Abstract
Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect—that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.
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Change history
08 July 2009
In the version of this article initially published, Figure 2f is not referenced in the figure legend, and is referenced as Figure 2e in the main text. Also, on p.5, right col., para. 1, line 8, miR-21 should be miR-122. The errors have been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank N. Stroustrup, Y. Merbl, G. Altan-Bonnet, A. Arvey and N. Gauthier for useful discussions. This work was supported in part by National Institutes of Health (NIH) grant CA124380 and NIH grant CA121852.
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A.A.K. performed the statistical and computational analysis and contributed to the manuscript. D.B. and M.L.M contributed to the computational analysis. C.S. contributed to discussions and the manuscript. C.S.L. designed the statistical and computational methods. D.S.M. conceived the idea for the project and contributed to the analysis. D.S.M. and C.S.L jointly supervised the research and wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–3 (PDF 9887 kb)
Supplementary Text and Figures
Supplementary Figures 4–6 (PDF 12031 kb)
Supplementary Table 1
Expression changes for all genes in 151 experiments. Transfection of small RNAs globally perturbs gene regulation by endogeneous microRNAs. (ZIP 7766 kb)
Supplementary Table 2
KS statistics for all experiments- 11 worksheets. Transfection of small RNAs globally perturbs gene regulation by endogeneous microRNAs. (XLS 111 kb)
Supplementary Table 3
Predicted sites in all genes for exogenous and endogenous microRNAs for each experiment. Transfection of small RNAs globally perturbs gene regulation by endogeneous microRNAs. (ZIP 8607 kb)
Supplementary Table 4
Regression analysis. Transfection of small RNAs globally perturbs gene regulation by endogeneous microRNAs. (XLS 349 kb)
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Khan, A., Betel, D., Miller, M. et al. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat Biotechnol 27, 549–555 (2009). https://doi.org/10.1038/nbt.1543
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DOI: https://doi.org/10.1038/nbt.1543
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