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。

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.