Fig. 5: M1-Ubi of AGO2 inhibits the miRISC activity.

a HOIP/HOIL-1L, but not HOIP-C885A/HOIL-1L and HOIP-C916A/HOIL-1L reduced the inhibition of luciferase activity mediated by AGO2. b OTULIN recovered the reduced inhibition of luciferase activity by LUBAC. c, d Knockdown HOIP by siRNAs in 293T cell (c) or by shRNA in HeLa cell (d) increased the inhibition of luciferase activity mediated by AGO2. 293T cells (a–c) or HeLa-shHOIP cells (d) transfected with indicated plasmids were harvested for the psiCHECK2-4xlet-7a-BS of dual-luciferase assay. Dual-luciferase reporter assay data were mean ± s.e.m., n = 3 (a, c, d) or n = 4 (b) biologically independent experiments, and P-values were determined by unpaired two-sided t-test. e, f Knockdown of HOIP increased let-7a-targeted GFP protein and mRNA levels. HeLa cells were transfected with indicated plasmids and siRNAs, the GFP protein expression was detected by WB (e). 293T cell stably expressing GFP-4x-let-7a-BS was transfected with HOIP siRNAs, and then total RNAs were extracted for detection of GFP mRNA levels by qRT-PCR. Data were mean ± s.d., n = 4 biologically independent samples, and P-values were determined by unpaired two-sided t-test (f). g, h Knockdown of OTULIN decreased let-7a-targeted GFP and c-MYC decay. HeLa cells were transfected with indicated plasmids and siRNAs, the proteins GFP (g) and c-MYC (h) expression were measured by WB analysis.