Coordination of SR Protein Phosphorylation and RNA Splicing

描述(申请人提供)：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，而是通过将基因的不同部分连接在一起产生不同的蛋白质的替代剪接事件来实现的。正确的剪接允许正常的健康功能，然而，错误的剪接与许多人类疾病有关。例如，肌肉营养不良、共济失调、帕金森氏症、神经纤维瘤病、精神障碍和癌症都源于拼接错误。剪接反应是由一个被称为剪接体的大分子机器催化的。剪接体由RNA和蛋白质组成，可以从健康细胞中相当大的前体mRNA中准确地选择合适的剪接位点。剪接体的组装、正确的5‘和3’剪接位点的识别以及化学剪接反应本身都受一大类称为SR蛋白的剪接因子的调控。SR蛋白含有一个或两个RNA识别基序和一个长的C-末端结构域，富含大量的精氨酸-丝氨酸二肽重复。RS结构域的磷酸化具有许多RNA加工功能，包括剪接位点选择、SR蛋白进入细胞核和将成熟的mRNA输出到细胞质。这个项目将研究两个主要的剪接酶家族如何通过区域特异性的RS结构域的多位点磷酸化来独特地影响SR蛋白的功能。使用工程足迹方法，剪接酶的方向性将被定义和显示，以控制RS结构域中的哪些丝氨酸被修饰。这些选择性的磷酸化反应对SR蛋白结构和剪接体内的相互作用/功能的影响将通过动力学、结构、剪接和细胞分析来评估。目的是确定剪接蛋白是如何识别和磷酸化RS结构域的特定区域，并确定这些化学修饰是如何影响剪接成分的。公共卫生相关性：健康相关性声明对人类和低等生物的基因组进行比较表明，人类的复杂性不是通过基因数量的急剧增加实现的，而是通过将基因的不同部分拼接在一起产生不同的蛋白质来实现的。正确的剪接可以实现正常的健康功能，然而，错误的剪接与许多人类神经退行性疾病和癌症有关。我们正在研究新发现的针对各种疾病的药物靶点-剪接酶(称为SR-激酶)如何调节重要的剪接因子(一种称为SR蛋白的特定蛋白质家族)，这些剪接因子在控制对健康和疾病都很重要的选择性剪接反应中发挥作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Health Relevance Statement 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 genes but by 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 neurodegenerative diseases and cancer. We are investigating how the newly identified drug targets for diverse diseases known as splicing enzymes (named SR-kinases) regulate important splicing factors (a specific family of proteins known as SR proteins) which cooperate in the control of alternative splicing reactions important in both health and disease.