Figure 4.

NF90 cooperates with miR-15a to suppress BAFF translation. (A) Schematic of BAFF-WT and BAFF-var mRNAs, with the location of miR-15a and NF90 indicated. (B, C) The levels of BAFF, NF90 and sBAFF produced by THP-1 cells were assessed by western blot analysis (B) or ELISA (C) 48 h after overexpressing miR-15a by transfecting a miR-15a mimic (left) or neutralizing miR-15a by transfecting a miR-15a antagomir (right). (D) BAFF mRNA levels in THP-1 cells transfected with miR-15a mimic as in panel (B) were measured 48 h later by RT-qPCR analysis. (E) AGO2 RIP analysis was performed in cells expressing normal (Ctrl shRNA) or reduced (NF90 shRNA) NF90 levels to identify AGO2-bound BAFF mRNA. (F) The steady-state levels of miR-15a in the cells described in panel (E) were assessed by RT-qPCR analysis. (G) The cells described in (E) were transfected with biotinylated miR-15a or a biotinylated control RNA; 48 h later, cells were lysed and the levels of BAFF mRNA enrichment in the biot-miR-15a pulldown were assessed by RT-qPCR analysis. (H) THP-1 cells described in (E) were co-transfected with a chimeric reporter construct expressing EGFP-BAFF-WT 3′UTR mRNA and either Ctrl miRNA or miRNA-15a mimic; 24 h later, EGFP expression levels were assessed by western blot analysis (left). EGFP signals were quantified using ImageJ and plotted (right). (I) Forty-eight hours after transfecting THP-1 cells with Ctrl miRNA or miR-15a mimic, the interaction of NF90 with BAFF mRNA was assessed by RIP followed by RT-qPCR analysis. (J) Model summarizing the results of this study. The wild-type TNFSF13B gene uses the constitutive polyadenylation site (blue, PA) to give rise to BAFF-WT mRNA (left), bearing a long 3′UTR with two miR-15a sites and an NF90 binding site; the cooperative actions of these factors contribute to maintaining low BAFF translation levels and overall BAFF production. The variant TNFSF13B gene uses with high frequency an upstream polyadenylation site (green, APA) that gives rise to a shorter transcript, BAFF-var mRNA (right), bearing a shorter 3′UTR that lacks the distal miR-15a site and the NF90 binding site; consequently, BAFF-var mRNA is translated at aberrantly higher levels and results in the production of higher BAFF in individuals bearing this variant sequence (8). In (C, D, G, H, I), the data represent the means and SEM from three independent experiments (*P < 0.05, ***P< 0.001; significance was determined by Student's t-test).