Mechanism of SR Protein Binding to the Splicing Kinase SRPK1

SR 蛋白与剪接激酶 SRPK1 结合的机制

• 批准号: 8211753
• 负责人: Ryan Matthew Plocinik
• 金额: $ 5.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8211753
• 关键词: Active Sites; Address; Affect; Binding; Binding Sites; Biological; Biological Assay; C-terminal; Communication; Complex; Cytoplasm; Data; Deuterium; Disease; Docking; Enzymes; Equilibrium; Excision; Face; Fluorescence; Genes; Goals; Gold; Gray unit of radiation dose; Human; Hydrogen; Investigation; Kinetics; Light; Link; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Modification; Molecular Chaperones; Molecular Conformation; Multienzyme Complexes; N Domain; N-terminal; Nature; Optics; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Process; Protein Binding; Protein Family; Proteins; RNA Recognition Motif; RNA Splicing; RRM1 gene; RRM2 gene; Reaction; Roentgen Rays; Role; Site; Spliceosomes; Stretching; Structure; Surface; Techniques; acrosome stabilizing factor; cell growth regulation; enzyme substrate complex; human disease; mRNA Precursor; novel; protein function; public health relevance; research study; stopped-flow fluorescence

DESCRIPTION (provided by applicant): Many human diseases result from irregularities in mRNA splicing, a process catalyzed by the spliceosome. As vital components of the spliceosome, SR proteins establish splice sites in the precursor mRNA. They contain RNA recognition motifs (RRMs) and an RS domain that is polyphosphorylated by SRPK1. The latter modifications control SR protein function, thereby regulating the splicing of human genes. Recent kinetic and crystallographic studies indicate that SRPK1 phosphorylates the RS domain of the SR protein, ASF/SF2, using a mechanism that is directional, regiospecific, and processive. As the interaction of ASF/SF2 with SRPK1 is necessary for splicing activity, understanding how SRPK1 recognizes the SR protein subdomains is important for gaining a better understanding of mRNA splicing. In this proposal, the mechanism of SRPK1-ASF/SF2 complex formation will be investigated using a variety of kinetic and spectrometric techniques. As the domains of ASF/SF2 make numerous contacts with SRPK1, guide regiospecific phosphorylation and link to splicing activity, the binding order of these domains will be studied using fluorescence and autoradiographic methods under equilibrium and transient-state conditions. A new fluorescence assay for SR protein phosphorylation has been developed and will be explored to address how RRM and RS domain binding are structurally linked. Recent data show that a large insert domain in SRPK1 functions as a cytoplasmic anchor by interacting with chaperone proteins. It also acts as an allosteric regulator that promotes cross-talk between a docking groove in SRPK1 that binds the RS domain and a region that interacts with one of the RRMs (RRM2). This allosteric phenomenon will be investigated using hydrogen-deuterium exchange mass spectrometry, fluorescence, and nonlinear optical spectroscopic methods. As the insert also offers a docking surface for regulatory chaperones, the effects of these proteins on SRPK1 function will be explored. The broader goal is to understand the SRPK1-ASF/SF2 assembly mechanism and to establish fundamental principles for splicing factor recognition and biological control within the larger SR protein family. PUBLIC HEALTH RELEVANCE: Since abnormalities in mRNA splicing have been linked to human disease, understanding the role of factors that actively participate in the splicing reaction may help us to develop treatments directed at the splicing machinery that can alleviate human suffering. SR proteins are critical factors whose phosphorylation controls where splicing takes place. Through analyses of the SR protein-enzyme complex, a better understanding of the link between SR proteins and disease may be attained.

描述（由申请人提供）：许多人类疾病是由mRNA剪接中的不规则性引起的，这是由剪接体催化的过程。作为剪接体的重要成分，SR蛋白在前体mRNA中建立剪接位点。它们包含RNA识别基序（RRMS）和由SRPK1多磷酸化的RS结构域。后者的修饰控制SR蛋白功能，从而调节了人类基因的剪接。最近的动力学研究和晶体学研究表明，SRPK1使用定向，重新特异性和过程的机制磷酸化SR蛋白ASF/SF2的RS结构域。由于ASF/SF2与SRPK1的相互作用对于剪接活性是必需的，因此了解SRPK1如何识别SR蛋白子域如何获得更好地了解mRNA剪接的重要性。在此提案中，将使用各种动力学和光谱技术研究SRPK1-ASF/SF2复合物的机理。由于ASF/SF2的结构域与SRPK1建立了许多接触，指导型磷酸化并与剪接活性联系在一起，因此将使用荧光和自显影方法在平衡和瞬态条件下使用荧光和自显影方法研究这些结构域的结合顺序。已经开发了用于SR蛋白磷酸化的新荧光测定法，并将探讨以解决RRM和RS结构域结合的结构如何在结构上连接。最近的数据表明，SRPK1中的一个大插入结构域通过与伴侣蛋白相互作用来充当细胞质锚。它也充当了变构调节器，可在SRPK1中促进对接凹槽之间的串扰，该凹槽结合了RS域和与其中一个RRMS（RRM2）相互作用的区域。这种变构现象将使用氢 - 居民交换质谱，荧光和非线性光谱法进行研究。由于该插入物还提供了用于调节伴侣的对接表面，因此将探索这些蛋白质对SRPK1功能的影响。更广泛的目标是了解SRPK1-ASF/SF2组装机制，并在较大的SR蛋白家族中建立剪接因子识别和生物控制的基本原理。 公共卫生相关性：由于mRNA剪接中的异常与人类疾病有关，因此了解积极参与剪接反应的因素的作用可能有助于我们开发针对可以减轻人类苦难的剪接机制的治疗方法。 SR蛋白是关键因素，其磷酸化控制发生剪接的地方。通过对SR蛋白酶复合物的分析，可以更好地了解SR蛋白与疾病之间的联系。