Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-[beta] pathway and reveal microRNA regulation of TGFBR2

• Published in: Silence 2011-03, Vol.2, p.3
• Main Authors: Schultz, Nikolaus, Marenstein, Dina R, De Angelis, Dino A, Wang, Wei-Qing, Nelander, Sven, Jacobsen, Anders, Marks, Debora S, Massagué, Joan, Sander, Chris
• Format: Article
• Language: English
• Subjects: Growth factor receptors; MicroRNA; Physiological aspects; Transforming growth factors
• ISSN: 1758-907X; 1758-907X
• DOI: 10.1186/1758-907X-2-3

Background RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-[beta] pathway in a human keratinocyte cell line. The TGF-[beta] pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-[beta] responses have been strongly implicated in cancer. Results We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-[beta] pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-[beta] receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression. Conclusions Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-[beta] receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.