Stability of RNAi effectors: Dicer and human Ago2 proteins

• Main Authors: Z.C Chen, 陳稚晰
• Other Authors: Shih-Yen Lo, ph. D.
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/79320350550478395733

碩士 === 慈濟大學 === 醫學生物技術研究所 === 95 === The mechanism of RNA interference (RNAi)-gene silencing was first described in C. Elegans (1998 Fire .et al.). Though they started out as somewhat mysterious components of the RNAi effector complexes, Dicer and human Argonaute2 proteins have since taken center stage in RNAi gene silencing of Homo sapiens. Recent study shows that RNAi-gene silencing process fulfils fundamental regulatory roles, as well as antiviral functions. Our data shows that both Dicer and HsAgo2 will be cleaved by wild-type vaccinia virus infection. The cleavage of HsAgo2 will generate an N-terminal product (~50kDa contains PAZ domain) and C-terminal product (~65kDa contains PIWI domain) from full-length HsAgo2. Moreover, eGFP RNAi assay shows that N-terminal 1-349 amino acids of HsAgo2 slightly repress the efficiency of RNAi. RNAi regulates the expression of target genes, but the interference itself is also under regulation by unknown mechanisms. Our data shows that full-length HsAgo2 expressed in HeLa cells tends to be cleaved. HsAgo2 was separated into two parts: the N-terminus contains PAZ domain and the C-terminus contains PIWIs domain (~40kDa). Furthermore, HsAgo2 cleavage is an RNAi-dependent phenomenon using a reporter system for the study of RNA interference. These studies have not only provided the evidence to imply the relationships between host and viccinia virus, but also show a possible negative regulation of RNAi gene silencing pathways.