Figure 6: SUMOylation of TARBP2 promotes its binding with pre-miRNAs.

(a) The mutation K52R of TARBP2 slightly influences mature microRNA production. Differential expression analysis of mature miRNA profiles was scatter-plotted based on high-throughput small RNA-sequencing data of A549^luc stable cell lines expressing Flag-TARBP2-WT (x axis) and -K52R (y axis). (b) The expression levels of mature miRNAs are not much different between TARBP2-WT and -K52R expressed in cells. Quantitative PCR was performed to assess the expression of let-7a-1, let-7c, miR-10b, miR-19b-3p, miR-27ac, miR125b-1, miR-146, miR-296-3p and miR-331-3p in A549^luc stable cell lines expressing Flag-TARBP2-WT and -K52R. (c–e) SUMOylation of TARBP2 enhances the binding between TARBP2 and pre-miRNAs. (c) Stable 293T cell lines expressing the control vector, Flag-TARBP2-WT or -K52R were transiently transfected with pri-miR21. Thirty-six hours after transfection, cells were lysed for IP with anti-Flag antibody to pull down RNAs. Bound RNAs were extracted and analysed using real-time quantitative PCR for pre-miR21. Left panel: relative fold of pre-miR21 binding with Flag-TARBP2-WT or -K52R. Middle panel: efficiency of IP was detected with western blot analysis. Right: as a control, the expression levels of mature miR21 were analysed with total RNAs using quantitative PCR. (d) Stable 293T cell lines expressing control vector, Flag-TARBP2-WT or -K52R were transiently transfected with pre-miR21. The RIP assay was performed as c. (e) Stable control or Senp1 shRNA knockdown 293T cell lines were transfected with indicated plasmids. Thirty-six hours after transfection, cells were lysed and the RNA-binding assay was performed. The relative fold of pre-miR125b1 binding with TARBP2 was shown with quantitative PCR. Differences between individual groups as indicated were analysed using the t-test (two-tailed and unpaired), and P values of <0.01 (**) or <0.001 (***) are considered significant.