It has been reported that certain chemically synthesized and phage polymerase in vitro transcribed siRNAs can non-specifically induce IFN responses and produce offtarget effect via various PRRs, including TLRs. However, the induction of IFN response by shRNAs and its underlying mechanisms have not been as well studied. The actual number of shRNAs that are capable of triggering IFN response will certainly be larger than the few that have been reported in the literature, yet very little is known about the unique characteristics of the select shRNAs and the pathway that they use to activate IFN production. The present study identifies RIG-I, but not MDA5 or TLR3, as the mediator for activation of IFN responses by two shRNAs that are distinct in sequence and structure but both capable of IFN induction in human cells. This was demonstrated by induction of IRF-3 dimerization, activation of IFN promoters, induction of endogenous ISGs (ISG15, OAS and RIG-I), and secretion of IFN, all of which depended on RIG-I and its downstream adaptor, MAVS. In addition, we show that delivery of these shRNAs via lentiviral transduction does not reduce their IFN-inducing capacity, indicating that the ability of lentiviral vector transduction to avoid IFN induction by shRNAs, as reported previously (48), may not be universally applicable to all the shRNAs. Specific recognition of dsRNAs or ssRNAs bearing 5 0 -triphosphates by RIG-I is presumably determined mostly by structural features other than the nucleotide sequence of the RNA. Yet IFN activation by sh-B971 exhibited a stringent dependence on specific nucleotides at multiple positions of the shRNA. An AA dinucleotide at the beginning of the U6 transcript has previously been suggested to result in aberrant transcription, and preserving a C/G sequence at positions À1/+1 suggested to avert IFN induction (38) . We indeed observed a strict requirement for an adenylate at the +1 position of sh-B971 for RIG-I recognition and IFN activation, but we observed no difference in expression levels or the apparent sizes of the sh-B971 RNAs bearing either an A or a G at the +1 position. Furthermore, mutations introduced elsewhere in the shRNA also abolished or diminished sh-B971's ability to activate IFN, suggesting additional sequence requirement for efficient RIG-I recognition and IFN triggering. Despite these results, because we were not successfully in cloning and sequencing the vectorexpressed siRNA, we cannot exclude the possibility that the adenylate at the +1 position interferes with transcription and that the resultant abnormal transcript contributes to IFN induction.