br Fig IGF BP binds
Fig. 3. IGF2BP3 binds to TRIM25 mRNA and inhibits miR-3614 maturation in BC cells. (a) Proposed model of IGF2BP3, HuR and miR-3614 Trichostatin A (TSA) at the TRIM25 mRNA 3′-UTR. (b–e) The levels of IGF2BP3 mRNA, TRIM25 mRNA, pri-miR-3614 and miR-3614-3p were determined by qRT–PCR after BC cells transfection with si-IGF2BP3 and related controls. Data are presented as the mean ± SEM. (*P b 0.05, **P b 0.01, ***P b 0.001, ANOVA analysis). (f) Forty-eight hours after transfection with si-IGF2BP3 and treatment with E2 (100 nM), the levels of TRIM25, IGF2BP3, and β-actin (loading control) were assessed by Western blot analysis. (g) qRT–PCR analysis of TRIM25 mRNA stability in control or IGF2BP3-depleted BC cells. Data are presented as the mean ± SEM. (*P b 0.05, **P b 0.01, ***P b 0.001, Student's t-test) (h, i) RNA immunoprecipitation (RIP) combined with qRT–PCR assays of IGF2BP3 binding to TRIM25 mRNA in BC cells. Nonspecific rabbit IgG was used as a negative control. Input was used as a positive control. Western blot analysis of IGF2BP3 immunoprecipitated from control or IGF2BP3-overexpressed in MCF-7 cells or IGF2BP3-depleted in MDA-MB-231 cells (H). RT–PCR analysis of RNA precipitated with IGF2BP3 antibody or control IgG (I). (j) Schematic de-piction of the TRIM25 (A-D) biotinylated probes used for biotin pull-down analysis. (k) IGF2BP3 in the biotin pull-down samples was detected by Western blot analysis. (l) Western blot of TRIM25 and AGO2 expression in control or IGF2BP3-depleted (sh-IGF2BP3) MDA-MB-231 cells. (m-o) qRT-PCR analysis of RNA precipitated with a-Ago2 (Ago2 RIP) or nonspecific rabbit IgG (IgG RIP). Relative quantification for TRIM25 and IGF2BP2 transcript was performed using β-actin mRNA as a reference gene. Relative quantification for pri-miR-3614 and miR-3614-3p were performed using U6 as a reference gene. Statistical significance was estimated for each comparison using an unpaired t-test (***P b 0.001).
(Fig. 3g). However, silencing of HuR did not affect the expression of TRIM25 (Fig. S2). These results indicate that IGF2BP3 can promote TRIM25 expression and suppress miR-3614 maturation.
To confirm the predicted binding sites, we performed RBP immu-noprecipitation (RIP) and biotin pull-down analysis. As shown in Fig. 3h and i, TRIM25 mRNA was enriched in IGF2BP3 IP samples compared to the IgG IP samples, suggesting that IGF2BP3 specifically binds to TRIM25 mRNA. Furthermore, we prepared biotinylated probes (~50-nt fragments) spanning the 3′-UTRs at a site proximal and even overlapping the miR-3614-3p binding site, and examined their association with IGF2BP3 using biotin pull-down analysis. Bio-tinylated TRIM25 RNAs probes were incubated with the cell lysates, and the RBPs from the biotin pull-down were detected by western blot. The results indicate that IGF2BP3 binds to four regions with dif-ferent affinities. Among them, IGF2BP3 was found to bind preferen-tially to region A in MCF-7 cells, and to regions B and D in MDA-MB-231 cells (Fig. 3j and k).
Together, these results suggest that IGF2BP3 suppresses the forma-tion of mature miR-3614, thereby protecting TRIM25 mRNA stability.
3.4. IGF2BP3 modulates the interaction of miR-3614 and TRIM25 transcript associated with RISC
These data presented above suggest that IGF2BP3 may attenuate miR-3614-mediated TRIM25 mRNA decay. To further determine if IGF2BP3 protect TRIM25 transcript is associated with RISC, we tested this hypothesis by RIP with Ago2, a RISC component, from control or IGF2BP3-depleted MDA-MB-231 cells and further quantified co-purified RNA by qRT-PCR. Western blot showed IGF2BP3 depletion sig-nificantly repressed TRIM25 expression, however unaffected Ago2 ex-pression (Fig. 3l). As expected, RIP showed both of TRIM25 mRNA, pri-miR-3614 and miR-3614-3p co-precipitated with Ago2 from both con-trol and knockdown cells, and enriched concertation with Ago2 in-creased in IGF2BP3-depleted cells (Fig. 3m–o). The data suggest that IGF2BP3 stabilized TRIM25 mRNA associated with RISC.
We used Targetscan and RegRNA prediction programs to identify putative miR-3614-3p binding sites in the TRIM25 transcript and found potential miR-3614-3p binding motif which is in the 3′-UTR re-gion. To validate the theoretical relationship between miR-3614-3p and TRIM25, we constructed luciferase reporters by cloning the wild-type 3′-UTRs of TRIM25 (TRIM25-WT) or its mutant version (TRIM25-MUT) into the pmiRGLO dual-luciferase reporter vector (Fig. 4a). Pre-miR-3614-overexpressed plasmid and TRIM25-WT- or MUT-3′-UTR vectors were co-transfected into HEK293T cells. We found that pre-miR-3614 significantly reduced luciferase activity in TRIM25-WT sam-ples but did not significantly affect the TRIM25-MUT samples (Fig. 4b), indicating that miR-3614-3p binds directly to the 3′-UTR of TRIM25.