The central roles of SRSF1 in early-stage spliceosome assembly

SRSF1 在早期剪接体组装中的核心作用

• 批准号: 10678784
• 负责人: Jun Zhang
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-08 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678784
• 关键词: Affinity; Alternative Splicing; Binding; Complex; Disease; Enhancers; Equilibrium; Event; Exons; Family; Genes; Goals; Human; Knowledge; Length; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Modeling; Nuclear; Pattern; Phase; Phosphorylation; Phosphorylation Inhibition; Process; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Repetitive Sequence; Ribonucleoproteins; Role; Site; Solubility; Specificity; Spliceosome Assembly Pathway; biophysical techniques; human disease; member; molecular dynamics; preference; prototype; recruit; success; therapy development

Project Summary: Alternative splicing processes over 95% of human mRNA and enables a single gene to encode distinct protein isoforms of different functions. Dysregulation of alternative splicing causes incorrect selection of exons and consequently various human diseases. Alternative exons are selected in early-stage spliceosome assembly. Due to our limited knowledge about early-stage spliceosome assembly, it is still challenging to develop therapies for diseases related to aberrant RNA splicing. Early-stage spliceosome assembly involves selection of exons and recruitment to the splicing sites of ribonucleoprotein complexes U1 and U2. These processes depend on the interplay of Ser/Arg-rich proteins (SR), U1-70K and U2AF-35. SR proteins are the key factors that coordinate all these events. The SR family consists of 12 members and shares Arg-Ser repetitive regions (RS) that are subjected to phosphorylation. In this proposal, we have selected the prototype of the family, SRSF1, as a model to investigate the central roles of SR proteins in spliceosome assembly. Mounting cellular studies have shown that SRSF1 promotes inclusion of exons by binding to exonic splicing enhancer RNA motifs, and phosphorylation of SRSF1 regulates not only the overall splicing pattern, but also the spliceosome assembly. Despite the progress in cellular studies, elucidating the mechanisms by which phosphorylation of SRSF1 regulates exon selection and spliceosome assembly is challenging due to low solubility of SR proteins, U1-70K and U2AF-35. Our lab has obtained all three of these proteins in the soluble full-length form. With this success, we have found that the SRSF1 RS region (a) displays RNA-binding preference and its phosphorylation inhibits RNA binding; (b) is essential for interaction with U1-70K and U2AF-35, which are responsible for recruitment of U1 and U2 complexes, respectively; (c) mediates phase separation, which is consistent with its role in organizing nuclear speckles. This proposal will (1) use the high- throughput method RNA Bind-n-Seq to systematically investigate how phosphorylation regulates RNA-binding specificity of SRSF1; (2) use a combination of NMR, molecular dynamic simulations and other biophysical methods to elucidate the structural mechanism by which SRSF1 interacts with U1-70K and U2AF-35; (3) investigate how the RS region balance its roles in modulating RNA-binding affinity, mediating protein interactions, and organizing phase separation in the phase-separated state. In summary, our proposal will advance our knowledge of exon selection and splicing factor interaction during early-stage spliceosome assembly.

项目摘要： 选择性剪接处理超过95%的人类mRNA，并使单个基因能够编码 不同功能的不同蛋白质亚型。选择性剪接调节异常的原因 不正确的外显子选择，从而导致各种人类疾病。替代外显子是 在早期拼接体组装中选择。由于我们对早期阶段的知识有限， 尽管剪接体组装是一个复杂的过程，但开发用于与剪接体组装相关的疾病的疗法仍然具有挑战性。 RNA剪接异常早期剪接体组装涉及外显子的选择， 募集到核糖核蛋白复合物U1和U2的剪接位点。这些过程 依赖于富含Ser/Arg的蛋白质（SR）、U1- 70 K和U2 AF-35的相互作用。SR蛋白是 协调所有这些事件的关键因素。SR家族由12个成员组成， 共享经历磷酸化的Arg-Ser重复区域（RS）。在这一提议中， 我们已经选择了家庭的原型，SRSF 1，作为模型来调查的核心作用， SR蛋白在剪接体组装中的作用。越来越多的细胞研究表明，SRSF 1 通过结合外显子剪接增强子RNA基序促进外显子的包含，和 SRSF 1的磷酸化不仅调节整个剪接模式，还调节 剪接体组装。尽管在细胞研究中取得了进展，但通过以下方法来阐明机制： SRSF 1的磷酸化调节外显子选择和剪接体组装， 由于SR蛋白质U1- 70 K和U2 AF-35的低溶解度而具有挑战性。我们的实验室已经获得了所有 这些蛋白质中的三种以可溶性全长形式存在。有了这个成功，我们发现， SRSF 1 RS区（a）显示RNA结合偏好，其磷酸化抑制RNA结合。 结合;（B）对于与U1- 70 K和U2 AF-35的相互作用是必不可少的，它们负责 募集的U1和U2复合物，分别;（c）介导相分离，这是 与其在组织核斑点中的作用一致。本建议将（1）使用高- 通过RNA Bind-n-Seq方法系统地研究磷酸化 调节SRSF 1的RNA结合特异性;（2）使用NMR、分子动力学 模拟和其他生物物理方法来阐明结构机制， SRSF 1与U1- 70 K和U2 AF-35相互作用;（3）研究RS区如何平衡其作用 在调节RNA结合亲和力、介导蛋白质相互作用和组织相 在相分离状态下分离。总而言之，我们的建议将促进我们的知识 外显子选择和剪接因子的相互作用在早期剪接体组装。