质。正确的拼接允许正常的健康功能，但错误的拼接会链接 对许多人类疾病都有影响。例如，肌肉营养不良，共济失调，帕金森氏症， 神经纤维瘤病、精神疾病和癌症都源于拼接错误。 剪接反应是由一个大型的大分子机器催化的，称为 剪接体。剪接体由RNA和蛋白质组成，可以准确地选择 健康细胞中相当大的前体mRNA的适当剪接位置。该组件 剪接体，正确的5‘和3’剪接位点的鉴定和化学成分 剪接反应本身受一大类称为SR蛋白的剪接因子的调控。 SR蛋白含有一个或两个RNA识别基序和一个富含 大量的精氨酸-丝氨酸二肽重复。RS结构域的磷酸化服务于 许多RNA处理功能包括剪接位点选择、将SR蛋白导入到 并将成熟的mRNA输出到细胞质中。这个项目将调查两个 剪接酶主要家族通过以下途径影响SR蛋白的功能 RS结构域的区域特异性、多位点磷酸化。使用工程足迹 方法，剪接酶的方向性将被定义并显示以控制 RS结构域中的丝氨酸被修饰。这些选择性磷酸化的影响 然后在剪接体内对SR蛋白质结构和相互作用/功能的反应将是 使用动力学、结构、剪接和细胞分析进行评估。目标是确定如何 剪接激酶识别和磷酸化RS结构域的特定区域和 确定这些化学修饰如何影响剪接组件。

Project Summary/Abstract: Comparisons of the genomes from humans and lower organisms reveal that the complexity in humans is achieved not by a dramatic increase in the number of genes but by alternative splicing events that stitch together different portions of genes to generate diverse proteins. Correct splicing allows normal healthy function, however, incorrect splicing is linked to many human diseases. For example, muscular dystrophy, ataxias, parkinsonism, neurofibromatosis, psychiatric disorders and cancer have their origins in splicing errors. Splicing reactions are catalyzed by a large macromolecular machine known as the spliceosome. Composed of both RNA and protein, the spliceosome can accurately select the proper splice sites from a considerably large precursor mRNA in healthy cells. The assembly of the spliceosome, the identification of the correct 5' and 3' splice sites and the chemical splicing reaction itself is regulated by a large class of splicing factors known as SR proteins. SR proteins contain one or two RNA recognition motifs and a long C-terminal domain rich in numerous arginine-serine dipeptide repeats. The phosphorylation of the RS domain serves many RNA processing functions including splice-site selection, import of SR proteins into the nucleus and export of mature mRNA to the cytoplasm. This project will investigate how two principal families of splicing enzymes uniquely impact SR protein function through regiospecific, multi-site phosphorylation of the RS domains. Using engineered footprinting methods, the directionality of the splicing enzymes will be defined and shown to control which serines in the RS domain are modified. The effects of these selective phosphorylation reactions on SR protein structure and interaction/function within the spliceosome will then be evaluated using kinetic, structural, splicing and cellular assays. The goal is to identify how splicing kinases recognize and phosphorylate specific regions of the RS domains and determine how these chemical modifications impact splicing componentry.