MIS复合物介导酵母孢子形成的分子机制研究

RNA的m6A甲基化修饰与代谢和发育等多种生命过程密切相关。研究表明，酵母mRNA的m6A修饰由MIS（MUM2-IME4-SLZ1）复合物催化生成，是其减数分裂形成孢子的必要条件，但目前对MIS复合物介导酵母孢子形成的分子机制还知之甚少。前期，我们已初步鉴定IME4蛋白分别结合MUM2和SLZ1的C端结构，并获得了MUM2的初筛晶体。在此基础上，我们拟通过X-射线晶体衍射、蛋白质化学交联结合质谱鉴定技术、pull-down及双分子荧光技术揭示MIS复合物蛋白组分间的互作模式及其结构特点；通过定点突变、MALDI-TOF质谱、酵母突变株构建、免疫荧光技术分析IME4、MUM2和SLZ1调节MIS复合物甲基转移酶功能及其在核定位中的重要作用；研究结果将试图阐明MIS复合物介导酵母孢子形成的分子机制，亦可为进一步推进RNA甲基化修饰调节配子发生相关疾病的研究奠定基础。

RNA m6A methylation involves a variety of life processes, including metabolism and development. The m6A modification of mRNA is catalyzed by the MIS (MUM2-IME4-SLZ1) complex in yeast, which is necessary for the meiotic induction and sporogenesis. However, the molecular mechanism in which the MIS complex catalyzes mRNA to induce the formation of spores is as yet unclear. In the early stage, we have identified that IME4 recognizes the C- terminus of MUM2 and SLZ1, respectively, and also obtained the primary crystal of MUM2. Based on our studies, we plan to reveal the interaction patterns and structural characteristics of MIS complex by X-ray crystallography, chemical cross-linking of proteins coupled with mass spectrometry analysis, pull -down and bimolecular fluorescence; To further elaborate the important roles of IME4, MUM2 and SLZ1 in regulating the methylation functions and intracellular localization of MIS complex, we will carry out mutagenesis, MALDI-TOF mass spectrometry, construction of yeast mutants, immunofluorescence; Finally, our results will shed light on the molecular mechanism of MIS complex mediating the sporogenesis of yeast; Meanwhile, it may also provide clues for the further study about the important roles of RNA methylation in modulating gametogenesis related diseases.