Figure 8: SUMOylation of TARBP2 controls miRNA/siRNA efficiency.

(a,b) SUMOylation of TARBP2 is required for efficient RNA-induced gene silencing by recruiting more Ago2. A549^luc cell lines stably expressing pri-miRNA21 and Flag-TARBP2-WT or -K52R (a, named as miR21-WT or miR21-K52R, respectively), or pri-miR130b and Flag-TARBP2-WT or -K52R (b, named as miR130b-WT and miR130b-K52R, respectively) were generated by the lentiviral system. Cell lysates were used for IP with anti-Flag antibody and then detected with anti-Ago2 antibody. Cell lysates were used for immunoblotting with anti-Ago2, -GAPDH, -Flag and -PTEN (a) or -ZEB1 (b) antibodies. For full scans of western blots, see Supplementary Fig. 8. (c) The SUMO-site mutation K52R of TARBP2 dominant-negatively abolishes inhibition of cell migration mediated by miR130b-ZEB1. Cell motilities of stable A549^luc cell lines including Control, miR130b-con, miR130b-WT and miR130b-K52R were analysed by a wound-healing assay with μ-Dish. (d) RIP assay was performed with 293T cells transfected with pri-miR21, myc-Ago2 and Flag-TARBP2-WT or -K52R. Thirty-six hours after transfection, cells were lysed for IP with anti-myc antibody to pull down RNA. Ago2-bound mature miR21 was extracted and analysed by real-time quantitative PCR. (e–g) SUMOylation of TARBP2 influences the efficiency of miRNA or siRNA mimic duplexes via the formation of the functional RISC. TARBP2-WT or -K52R with/without Ago2, together with miR21 (e), miR130b (f), mimic duplexes or siPTEN (siRNA for PTEN; g) were co-transfected into HeLa cells, and the expression levels of the corresponding targets PTEN, ZEB1 and PTEN were determined. Immunoblotting was performed with anti-PTEN, anti-ZEB1, anti-Myc, anti-Flag and anti-GAPDH antibodies. (h) A model for SUMOylation of TARBP2 controls the efficiency of RNA-induced gene silencing by increasing its interaction with Ago2 and precursor miRNAs/siRNAs. ‘S’—SUMOylation; ‘P’—Phosphorylation.