Noise amidst the silence: Off-target effects of siRNAs?

Rosetta Inpharmatics LLC, 401 Terry Avenue N., Seattle, WA 98109, USA.
Trends in Genetics (Impact Factor: 11.6). 12/2004; 20(11):521-4. DOI: 10.1016/j.tig.2004.08.006
Source: PubMed

ABSTRACT RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is widely used to silence gene expression and to define gene function in mammalian cells. Initially, this gene silencing via transcript degradation was believed to be exquisitely specific, requiring near-identity between the siRNA and the target mRNA. However, several recent reports have suggested that non-specific effects can be induced by siRNAs, both at the level of mRNA and protein. These findings suggest that siRNAs can regulate the expression of unintended targets, and argue for further experiments on the mechanism and extent of off-target gene regulation(s). In the meantime, caution is warranted in interpreting gene function and phenotypes resulting from RNAi experiments.

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    • "We were concerned that the silencing of Cm-eIF4E could also result in the indirect silencing of Cm-eIF(iso)4E because these two genes share 75% of their nucleotide sequences. Nonspecific silencing, which is based on partial sequence homology, has been described previously (Jackson and Linsley, 2004; Scacheri et al., 2004). Recently, it has been shown that the silencing of HC-Pro from ZYMV in cucumber and melon resulted in resistance to ZYMV, but also to PRSV and WMV (Leibman et al., 2011). "
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    ABSTRACT: Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.
    Molecular Plant Pathology 02/2012; 13(7):755-63. DOI:10.1111/j.1364-3703.2012.00785.x · 4.49 Impact Factor
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    • "It must be noted that there appears to be some effect when comparing NS siRNA with Mock treated controls. Other groups have documented effects on cellular processes including cell viability, proliferation, cell cycle distribution, apoptosis, and migration (Jackson and Linsley, 2004, Tschahatganeh, 2007). We suggest here that this could perhaps to due to the increased nucleic acid ratio in NS siRNA treated cells versus Mock transfected controls, causing the cells some stress. "
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    ABSTRACT: The stress-inducible transcription factor, nuclear factor (NF)-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 small interfering RNA (siRNA), PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65(-/-) cells were more sensitive to ionizing radiation (IR) than p65(+/+) cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65(+/+), but not p65(-/-) cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single-strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65(+/+) and p65(-/-) cells. As preventing SSB repair did not radio-sensitize p65(-/-) cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for tumor necrosis factor (TNF)-α-treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA damage-activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.
    Oncogene 06/2011; 31(2):251-64. DOI:10.1038/onc.2011.229 · 8.56 Impact Factor
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    • "The RNAi approach offers the flexibility of using immortalized cell lines or primary neurons from rats, both of which can yield more cells in culture than the primary neurons isolated from mice. However, two well-known caveats that are intrinsic to the RNAi approach need to be addressed with appropriate controls (Jackson and Linsley, 2004). The first caveat pertains to the efficacy of expression knockdown of the target gene product, as such efficacy may vary significantly depending on the specific reagent and the experimental conditions used. "
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    ABSTRACT: Dysbindin (also known as dysbindin-1 or dystrobrevin-binding protein 1) was identified 10 years ago as a ubiquitously expressed protein of unknown function. In the following years, the protein and its encoding gene, DTNBP1, have become the focus of intensive research owing to genetic and histopathological evidence suggesting a potential role in the pathogenesis of schizophrenia. In this review, we discuss published results demonstrating that dysbindin function is required for normal physiology of the mammalian central nervous system. In tissues other than brain and in non-neuronal cell types, the protein has been characterized as a stable component of a multi-subunit complex, named BLOC-1 (biogenesis of lysosome-related organelles complex-1), which has been implicated in intracellular protein trafficking and the biogenesis of specialized organelles of the endosomal-lysosomal system. In the brain, however, dysbindin has been proposed to associate into multiple complexes with alternative binding partners, and to play a surprisingly wide variety of functions including transcriptional regulation, neurite and dendritic spine formation, synaptic vesicle biogenesis and exocytosis, and trafficking of glutamate and dopamine receptors. This puzzling array of molecular and functional properties ascribed to the dysbindin protein from brain underscores the need of further research aimed at ascertaining its biological significance in health and disease.
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