Fig. 3

PSMC3 is required for efficient RNAi. a Depletion of PSMC3 abolishes the siRNA-mediated cleavage of EGFP mRNA. Overview of the siRNA-mediated cleavage of target mRNA (left). A stable HeLa cell line expressing EGFP was either untransfected or transfected with the indicated siRNAs. pDsRed2-N1, a plasmid expressing RFP (red fluorescent protein), was also included for normalization. After 48 h, expression ratios between the EGFP and RFP reporters were calculated on an F-4500 fluorescence spectrophotometer (right). b Expression of the siRNA-resistant PSMC3 mutant overrides the effect of PSMC3 depletion on RNAi. EGFP-expressed HeLa cells were transfected with siNC or siPSMC3, together with pcDNA3 plasmid only or vectors expressing wild-type (wt) or siRNA-resistant (mut) PSMC3. After 24 h, cells were re-transfected with EGFP siRNA. The fluorescence value in the siNC treatment was set to 1. c PSMC3 is required for miR-21-mediated mRNA cleavage. Schematic of the miRNA-mediated cleavage of target mRNA (left). A stable HeLa cell line expressing EGFP-miR-21 (which contains a sequence with 1× perfect complementarity to miR-21 in its 3′ UTR) was transfected with the indicated siRNAs. The ratio of EGFP to RFP was normalized to quantify the effect of depleting AGO2 and PSMC3 on RNAi (right). d Expression of siRNA-resistant PSMC3 mutant rescues the cleavage activity of miRISC. EGFP-miR-21-expressing HeLa cells were transfected with control or PSMC3 siRNAs together with pcDNA3 plasmid only or vectors expressing wild-type (wt) or siRNA-resistant mutant (mut) PSMC3. The fluorescence value was detected on an F-4500 fluorescence spectrophotometer. e Depletion of PSMC3 has no effect on translational repression. Diagram of the siRNA-mediated translational repression of target mRNA (left). A stable HeLa cell line expressing EGFP-CXCR4 (which contains a sequence with 4× bulged CXCR4 binding sites in its 3′ UTR) was transfected with siRNAs targeting AGO2 or PSMC3. After 24 h, cells were re-transfected with control siRNA or CXCR4 siRNA. EGFP protein levels were measured and normalized to RFP as a control (right). In all statistical comparisons, three independent experiments were performed (mean ± SD, n = 3, Student’s t-test). *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001