Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 703-708 |
| Number of pages | 6 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 368 |
| Issue number | 3 |
| DOIs | |
| State | Published - Apr 11 2008 |
| Externally published | Yes |
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Keywords
- RISC assembly
- Silencing activity
- siRNA
- Thermodynamic stability
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Focusing on RISC assembly in mammalian cells. / Hong, Junmei; Wei, Na; Chalk, Alistair; Wang, Jue; Song, Yutong; Yi, Fan; Qiao, Ren Ping; Sonnhammer, Erik L L; Wahlestedt, Claes R; Liang, Zicai; Du, Quan.
In: Biochemical and Biophysical Research Communications, Vol. 368, No. 3, 11.04.2008, p. 703-708.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Focusing on RISC assembly in mammalian cells
AU - Hong, Junmei
AU - Wei, Na
AU - Chalk, Alistair
AU - Wang, Jue
AU - Song, Yutong
AU - Yi, Fan
AU - Qiao, Ren Ping
AU - Sonnhammer, Erik L L
AU - Wahlestedt, Claes R
AU - Liang, Zicai
AU - Du, Quan
PY - 2008/4/11
Y1 - 2008/4/11
N2 - 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.
AB - 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.
KW - RISC assembly
KW - Silencing activity
KW - siRNA
KW - Thermodynamic stability
UR - http://www.scopus.com/inward/record.url?scp=39749200012&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39749200012&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2008.01.116
DO - 10.1016/j.bbrc.2008.01.116
M3 - Article
C2 - 18252196
AN - SCOPUS:39749200012
VL - 368
SP - 703
EP - 708
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -