Abstract
BackgroundRNA 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)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.ResultsWe 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-β 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-β 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.ConclusionsOur findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β 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.
Highlights
RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms
A 6,000-gene siRNA screen to look for transforming growth factor (TGF)-b pathway components The screen used a nuclear translocation assay consisting of a green fluorescent protein (GFP)-SMAD2 fusion protein stably expressed in HaCaT keratinocytes (Figure 1A) [32]
A change in the N:C ratio can be caused by facilitation or inhibition of TGF-b-induced GFP-SMAD2 phosphorylation or nuclear translocation
Summary
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)-b pathway in a human keratinocyte cell line. RNAi screens, using either double-stranded RNA in Drosophila cells, or small hairpin (sh)RNA or small interfering (si)RNA libraries in mammalian cells, have been used for the identification of novel components of a variety of signal-transduction pathways [1]. Individual siRNAs have been shown to downregulate tens or even hundreds of genes by binding in a micro (mi)RNA-like manner to the 3’ untranslated regions (UTRs) of off-target mRNAs [18,19,20,21,22]. Screens attempt in a number of ways to control for these off-target effects [23], but results must still be interpreted cautiously
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