The central roles of SRSF1 in early-stage spliceosome assembly

项目总结： 选择性剪接处理超过95%的人类mRNA，使单个基因能够编码 不同功能的不同蛋白质亚型。选择性剪接原因的调节失调 不正确的外显子选择，从而导致各种人类疾病。备选外显子有 在早期剪接体组装中被选中。由于我们对早期阶段的了解有限 剪接体组装，但开发与之相关的疾病的治疗方法仍然具有挑战性 异常的RNA剪接。早期剪接体组装涉及外显子和 募集到核糖核蛋白复合体U1和U2的剪接位点。这些过程 依赖于富含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名成员和 分享被磷酸化的Arg-Ser重复区(RS)。在这份提案中， 我们选择了该家族的原型SRSF1作为模型来研究核心角色 剪接体组装中的SR蛋白。越来越多的细胞研究表明，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性，以及 SRSF1的磷酸化不仅调节整个剪接模式，而且还调节 剪接体组装。尽管在细胞研究方面取得了进展，但通过 SRSF1的哪一种磷酸化调控外显子选择和剪接体组装 由于SR蛋白、U1-70K和U2AF-35的低溶解性而具有挑战性。我们的实验室已经得到了所有 这些蛋白质中有三种是可溶的全长形式。随着这一成功，我们发现 SRSF1 RS区(A)表现出与RNA结合的偏好，其磷酸化抑制RNA 结合；(B)对于与U1-70K和U2AF-35的相互作用是必不可少的，它们负责 分别招募U1和U2复合体；(C)调节相分离，这是 这与它在组织核斑点方面的作用是一致的。这项建议将(1)使用高- 吞吐量法RNA Bind-n-Seq系统研究磷酸化 调节SRSF1的RNA结合特异性；(2)结合使用核磁共振、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交互；(3)研究RS区如何平衡其角色 在调节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。总而言之，我们的建议将增进我们的知识 剪接体组装早期的外显子选择和剪接因子相互作用的研究。

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.