Despite the abilities of both sh-B971 and sh-PCAF to activate the RIG-I pathway, the two shRNAs are unrelated in sequence. Two short stretches of siRNA sequences, GUCCUUCCAA and UGUGU, that have been previously defined as IFN-or cytokine-activating motifs (8, 9) are not found in either sh-B971 or sh-PCAF. Any common sequence motifs of IFN-activating shRNAs, if any, remain to be defined. The two shRNAs also differ in that one is predicted to contain one blunt end and the other two ends with overhangs. These results suggest that, although blunt ends may increase siRNA's ability to be recognized by RIG-I (47), they are not required for IFN activation by an endogenously expressed shRNA. The best-characterized RNA structure motif recognized by RIG-I is the 5 0 -ppp, which is absent from virtually all the cellular RNAs as a result of either 5 0 -capping or internal cleavage before their appearance in the cytoplasm. A synthetic shRNA that has the same sequence as sh-B971 but lacks the 5 0 -ppp failed to induce IFN, suggesting the 5 0 -end status of the intracellularly expressed sh-B971 contributes to IFN activation. Whether or not the 5 0 -end of an shRNA is capped has not been investigated. Murine U6 RNA does not contain the trimethylguanosine cap that is present on mRNAs and other U small nuclear RNAs; instead it contains a g-monomethyl phosphate cap at its 5 0 -end (49) . Capping of heterologous transcripts produced from the mU6 promoter, however, requires a stem loop at the 5 0 -end of the transcript and an AUAUAC sequence immediately after (50) . Most shRNAs, including sh-B971 and sh-PCAF, would not meet these requirements and thus should contain unmodified 5 0 -ppp. Similarly, no evidence of a cap structure for H1 transcripts could be found in the literature. We attempted to express sh-B971 using a miRNA expression cassette and the pol II promoter (51) . The primary transcript generated with this construct would be capped at 5 0 -end by a trimethylguanosine cap and the final siRNA duplex would bear a monophosphate at the 5 0 -ends of both strands because of Drosha and Dicer cleavage. This version of the sh-B971 vector was much weaker in its ability to trigger IFN activation. Unfortunately the intracellular expression of the RNA duplex was also much weaker and barely detectable by northern blotting. In addition, no knockdown of the target CyPB mRNA was seen with this miRNA-based sh-B971 (data not shown). As a result, whether sh-B971, if expressed at higher level from this construct, could effectively activate IFN remains unclear.