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Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

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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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Figure 1: Schematic of the hypothesis that transfected si/miRNAs and the cell's endogenous miRNAs compete for RISC machinery.
Figure 2: Genes with predicted target sites for endogenous miRNAs are significantly dysregulated after si/miRNA transfections.
Figure 3: Quantitative model predicting expression change after transfection.
Figure 4: Competition effect shows dose-response and temporal dynamics proportional in magnitude, but opposite in direction, to targeted effect.

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Christina S Leslie or Debora S Marks.

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)

Supplementary Figure Legends (DOC 353 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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