隐球酵母WG/GW Argonaute结合蛋白的功能表征

Small non-coding RNAs (sRNAs) ranging from 20 to 30 nt in length is distinctly abundant in Cryptococcus neoformans which is clinically one of the most encountered human fungal pathogens. Therefore, it is regarded as the ideal research system to investigate the RNAi pathway in fungi. However, the biological function of sRNAs and the mechanism of RNAi have not been thoroughly elucidated in C. neoformans. Preliminary results indicate that cryptococcal GWC1 plays important roles in the biogenesis of total sRNAs. According to the computational analysis, we speculate that GWC1 encodes for the WG/GW Argonaute-binding protein. In this project, the primary task is to study clearly whether Gwc1 works as the WG/GW Argonaute-binding protein in C. neoformans using multiple molecular biological technologies, such as gene editing, immunoprecipitation, Northern blot and mass spectrometry. On the basis of the first part of the project, the effect of Gwc1 on RNAi and its molecular mechanisms of action will be explored thoroughly. Furthermore, the amino acids that play a key role in the interaction between Gwc1 and Argonaute, the accessory factors and target proteins linking to Argonaute via Gwc1 scaffold, as well as the regulation of GWC1 expression will be discussed further in the study. Consequently, RNAi regulatory networks centering on the Argonaute protein will be enriched and extended. This project is expected to provide new insights for understanding the assembly of RNA-induced silencing complex and the regulatory mechanism of the RNAi process.

新型隐球酵母是临床常见人类条件致病真菌，生成大量内源小分子RNA（sRNA），是研究真菌RNAi过程的良好材料。目前仅少数隐球酵母sRNA的生物学功能得到揭示，其RNAi机制有待详细阐述。课题组前期研究发现GWC1对隐球酵母sRNA合成至关重要。生物信息学分析推测GWC1编码隐球酵母WG/GW Argonaute结合蛋白。本项目拟通过基因编辑、免疫共沉淀、Northern blot、蛋白质谱分析等技术，阐明Gwc1是否为隐球酵母WG/GW Argonaute结合蛋白，并在此基础上深入探讨Gwc1作用机制，揭示Gwc1与Argonaute相互作用的关键区域和关键位点，探究Gwc1连接的RNAi辅助因子及下游靶蛋白，并探索Gwc1的表达调控机制，进而充实新型隐球酵母以Argonaute为核心的RNAi调控网络。该研究有望为理解RNA诱导的沉默复合物的组装、RNAi进程的调控机制提供新的认识。

新生隐球菌是临床常见条件致病真菌，能够生成大量内源性非编码小RNA，是研究真菌RNA干扰过程的良好材料，但是其sRNA生成机制并未完全阐述清楚。课题组前期发现并确认了sRNA生成途径中的关键蛋白Gwc1。GWC1基因缺失显著影响sRNA的生成，推测GWC1编码隐球菌WG/GW Argonaute结合蛋白。为详细分析隐球菌Gwc1蛋白参与sRNA生成的分子机制，本研究通过更换启动子和异源表达的方式，将裂殖酵母中已被证实为WG/GW Argonaute蛋白的Tas3转化至gwc1∆突变菌株中，结果发现gwc1Δ::Tas3异源表达菌株无法恢复sRNA表达。同时，免疫共沉淀结果并未检测到Gwc1与Argonaute蛋白的互作关系。因此，我们推测Gwc1不是新生隐球菌的WG/GW Argonaute结合蛋白，而是隐球菌sRNA生成途径中的一个新的作用分子。在对Gwc1的互作蛋白进行深入分析时，我们发现Gwc1与Qip1、多种热激蛋白相互作用。生物信息学分析表明QIP1编码Argonaute互作蛋白，猜测Gwc1通过Qip1与Argonaute相互作用。利用CRISPR-Cas9基因编辑工具，我们构建了QIP1缺失突变菌株qip1∆。Qip1缺失显著阻碍sRNA生成，说明Qip1能够调控sRNA表达。利用荧光标记技术对Gwc1和Qip1的亚细胞定位进行分析，进一步证明Gwc1与Qip1相互作用参与sRNA生成。同时，我们还发现Gwc1和Qip1蛋白不参与毒力因子产生，但Gwc1能通过改变亚细胞定位对环境压力应答。此外，我们也发现隐球菌RNAi系统参与细胞营养代谢及对氟康唑的抗性应答。综上，本研究通过蛋白质异源表达、免疫共沉淀、CRISPR-Cas9等技术初步揭示Gwc1通过与Qip1互作参与调控隐球菌sRNA生成，为新生隐球菌甚至真菌领域中RNAi机制的深入研究奠定了理论基础。