5162 Nucleic Acids Research, 2007, Vol. 35, No. 15 Figure 6. (a) Gel shift assay shows loss of complex formation when TRBP depleted cellular extracts are mixed with labeled siRNAs. A 32P end- labeled anti-EGFP siRNA (without cellular extracts in lane 1) was mixed with: lane 2, total cellular extract; lane 3, extracts depleted of b-tubulin; lane 4 extracts depleted of TRBP. (b) Western detection of TRBP and b-tubulin after immunodepletion. Extracts were treated with TRBP or b-tubulin antibodies and precipitated as described in the Materials and Methods section. S, supernatant; P, pellet. The supernatant fractions show to be substantially depleted of TRBP or b-tubulin and were subsequently used in the gel shift assay for detection of complex formation (a). (c) Western analyses of lysates from cells treated with (+) or without ( ) TRBP siRNA as described in Materials and Methods section. Lysates from cells transfected with a TRBP-expressing plasmid (TRBP) were used for size determination. Detection of b-tubulin was used as an internal control. (d) TRBP down-regulation eliminates the diﬀerential competition among interfering RNAs. Twenty nanomolar of each synthetic siRNA (siH3, siH6, siH1) or 50 ng of each shRNA construct (shL, shVif, shSII, shTAT, shSI) were co-transfected with the ﬁreﬂy luciferase target (FLT) and the Luc shRNA in cells that were pre-treated for 5 days with a siRNAs targeted against TRBP. Fireﬂy luciferase units were calculated as previously described. It has been proposed that TRBP couples the initiation and executions steps of RNAi (26) and that it identiﬁes the guide strand prior for passage on to Ago-2 (34). Our data reveal that cellular down-regulation of TRBP results in loss of diﬀerential competition by the various sh/siRNAs, although competition per se still persists (Figure 6d), perhaps via a bypass mechanism for incorporation into the Ago2-binding domain. Our results support the role of TRBP as a sensor for the RISC loading or Ago-2 handoﬀ (26). However, it is possible that TRBP does not act alone and loading may include other RISC proteins. In our experimental system, sequences expressed from a microRNA backbone are poor competitors relative to shRNA or siRNAs of the same sequences (Figure 4). Two diﬀerent sequences were expressed from three diﬀerent microRNA backbones and none of them was able to compete (Figure 4 and data not shown) despite the fact that these sequences were still eﬀective in RNAi. The overall level of shRNA expression does not seem to be the primary reason for this lack of competition (Figure 4c), which is probably determined by the overall kinetics of incorporation. Since miRNAs are being shunted through the endogenous miRNA-processing pathway, this may slow their entry into RISC relative to highly expressed shRNAs and transfected siRNAs. Si and sh-RNAs are routinely used to down-regulate cellular genes, siRNA libraries have been widely employed to screen for gene function (9–11), and siRNAs are