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Title: Focusing on RISC assembly in mammalian cells

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 thatmore » instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.« less

Authors:
;  [1];  [2];  [1];  [3];  [1];  [4];  [5];  [6];  [1];  [1]
  1. Institute of Molecular Medicine, Peking University, 100871 Beijing (China)
  2. Department of Molecular Medicine and Surgery, Karolinska Institute, 171 76 Stockholm (Sweden)
  3. Department of Woman and Child Health, Karolinska Institute, 171 76 Stockholm (Sweden)
  4. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Science, Peking University, Beijing 100083 (China)
  5. Stockholm Bioinformatics Center, 171 74 Stockholm (Sweden)
  6. Scripps Florida, Jupiter, FL 33458 (United States)
Publication Date:
OSTI Identifier:
21043705
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 368; Journal Issue: 3; Other Information: DOI: 10.1016/j.bbrc.2008.01.116; PII: S0006-291X(08)00201-5; Copyright (c) 2008 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ASYMMETRY; DATA ANALYSIS; DROSOPHILA; EMBRYOS; GENES; IN VITRO; MELTING POINTS; PROTEINS; RNA; STABILITY

Citation Formats

Junmei, Hong, Na, Wei, Chalk, Alistair, Jue, Wang, Song, Yutong, Fan, Yi, Renping, Qiao, Sonnhammer, Erik L.L., Wahlestedt, Claes, Zicai, Liang, Department of Molecular Medicine and Surgery, Karolinska Institute, 171 76 Stockholm, and Du, Quan. Focusing on RISC assembly in mammalian cells. United States: N. p., 2008. Web. doi:10.1016/j.bbrc.2008.01.116.
Junmei, Hong, Na, Wei, Chalk, Alistair, Jue, Wang, Song, Yutong, Fan, Yi, Renping, Qiao, Sonnhammer, Erik L.L., Wahlestedt, Claes, Zicai, Liang, Department of Molecular Medicine and Surgery, Karolinska Institute, 171 76 Stockholm, & Du, Quan. Focusing on RISC assembly in mammalian cells. United States. https://doi.org/10.1016/j.bbrc.2008.01.116
Junmei, Hong, Na, Wei, Chalk, Alistair, Jue, Wang, Song, Yutong, Fan, Yi, Renping, Qiao, Sonnhammer, Erik L.L., Wahlestedt, Claes, Zicai, Liang, Department of Molecular Medicine and Surgery, Karolinska Institute, 171 76 Stockholm, and Du, Quan. 2008. "Focusing on RISC assembly in mammalian cells". United States. https://doi.org/10.1016/j.bbrc.2008.01.116.
@article{osti_21043705,
title = {Focusing on RISC assembly in mammalian cells},
author = {Junmei, Hong and Na, Wei and Chalk, Alistair and Jue, Wang and Song, Yutong and Fan, Yi and Renping, Qiao and Sonnhammer, Erik L.L. and Wahlestedt, Claes and Zicai, Liang and Department of Molecular Medicine and Surgery, Karolinska Institute, 171 76 Stockholm and Du, Quan},
abstractNote = {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.},
doi = {10.1016/j.bbrc.2008.01.116},
url = {https://www.osti.gov/biblio/21043705}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 3,
volume = 368,
place = {United States},
year = {Fri Apr 11 00:00:00 EDT 2008},
month = {Fri Apr 11 00:00:00 EDT 2008}
}