Figure 5. (a) p24 HIV challenge assay shows escape from RNAi when multiple shRNAs are simultaneously expressed. Stable CEM cells expressing shSII (shSII) are protected from HIV infection. This protection is lost when two other shRNAs are expressed simulta- neously. Cells transduced with the vector backbone (Vect) are used as control. (b) Northern analysis shows comparable expression of shSII in transduced cells when expressed alone (shSII) or in combination with two other shRNAs (shSII + 2). A probe complementary to the mature SII RNA sequence was used to detect the processed siRNA. Probes for the other two shRNAs also revealed robust expression (data not shown). TRBP contributes to the preferential loading of different sequences into RISC Our experiments show that each siRNA sequence competes to a diﬀerent extent with other siRNAs and shRNAs. Incubation of 32P end-labeled siRNAs in HEK293 cytoplasmic extracts results in the formation of a gel-shifted complex using native polyacrylamide gel electrophoresis (Figure 6a, lane 2). The amount of binding in this assay is directly proportional to the intracellular eﬃcacy of the siRNAs analyzed (Sakurai et al., unpub- lished data). We speculated that TRBP, a double-stranded RNA-binding protein that has been shown to be part of the minimal RISC (26,32), could contribute to the loading of siRNA guide sequences into RISC and that it would be part of this complex. Depletion of TRBP from the cellular extracts (anti-TRBP) resulted in a marked reduction of siRNA complex formation (Figure 6a, lanes 4), while depletion of b-tubulin (anti-b-tubulin), which was used as control, had no eﬀect (Figure 6a, compare lanes 2 and 3). Identical results were obtained when depletion for b-actin was used as control (data not shown). Western analyses of the extracts showed that depletion of both TRBP and b-tubulin were nearly complete (Figure 6b). However, the small amount of residual gel-shifted complex may reﬂect incomplete removal of TRBP or could be due to binding of the siRNAs by other components of RISC. We repeated our competition experiment using cells in which Nucleic Acids Research, 2007, Vol. 35, No. 15 5161 TRBP levels were reduced by siRNA-mediated targeting of the TRBP2 mRNA (see Materials and Methods section). TRBP levels were reduced by 80% as deter- mined by western (Figure 6c) and northern (data not shown) analyses. Depletion of TRBP resulted in more equivalent competition by each shRNA sequence (shL, shVif, shSII, shTAT, shSI) or siRNA sequence (siH3, siH6, siH1), but competition with shLuc still persisted (Figure 6d). DISCUSSION We have shown competition between siRNAs and shRNAs when they are co-transfected into cultured cells. This competition is partially dependent upon saturation of Exportin-5 by the shRNAs and is largely sequence independent. The activity of a luciferase-targeted si or shRNA can be compromised by the ability of another siRNA to be preferentially loaded into RISC. The competition among shRNAs raises some important issues. Our data show that both siRNAs and shRNAs can compete with the endogenous microRNA pathway (Figure 4d) thus posing the potential for perturbation of endogenous cellular miRNA processes. In a recent publication, Grimm et al. (19) reported that some shRNA sequences were able to block the activity of the liver- speciﬁc miR122 and additionally caused severe liver toxicity and death of some of the treated mice. The authors attributed this adverse eﬀect to saturation of Exportin-5, postulating that despite being expressed from the same AAV vector backbone, some sequences were expressed at higher levels and were more toxic. Our results demonstrate that competition with endogenous microRNAs can be a general phenomenon and that Exportin-5 is only one of the components involved, as shown by the inability to fully restore the U6sh-luciferase activity when Exportin-5 was ectopically expressed (Figure 3b). Moreover, synthetic siRNAs, which do not rely on Exportin-5 for their transport, can still aﬀect the activity of both shRNAs (Figure 3a) and microRNAs (Figure 4d). These results indicate that the ability of a speciﬁc sequence to compete is also determined by its ability to be loaded into RISC. Interestingly, the relative competitive strength of the synthetic siRNAs with the Luc shRNA correlated with their eﬃcacy to down-regulate their cognate targets, which in turn is directly proportional to the complex formation in cell extracts (Sakurai et al., unpublished data). These results are consistent with a previously published study (33) in which competition was detected between heavily modiﬁed and unmodiﬁed syn- thetic siRNAs. The competition was found to correlate with the potency of the siRNAs and it was attributed to a possible saturation of the export system or competition for other RISC components. The aﬃnity of each sequence for one or more RNAi component(s) may determine the eﬃciency of loading of that sequence into RISC, thereby potentiating the competition with other sh/siRNAs and miRNAs. TRBP is a double-stranded RNA-binding protein that has been shown to associate with both Dicer and Ago-2 (26,32).