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Published on: March 24, 2023
MiR-219 represses expression of dFMR1 in Drosophila melanogaster
Chao Wang1, Liang Ge2, Jianban Wu2
1Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, China; State Key Laboratory for Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, China.
Aims:
Fragile X mental retardation protein (FMRP) plays a vital role in mRNA trafficking and translation inhibition to regulate the synthesis of local proteins in neuronal axons and dendritic terminals. However, there are no reports on microRNA (miRNA)-mediated regulation of FMRP levels in Drosophila. Here, we aimed to identify miRNAs regulating FMRP levels in Drosophila.
Main Methods:
Using online software, we predicted and selected 11 miRNAs potentially acting on the Drosophila fragile X mental retardation 1 (dFMR1) transcript. These candidates were screened for modulation of dFMR1 transcript levels at the cellular level using a dual luciferase reporter system. In addition, we constructed a transgenic Drosophila model overexpressing miR-219 in the nervous system and quantified dFMRP by western blotting. The neuromuscular junction phenotype in the model was studied by immunofluorescence staining.
Key Findings:
Among the 11 miRNAs screened, miR-219 and miR-960 reduced luciferase gene activity by binding to the 3'-UTR of the dFMR1 transcript. Mutation of the miR-219 or miR-960 binding sites on the transcript resulted in complete or partial elimination of the miRNA-induced repression. Western blots revealed that dFMRP expression was decreased in the miR-219 overexpression model (Elav>miR-219). Drosophila larvae overexpressing miR-219 showed morphological abnormalities at the neuromuscular junction (increased synaptic boutons and synaptic branches). This finding is consistent with some phenotypes observed in dfmr1 mutants.
Significance:
Our results suggest that miR-219 regulates dFMR1 expression in Drosophila and is involved in fragile X syndrome pathogenesis. Collectively, these findings expand the current understanding of miRNA-mediated regulation of target molecule-related functions.
Insights
MicroRNA-219 (miR-219) regulates fragile X mental retardation protein (FMRP) levels in Drosophila. This discovery sheds light on fragile X syndrome pathogenesis and miRNA-mediated gene regulation.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Fragile X mental retardation protein (FMRP) is crucial for neuronal protein synthesis regulation.
- MicroRNA (miRNA)-mediated regulation of FMRP in Drosophila is not well understood.
Purpose of the Study:
- To identify specific miRNAs that regulate FMRP levels in Drosophila.
- To investigate the role of identified miRNAs in fragile X syndrome pathogenesis.
Main Methods:
- Bioinformatic prediction of miRNAs targeting the dFMR1 transcript.
- Dual luciferase reporter assay to validate miRNA binding and repression.
- Generation of transgenic Drosophila overexpressing miR-219.
- Western blotting to quantify dFMRP levels.
- Immunofluorescence staining to analyze neuromuscular junction morphology.
Main Results:
- miR-219 and miR-960 were identified as direct regulators of the dFMR1 transcript.
- Overexpression of miR-219 in Drosophila led to decreased dFMRP levels.
- miR-219 overexpression caused neuromuscular junction abnormalities, mirroring dfmr1 mutant phenotypes.
Conclusions:
- miR-219 is a key regulator of dFMRP expression in Drosophila.
- This study implicates miR-219 in the pathogenesis of fragile X syndrome.
- Findings enhance understanding of miRNA-mediated gene regulation in biological functions.
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