禾谷镰刀菌FgPrp6调控剪接体激活的作用机制研究

Pre-mRNA splicing is an important cellular regulation process and is related to many human diseases including cancer. The process of splicing is catalyzed by spliceosome, the RNA and protein complex. Splicing is regulated by many non-SR proteins, among which Prp6 is a critical one. Prp6 is important for spliceosome assembly and its phosphorylation by Prp4 is required for spliceosome activation. Despite of its importance, the molecular mechanism of Prp6 in spliceosome activation is unknown. The Fgprp4 deletion mutant identified during systemic characterization of the kinome of Fusarium graminearum has severe growth defects and is unstable. Fast-growing sectors were frequently produced. Interestingly, previous results showed that spontaneous mutations occurred in the putative FgPrp4 phosphorylation region of FgPrp6 rescued the growth defects of Fgprp4 mutant, indicating the importance of this region for regulation of FgPrp6's function and spliceosome activation. Therefore, we proposed to identified the FgPrp4 phosphorylation sites of FgPrp6 and characterize the function of phosphorylation in spliceosome activation. To further elucidate the molecular mechanism of FgPrp6 action, we will study the effects of the spontaneous mutations and phosphorylation on the intra-molecular interaction of FgPrp6 and the interactions between FgPrp6 and other spliceosome proteins. Our study will reveal the molecular mechanism of Prp6 in regulation of spliceosome activation and further provide theoretical informations for cancer research.

前体RNA剪接是一种重要的转绿后修饰，多种人类疾病与剪接调控异常相关。剪接过程主要由一个核酸蛋白质复合体—剪接体催化完成。已知剪接体的核心组成蛋白质Prp6是调控剪接体组装的重要因子。它被Prp4磷酸化是剪接体激活的关键，但具体的分子机制未知。项目前期在对禾谷镰刀菌PRP4基因进行敲除研究时发现，突变体生长缓慢，易发生角突变，产生恢复表型的角突变子。经鉴定，部分角突变子是FgPrp6上预测为Prp4磷酸化位点的区域发生角突变引起的，表明该区域为Prp6功能的调控区，对剪接体的激活至关重要。本项目拟进一步确认FgPrp6上Prp4的磷酸化位点，明确磷酸化对该蛋白功能的调控作用。同时研究角突变与磷酸化之间的关系，分析角突变和磷酸化对Prp6自身分子内及其与其他剪接体蛋白间互作的影响。研究结果将阐明禾谷镰刀菌中Prp6调控剪接体激活的分子机制，为剪接相关疾病尤其是癌症的研究提供理论基础。

已结题项目从309个Fgprp4突变体的角变子中鉴定到20个在FgPrp6上有突变的角变子，并有多个角变子在FgPrp6的同一个位点发生突变的现象。其中，有6个角变子在E308位发生突变，各有两个角变子在R230和R282发生突变。总体来说，碱性氨基酸更易发生突变。对这些角变子进行表型分析发现它们都未能全部恢复Fgprp4突变体的缺陷，它们的共同特点是致病力大大下降。采用RNA-seq转录组测序技术分析S22和S202菌丝时期的剪接情况，发现菌丝生长正常的S22和S202的剪接效率与野生型一致。这些结果揭示禾谷镰刀菌在菌丝阶段与侵染阶段的剪接调控机制可能不同。通过在野生型菌株PH-1的FgPrp6上引入角突变的方法，验证角变子S47和S202确实是由于FgPrp6上的E308K或R230H突变部分恢复表型的。利用磷酸化anti-Ser抗体，明确了FgPrp6是FgPrp4的底物。通过将FgPrp6上与人类和酵母Prp6上Prp4磷酸化位点保守位点去磷酸化突变，明确T199、T200、T221、T261对FgPrp6在植物侵染中的功能重要。通过去分析磷酸化突变体侵染阶段的RNA-seq转录组数据，发现超过30%基因的剪接效率降低了2倍以上，因此这四个位点对FgPrp6调控禾谷镰刀菌侵染阶段的剪接具有一定作用。本项目研究结果不仅增进了人们对禾谷镰刀菌RNA剪接调控机理的认识，也为进一步研究侵染时期剪接调控机理提供好的实验材料（S22和S202）。