Coordination of SR Protein Phosphorylation and RNA Splicing

项目概要/摘要： 人类和低等生物的基因组比较显示， 人类的复杂性不是通过基因数量的急剧增加，而是通过 选择性剪接事件将基因的不同部分拼接在一起， proteins.正确的剪接允许正常的健康功能，然而，不正确的剪接与 许多人类疾病。例如，肌肉萎缩症，共济失调，帕金森症， 神经纤维瘤病、精神疾病和癌症都起源于剪接错误。 剪接反应是由一个大的大分子机器催化的， 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的适当剪接位点。大会 剪接体的识别、正确5'和3'剪接位点的识别以及化学物质 剪接反应本身受称为SR蛋白的一大类剪接因子调节。 SR蛋白含有一个或两个RNA识别基序和一个长的C-末端结构域， 许多丝氨酸-丝氨酸二肽重复。RS结构域的磷酸化 许多RNA加工功能，包括剪接位点的选择，SR蛋白的输入， 细胞核和输出成熟的mRNA到细胞质。该项目将研究如何两个 剪接酶的主要家族通过以下方式独特地影响SR蛋白功能： 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和显示控制， RS结构域中的丝氨酸被修饰。这些选择性磷酸化的作用 然后，在剪接体内SR蛋白质结构和相互作用/功能的反应将被 使用动力学、结构、剪接和细胞测定进行评价。我们的目标是确定 剪接激酶识别并磷酸化RS结构域的特定区域， 确定这些化学修饰如何影响拼接组件。

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.