title: Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2.
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.
authors.affiliation: Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Schultz
authors.affiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Dina R
authors.initials: DR
authors.lastname: Marenstein
authors.affiliation: High-Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Dino A
authors.initials: DA
authors.lastname: De Angelis
authors.affiliation: Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Wei-Qing
authors.initials: WQ
authors.lastname: Wang
authors.affiliation: Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.|Cancer Center Sahlgrenska, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Nelander
authors.affiliation: Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Anders
authors.initials: A
authors.lastname: Jacobsen
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Debora S
authors.initials: DS
authors.lastname: Marks
authors.affiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Joan
authors.initials: J
authors.lastname: Massagué
authors.affiliation: Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Sander