Nucleic Acids Research Advance Access originally published online on September 3, 2009
Nucleic Acids Research 2009 37(19):6587-6599; doi:10.1093/nar/gkp714
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Nucleic Acids Research, 2009, Vol. 37, No. 19 6587-6599
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation
1Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, 2Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, 3Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555 and 4Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32611, USA
*To whom correspondence should be addressed. Tel: +1 850 645 2402; Fax: +1 850 645 8447; Email: tang{at}bio.fsu.edu
Received May 15, 2009. Revised August 13, 2009. Accepted August 14, 2009.
Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short-hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify a shRNA that potently activates the IFN pathway in human cells in a sequence- and 5'-triphosphate-dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation.