RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21–23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities1,2. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function3,4,5,6,7,8,9. However, owing to a tolerance for mismatches and gaps in base-pairing with targets10,11,12, small RNAs could have up to hundreds of potential target sequences in a genome13,14, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets15,16. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such ‘off-target effects’ (OTEs)5. Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction17 in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.
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Valencia-Sanchez, M. A., Liu, J., Hannon, G. J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006)
Zamore, P. D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005)
Moffat, J. & Sabatini, D. M. Building mammalian signalling pathways with RNAi screens. Nature Rev. Mol. Cell Biol. 7, 177–187 (2006)
Armknecht, S. et al. High-throughput RNA interference screens in Drosophila tissue culture cells. Methods Enzymol. 392, 55–73 (2005)
Echeverri, C. J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nature Rev. Genet. 7, 373–384 (2006)
Fraser, A. G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 (2000)
Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004)
Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003)
Paddison, P. J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004)
Doench, J. G. & Sharp, P. A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004)
Haley, B. & Zamore, P. D. Kinetic analysis of the RNAi enzyme complex. Nature Struct. Mol. Biol. 11, 599–606 (2004)
Brennecke, J., Stark, A., Russell, R. B. & Cohen, S. M. Principles of microRNA-target recognition. PLoS Biol. 3, e85 (2005)
Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005)
Lim, L. P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005)
Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3, 199–204 (2006)
Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003)
Logan, C. Y. & Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004)
Veeman, M. T., Slusarski, D. C., Kaykas, A., Louie, S. H. & Moon, R. T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13, 680–685 (2003)
Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004)
Zeng, W. et al. naked cuticle encodes an inducible antagonist of Wnt signalling. Nature 403, 789–795 (2000)
DasGupta, R., Kaykas, A., Moon, R. T. & Perrimon, N. Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308, 826–833 (2005)
Mosimann, C., Hausmann, G. & Basler, K. Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125, 327–341 (2006)
Banziger, C. et al. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522 (2006)
Bartscherer, K., Pelte, N., Ingelfinger, D. & Boutros, M. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125, 523–533 (2006)
Jackson, A. L. & Linsley, P. S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20, 521–524 (2004)
Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005)
We thank N. Perrimon and colleagues for their role in producing the dsRNA library, S. Yao for initial assistance in the Hh screen, and R. Gong for technical assistance. We also thank all members of the Beachy laboratory, R. Green and S. Dorner for helpful discussions, and G. Seydoux and J. Taipale for critical comments on the manuscript.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
This file contains Supplementary Methods, Supplementary Figures 1–8, Supplementary Tables 1–5, and Supplementary List (Alignment between Wg screen candidates with known pathway components). (PDF 514 kb)
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