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Off-target effects ...
Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-beta pathway and reveal microRNA regulation of TGFBR2
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Schultz, Nikolaus (author)
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Marenstein, Dina R (author)
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De Angelis, Dino A (author)
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Wang, Wei-Qing (author)
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- Nelander, Sven, 1974 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin,Institute of Biomedicine
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Jacobsen, Anders (author)
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Marks, Debora S (author)
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Massagué, Joan (author)
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Sander, Chris (author)
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(creator_code:org_t)
- 2011
- 2011
- English.
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In: Silence. - 1758-907X. ; 14:2
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https://gup.ub.gu.se...
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https://doi.org/10.1...
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Abstract
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- 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.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Bioinformatik och systembiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)
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Silence
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- By the author/editor
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Schultz, Nikolau ...
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Marenstein, Dina ...
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De Angelis, Dino ...
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Wang, Wei-Qing
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Nelander, Sven, ...
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Jacobsen, Anders
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show more...
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Marks, Debora S
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Massagué, Joan
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Sander, Chris
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show less...
- About the subject
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Basic Medicine
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and Cell and Molecul ...
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Bioinformatics a ...
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Silence
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University of Gothenburg