%0 Journal Article
%T Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-¦Â pathway and reveal microRNA regulation of TGFBR2
%A Nikolaus Schultz
%A Dina R Marenstein
%A Dino A De Angelis
%A Wei-Qing Wang
%A Sven Nelander
%A Anders Jacobsen
%A Debora S Marks
%A Joan Massagu¨¦
%A Chris Sander
%J Silence
%D 2011
%I BioMed Central
%R 10.1186/1758-907x-2-3
%X 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-¦Â 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-¦Â 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.Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-¦Â 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.RNA interference (RNAi) has emerged as a central tool to analyze the function of mammalian genes, both in vitro and in vivo. The technology has been widely used in mammalian cells to suppress the expression level of individual genes, thus helping to define the functional roles of genes, particularly in disease. RNAi screens, using either double-stranded RNA in Drosophila cells, or small
%U http://www.silencejournal.com/content/2/1/3