FXR1P but not FMRP regulates the levels of mammalian brain-specific microRNA-9 and microRNA-124

Abstract

Mammalian brain-specific miR-9 and miR-124 have been implicated in several aspects of neuronal development and function. However, it is not known how their expression levels are regulated in vivo. We found that the levels of miR-9 and miR-124 are regulated by FXR1P but not by the loss of FXR2P or FMRP in vivo, a mouse model of fragile X syndrome. Surprisingly, the levels of miR-9 and miR-124 are elevated in fmr1/fxr2 double-knock-out mice, in part reflecting posttranscriptional upregulation of FXR1P. Indeed, FXR1P is required for efficient processing of pre-miR-9 and pre-miR-124 in vitro and forms a complex with Dicer and pre-miRNAs. These findings reveal differential roles of FMRP family proteins in controlling the expression levels of brain-specific miRNAs.

Figure 1.

The levels of brain-specific miR-9 and miR-124 are regulated by FXR1P but not FMRP or FXR2P in vivo. A, Brain levels of miR-124 are lower in E18 fxr1 mutant embryos than in WT embryos, as shown by Northern blot. B, miR-9 expression was also decreased in embryonic mouse brains in the absence of FXR1P. C, Relative levels of miR-124 and miR-9 normalized to U6 RNA in the brains of E18 embryos. Values are mean ± SEM of three embryos for each genotype. The experiment was repeated with similar results. ***p < 0.001 by Student's t test. D, Western blot analysis confirms the absence of Fmr1 in the fmr1 KO mice in E and F. E, Northern blot analysis showing the expression level of mature miR-124 in the brains of four WT and four fmr1 KO mice at 2 months of age. U6 RNA served as the loading control. F, MiR-9 levels in the brains of three WT and three fmr1 KO mice. G, Western blot analysis confirmed the absence of FXR2P in the fxr2 KO mice in H. H, Expression levels of miR-124 in the brains of four WT and four fxr2 KO mice at 2 months of age. I, Expression levels of miR-124 and miR-9 normalized to U6 RNA. Values are mean ± SEM of three independent experiments.

Figure 2.

The levels of brain-specific miR-9 and miR-124 are elevated in fmr1/fxr2 double-knock-out (KO) mice. A, The levels of miR-9 and miR-124 are significantly elevated in the brains of fmr1/fxr2 double-KO mice at 2 months of age. B, Quantification of the levels of miR-9 and miR-124 in the brains of 2-month-old WT, fmr1 KO, and fmr1/fxr2 double-KO mice. Values are mean ± SEM (n = 2–4 mice per genotype). p < 0.001 by Student's t test for fmr1/fxr2 double-KO mice compared with WT. The experiment was repeated with similar results. C, Western blot analysis confirmed the absence of Fmr1 in single-KO mice and FMRP and FXR2P in double-KO mice. D, Western blot analysis revealed a 2.4-fold increase in FXR1P expression in fmr1/fxr2 double-KO mice (p < 0.001). Equal amounts of protein lysates were loaded in each lane. E, Real-time RT-PCR showed that fxr1 mRNA levels remained the same in the brains of fmr1 and fxr2 single- or double-KO mice. Values are mean ± SEM of three independent experiments.

Figure 3.

FXR1P affects the processing efficiency of pre-miR124 or pre-miR-9 by Dicer in HEK293 cells. A, The relative expression levels of three miR-124 precursors in the adult mouse brain as measured by quantitative RT-PCR. B, Western blot analysis with anti-Flag antibody confirmed the ectopic expression of transfected FXR1P in HEK293 cells. C, Overexpression of Flag-FXR1P in HEK293 cells increased the steady-state level of miR-124 processed from transfected pre-miR-124-2. D, Relative miR-124 levels in transfected HEK293 cells with FXR1P overexpression. E, Overexpression of Flag-FXR1P in HEK293 cells increased the steady-state level of miR-9 processed from transfected pre-miR-9-2. F, Quantification of relative miR-124 levels in transfected HEK293 cells with FXR1P overexpression. Values are mean ± SEM of three independent experiments. **p < 0.01 by Student's t test. G, H, qRT-PCR analysis indicates that pre-miR-124-2 (G) or pre-miR-9-2 (H) formed a complex with FXR1P. I, Co-IP experiment demonstrates an association between Dicer and FXR1P in HEK293 cells. IP, Immunoprecipitation with Flag antibody. J, FXR1P does not affect the steady-state level of Dicer expression as shown by Western blot analysis.

Figure 4.

Loss of endogenous FXR1P affects the efficient processing of transfected brain-specific miRNA precursors. A, The level of mature miR-124 processed from transfected pre-miR-124-2 was reduced in the absence of FXR1P. Two independent siRNA knockdown experiments are presented in this panel. B, The level of mature miR-9 processed from transfected pre-miR-9-2 was reduced in the absence of FXR1P. Two independent siRNA knockdown experiments are presented in this panel. C, Quantification of the levels of mature miR-9 and miR-124 in the presence of absence of endogenous FXR1P. D, Fxr1 siRNA effectively decreased the expression of FXR1P in HEK293 cells, as shown by Western blot analysis. Asterisk indicates a nonspecific band recognized by the anti-FXR1P antibody. Lanes 3 and 4 are two independent knockdown experiments to show the effectiveness of the fxr1 siRNA.