剪接因子SFRS5的泛素化和乙酰化修饰调控研究

Alternative splicing makes fundermental contributions to the plethora of eukaryotic proteomics. The abnormality of alternative splicing has been demonstrated to be tightly correlated with malignant tumor,metabolic disorders, autoimmune disseases and many other clinical syndromes. The pervasive study of the regulatory mechanisms of alternative study is a significant and cutting-edge direction. SFRS5 is a classical member of splicing regulators. The current knowledge of its regulation comes from the studies on its transcriptional level, however the post-translational regulation of SFRS5 remains largely unclear. We recently found that SFRS5 can be ubiquitinated and acetylated,and the potential ubiquitin ligase for SFRS5 is the HECT-type ubiquitin ligase Smurf1. Interestingly,the acetylation of SFRS5 on K125 site competes the SFRS5 ubiquitination by Smurf1 and then stabilizes SFRS5. Upon apoptosis inducer treatment, the activity of SFRS5 changed, which was reflected by the ratio of two isoforms of its splicing substrates, Cell Cycle and Apoptosis Regulator 1 (CCAR1). This resulted in apoptosis in various cell types. In this project, we plan to delineate the intricate mechanisms of the ubiquitination and acetylation as well as their physiological significance. Meanwhile we plan to study the outcomes on the splicing ablity and classical translation-promoting function of SFRS5. We will generate an acetylated SFRS5-specific antibody to detect the level of acetylated SFRS5 in tumor cells and evaluate the function of K125 modification in metabolism and tumorgenesis. Parallelly, we will use cell proliferation, apoptosis, migration, invasion assays and tumor formation assay in nude mice to investigate the functions of SFRS5 in tumor progression. Finally, we plan to use the biological tool ESE finder to discover the new substrate of SFRS5 which biological roles are pivotal and multiformed to better understand how these two modifications influence the basic splicing events of SFRS5. This project will make contribution to understand the post-translational regulation of splicing factors.

可变剪接是形成真核生物蛋白质多样性的重要原因，其异常与肿瘤形成、代谢紊乱、免疫疾病发生等密切相关，探讨可变剪接的调节机制具有重要的生物学意义。SFRS5作为一种重要的剪接调节因子，已知的调节机制集中于转录水平，而翻译后水平的调节机制知之甚少。我们前期发现，SFRS5可被泛素化和乙酰化修饰;乙酰化在K125位点竞争Smurf1介导的泛素化、抑制降解从而增强SFRS5的稳定性。在凋亡信号刺激下，其剪接底物CCAR1剪接体比例下降,诱发凋亡。本项目拟深入探讨乙酰化和泛素化修饰的具体机制和发生的生理条件;明确这两种修饰对于SFRS5剪接功能的影响;制备特异性的乙酰化抗体，利用裸鼠成瘤、临床样本筛查、敲除、敲入小鼠模型研究K125位点修饰与肿瘤及正常细胞代谢的关系;发掘鉴定新的剪接底物以全面揭示两种修饰对SFRS5剪接活性调控的普遍性。本项目对于理解剪接因子家族的翻译后修饰机制具有重要的启示作用。

真核生物95% 的基因存在可变剪接，可变剪接决定了RNA 的多样性并对蛋白质的表达模式产生重要影响，剪接因子的精细调控对于剪接小体的组装和功能发挥尤为重要。葡萄糖是维持代谢稳态的主要生命组成物质，葡萄糖浓度的稳态维持具有重要的生理和病理学意义。最近研究表明，可变剪接可能以精细复杂的机制参与多种基础生命活动形式的调控，而目前对于葡萄糖浓度变化情况下引起的细胞命运决定过程和细胞状态响应的精细调节机制认识尚浅。. 本团队通过无偏向性筛选方式，发现剪接因子 SRSF5特异性的在葡萄糖诱导下高表达，其表达量升高促进细胞命运决定关键成分—细胞分裂周期和凋亡调节蛋白 1（CCAR1）的可变剪接。位于SRSF5同一赖氨酸位点K125的乙酰化和泛素化竞争决定了肺癌细胞在低糖和高糖条件下的细胞应答和命运选择。相关研究成果发表于Nature Communications 杂志，该研究不仅拓宽了剪接小体调控机制的认识，更是首次将葡萄糖代谢和可变剪接调节信号轴偶联起来；SRSF5-CCAR1调控轴的发现为肺癌的靶向治疗提供了新的参考思路和科学基础。