Mechanism of SR Protein Binding to the Splicing Kinase SRPK1

描述（由申请人提供）：许多人类疾病是由mRNA剪接的不规则性引起的，这是一个由剪接体催化的过程。作为剪接体的重要组成部分，SR蛋白在前体mRNA中建立剪接位点。它们含有RNA识别基序（RRM）和被SRPK 1多磷酸化的RS结构域。后一种修饰控制SR蛋白的功能，从而调节人类基因的剪接。最近的动力学和晶体学研究表明，SRPK 1磷酸化的SR蛋白，ASF/SF 2的RS结构域，使用的机制是定向的，区域特异性，和进行性。由于ASF/SF 2与SRPK 1的相互作用是剪接活性所必需的，因此了解SRPK 1如何识别SR蛋白亚结构域对于更好地理解mRNA剪接至关重要。在这个建议中，SRPK 1-ASF/SF 2复合物的形成机制将使用各种动力学和光谱技术进行研究。由于ASF/SF 2的结构域与SRPK 1进行大量接触，引导区域特异性磷酸化并与剪接活性相关联，因此将在平衡和瞬态条件下使用荧光和放射自显影方法研究这些结构域的结合顺序。已经开发了一种新的SR蛋白磷酸化的荧光测定法，并将探讨RRM和RS结构域结合在结构上是如何联系的。最近的数据表明，SRPK 1中的一个大的插入结构域通过与伴侣蛋白相互作用而起到细胞质锚的作用。它还作为一种变构调节剂，促进SRPK 1中结合RS结构域的对接槽与一个与RRM（RRM 2）相互作用的区域之间的串扰。这种变构现象将使用氢-氘交换质谱，荧光和非线性光学光谱方法进行研究。由于插入还提供了一个对接表面的监管分子伴侣，这些蛋白质的SRPK 1功能的影响将进行探讨。更广泛的目标是了解SRPK 1-ASF/SF 2组装机制，并建立剪接因子识别和生物控制的基本原则，在更大的SR蛋白家族。 公共卫生相关性：由于mRNA剪接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剪接机制的治疗方法，从而减轻人类的痛苦。SR蛋白是其磷酸化控制剪接发生的关键因素。通过对SR蛋白-酶复合物的分析，可以更好地理解SR蛋白与疾病之间的联系。

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.