Focusing on RISC assembly in mammalian cells
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- 作者：Junmei Hong ; Na Wei ; Alistair Chalk ; Jue Wang ; Yutong Song ; Fan Yi ; Ren-Ping Qiao ; Erik L.L. Sonnhammer ; Claes Wahlestedt ; Zicai Liang ; Quan Du
- 关键词：siRNA ; RISC assembly ; Silencing activity ; Thermodynamic stability
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：2008
- 出版时间：11 April 2008
- 年：2008
- 卷：368
- 期：3
- 页码：703-708
- 全文大小：199 K
文摘
RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5′ end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5′ end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.