Nucleic Acids Research Advance Access originally published online on September 13, 2006
Nucleic Acids Research 2006 34(17):4801-4815; doi:10.1093/nar/gkl646
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Nucleic Acids Research, 2006, Vol. 34, No. 17 4801-4815
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells
1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66 4002 Basel, Switzerland 2 Division of Bioinformatics, Biozentrum, University of Basel Klingelbergstrasse 50/70 CH-4056 Basel, Switzerland
*To whom correspondence should be addressed. Tel: +41 616974128; Fax: +41 616973976; Email: filipowi{at}fmi.ch
*Correspondence may also be addressed to Petr Svoboda. Tel: +41 616974128; Fax: +41 616973976; Email: svoboda{at}fmi.ch
Received June 10, 2006. Revised August 19, 2006. Accepted August 21, 2006.
RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic cleavage but it was Dicer- and Ago2-dependent. We used this feature of HEK293 to address the size of a pool of transcripts regulated by RNA silencing in a single cell type. We generated HEK293 cell lines depleted of Dicer or individual Ago proteins. The cell lines were used for microarray analyses to obtain a comprehensive picture of RNA silencing. The 3'-untranslated region sequences of a few hundred transcripts that were commonly up-regulated upon Ago2 and Dicer knock-downs showed a significant enrichment of putative miRNA-binding sites. The up-regulation upon Ago2 and Dicer knock-downs was moderate and we found no evidence, at the mRNA level, for activation of silenced genes. Taken together, our data suggest that, independent of the effect on translation, miRNAs affect levels of a few hundred mRNAs in HEK293 cells.
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