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Title: | Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-? pathway and reveal microRNA regulation of TGFBR2 | Authors: | Schultz, N Marenstein, D.R De Angelis, D.A Wang, W.-Q Nelander, S Jacobsen, A Marks, D.S Massagué, J Sander, C |
Keywords: | green fluorescent protein microRNA microRNA 20a microRNA 34a microRNA 373 Smad2 protein small interfering RNA transforming growth factor beta transforming growth factor beta receptor 1 transforming growth factor beta receptor 2 unclassified drug 3' untranslated region article CDKN1A gene cell proliferation cell survival controlled study gene gene identification gene sequence gene targeting gene translocation genetic analysis genetic regulation genetic screening genetic transcription human human cell keratinocyte nuclear localization signal open reading frame PAI 1 gene phenotype priority journal protein expression protein phosphorylation protein targeting RNA interference sequence analysis signal transduction SMAD7 gene TGFBR1 gene TGFBR2 gene |
Issue Date: | 2011 | Publisher: | BMC | Citation: | Schultz, N, Marenstein, D.R, De Angelis, D.A, Wang, W.-Q, Nelander, S, Jacobsen, A, Marks, D.S, Massagué, J, Sander, C (2011). Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-? pathway and reveal microRNA regulation of TGFBR2. Silence 2 (1) : 3. ScholarBank@NUS Repository. https://doi.org/10.1186/1758-907X-2-3 | Rights: | Attribution 4.0 International | 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)-? pathway in a human keratinocyte cell line. The TGF-? pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-? 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-? 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-? 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-? 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. © 2011 Schultz et al; licensee BioMed Central Ltd. | Source Title: | Silence | URI: | https://scholarbank.nus.edu.sg/handle/10635/178179 | ISSN: | 1758-907X | DOI: | 10.1186/1758-907X-2-3 | Rights: | Attribution 4.0 International |
Appears in Collections: | Staff Publications Elements |
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