The steady-state level of the nervous-system-specific microRNA-124a is regulated by dFMR1 in Drosophila - PubMed (original) (raw)

The steady-state level of the nervous-system-specific microRNA-124a is regulated by dFMR1 in Drosophila

Xia-Lian Xu et al. J Neurosci. 2008.

Abstract

Fragile X syndrome is the most common form of inherited mental retardation caused by loss of the fragile X mental retardation protein 1 (FMRP). The detailed molecular pathways underlying the pathogenesis of this disorder remain incompletely understood. Here, we show that miR-124a, a nervous-system-specific miRNA, is associated with the Drosophila homolog of FMRP (dFMR1) in vivo. Ectopic expression of wild-type but not mutant miR-124a precursors decreased dendritic branching of dendritic arborization sensory neurons, which was partially rescued by the loss of dFMR1 activity, suggesting that the biogenesis and/or function of miR-124a are partially dependent on dFMR1. Indeed, in contrast with the complete loss of mature miR-124a in Dicer-1 mutants, steady-state levels of endogenous or ectopically expressed mature miR-124a were partially reduced in dfmr1 mutants, whereas the level of pre-miR-124a increased. This effect could be explained in part by the reduced abundance of the Dicer-1-Ago1 complex in the absence of dFMR1. These findings suggest a modulatory role for dFMR1 to maintain proper levels of miRNAs during neuronal development.

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Figures

Figure 1.

Figure 1.

miR-124a and dFMR1 are present in the same RNP complex in the Drosophila nervous system. A, In situ analysis shows prominent miR-124a expression in the brain and ventral nerve cord in a stage 13 Drosophila embryo. B, _miR-124a_-Gal4 drives GFP expression specifically in the embryonic nervous system, and the expression persists to adulthood. The axon bundles from motor neurons are visible in this image. C, Immunoprecipitation (IP) experiments demonstrate that miR-124a and dFMR1 are present in the same RNP complex independent of polyribosomes. dFMR1-containing RNP complexes were pulled down with HA antibody, and miR-124a in the immunoisolates was detected with a 32P-labeled ribonucleotide probe. A portion of total RNA from control larvae before IP was loaded as a positive control. D, Western blot analysis of immunoisolates with HA antibody. HA-tagged dFMR1 was readily detectable in lysates isolated from larval brains or in immunoisolates after IP. The absence of tubulin in immunoisolates indicates the specificity of the IP experiment with HA antibody. In C and D, control larvae express _elav_-Gal4 alone, and other larvae express dFMR1 under the control of _elav_-Gal4.

Figure 2.

Figure 2.

miR-124a suppresses dendritic branching of DA neurons in Drosophila. A, Wild-type ddaE and ddaF neurons in the dorsal cluster were labeled with mCD8-GFP driven by Gal4221. B, Ectopic expression of pre-miR-124a reduced dendritic branching of DA neurons. C, Ectopic expression of pre-miR-9a increased dendritic branching. D, Quantification of dendritic ends in wild-type (WT) ddaE or ddaF neurons and in neurons expressing pre-miR-124a, pre-miR-9a, and mutant pre-miR-124a. The values are mean ± SEM. ***p < 0.001 versus wild type.

Figure 3.

Figure 3.

dFMR1 is required for pre-miR-124a to exert its effect on dendritic morphogenesis of DA neurons. A, A wild-type (WT) ddaC neuron is labeled with mCD8-GFP whose expression is driven by Gal4 477. B, Reduced dendritic branching and dendritic field size in a ddaC neuron expressing pre-miR-124a. C, Expression of pre-miR-124a in ddaC neurons in dfmr1 mutant background (dfmr1 4/dfmr1 4 or dfmr1 3/dfmr1 4) resulted in a dendritic field of nearly normal size. D, Quantification of dendritic ends of wild-type (WT) ddaC neurons and neurons expressing pre-miR-124a in normal or dfmr1 mutant backgrounds. The values are mean ± SEM. **p < 0.01. E, A MARCM-generated dcr-1 mutant ddaC neuron. F, A dcr-1 mutant ddaC neuron expressing pre-miR-124a. G, Northern blot analysis of miR-124a processing in WT or dcr-1 mutant larvae demonstrates that Dcr-1 is absolutely required for the production of mature miR-124a in vivo.

Figure 4.

Figure 4.

dFMR1 is required to ensure normal steady-state levels of mature miR-124a in the Drosophila nervous system. A, Northern blot analysis of miR-124a levels in Drosophila third instar larvae. Lane 1: Gal4 109(2)80, UAS-mCD8-GFP/± larvae (controls). Lane 2: Gal4 109(2)80, UAS-mCD8-GFP+; _UAS-pre-miR-124a/_+ larvae that express miR-124a in all MD neurons and a small number of CNS neurons. Lane 3: Gal4 109(2)80, UAS-mCD8-GFP/+; UAS-mutant pre-miR-124a/+ larvae. Lane 4: Gal4 109(2)80, UAS-mCD8-GFP/+; dfmr13/dfmr14 larvae. Lane 5: Gal4 109(2)80, UAS-mCD8-GFP/UAS-pre-miR-124a; dfmr13/dfmr14 larvae. Lane 6: Gal4 109(2)80, UAS-mCD8-GFP/UAS-mutant pre-miR-124a; dfmr13/dfmr14 larvae. Equal amounts of total RNAs were loaded for each genotype, and 2 s RNA was used as the internal control. B, Relative expression levels of miR-124a in larvae with different genetic backgrounds and transgene expression. The data (mean ± SEM) were derived from three independent experiments and normalized against the levels of 2s RNA. C, Western blot analysis verified the absence of dFMR1 in dfmr1 mutant larvae as described in A. D, Quantitative RT-PCR analysis of relative expression levels of pri-miR-124a, pre-miR-124a, and mature miR-124a in wild-type (WT) and dfmr1 mutant larvae. E, The Northern blot membrane used in A was stripped and reprobed for miR-1 and miR-7 which showed that the in vivo expression levels of miR-1 and miR-7 were also reduced in dfmr1 mutant larvae. The values are mean ± SEM. ***p < 0.001 versus wild type.

Figure 5.

Figure 5.

The Dcr-1-Ago1 complex is less abundant in the absence of dFMR1. A, Control mouse IgG or Ago1 antibody were used to immunoprecipitate Ago1-containing complexes from lysates of dissected wild-type (WT) or dfmr1 mutant larval brains. The presence or absence of Dcr-1 or dFMR1 was detected by Western blot. This experiment was repeated three times. B, Quantification of the abundance of Dicer-1 in Ago1 immunoprecipitates based on three independent experiments. The values are mean ± SEM. p < 0.01 versus wild type. C, The protein band recognized by the Dcr-1 antibody obtained from Abcam is absent in the dcr-1 mutants. D, An Ago1 antibody (Okamura et al., 2004) identifies Ago1 isoforms that are absent in ago1 mutants. Note that the expression levels of Dcr-1 and Ago1 are the same in dfmr1 mutants and wild-type larvae. IP, Immunoprecipation.

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