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: 10797788
• 负责人: Jun Zhang
• 金额: $ 13.61万
• 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/10797788
• 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.

项目概要： 选择性剪接可处理超过 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。选择性剪接失调的原因 外显子选择不正确导致各种人类疾病。替代外显子是 在早期剪接体组装中选择。由于我们对早期阶段的了解有限 剪接体组装，开发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和 募集到核糖核蛋白复合物 U1 和 U2 的剪接位点。这些过程 取决于富含 Ser/Arg 的蛋白 (SR)、U1-70K 和 U2AF-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名成员组成 共享经过磷酸化的 Arg-Ser 重复区 (RS)。在这个提案中， 我们选择了该家族的原型SRSF1作为模型来研究其核心角色 SR 蛋白在剪接体组装中的作用。细胞研究表明 SRSF1 通过与外显子剪接增强子RNA基序结合促进外显子的包含，并且 SRSF1 的磷酸化不仅调节整体剪接模式，还调节 剪接体组装。尽管细胞研究取得了进展，但通过以下方式阐明其机制 SRSF1 的磷酸化调节外显子选择和剪接体组装 由于 SR 蛋白 U1-70K 和 U2AF-35 的溶解度较低，因此具有挑战性。我们实验室已获得全部 其中三种蛋白质为可溶性全长形式。凭借这一成功，我们发现 SRSF1 RS 区域 (a) 显示 RNA 结合偏好，其磷酸化抑制 RNA 绑定； (b) 对于与 U1-70K 和 U2AF-35 的相互作用至关重要，它们负责 分别招募U1和U2复合体； (c) 介导相分离，即 与其在组织核斑点中的作用一致。该提案将（1）使用高 通量方法 RNA Bind-n-Seq 系统地研究磷酸化过程 调节 SRSF1 的 RNA 结合特异性； (2) 结合NMR、分子动力学 模拟和其他生物物理方法来阐明其结构机制 SRSF1与U1-70K和U2AF-35相互作用； (3)研究RS区域如何平衡其角色 调节 RNA 结合亲和力、介导蛋白质相互作用和组织阶段 相分离状态下的分离。总之，我们的建议将增进我们的知识 早期剪接体组装过程中外显子选择和剪接因子相互作用的研究。

项目成果

Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding.

• DOI: 10.1038/s41467-023-43098-1
• 发表时间: 2023-11-13
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Qiu, Chen;Zhang, Zihan;Wine, Robert N.;Campbell, Zachary T.;Zhang, Jun;Hall, Traci M. Tanaka
• 通讯作者: Hall, Traci M. Tanaka