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

Nikolaus Schultz; Dina R Marenstein; Dino A De Angelis; Wei-Qing Wang; Sven Nelander; Anders Jacobsen; Debora S Marks; Joan Massagué; Chris Sander

doi:10.1186/1758-907X-2-3

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

Silence. 2011 Mar 14:2:3. doi: 10.1186/1758-907X-2-3.

Authors

Affiliations

¹ Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
² Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
³ High-Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
⁴ Cancer Center Sahlgrenska, University of Gothenburg, Gothenburg, Sweden.
⁵ Department of Systems Biology, Harvard Medical School, Boston, MA, USA.

^# Contributed equally.

Abstract

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)-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.

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-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.

Conclusions: Our 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.