State Key Laboratory for Pharmaceutical Biotechnology of Nanjing University, Nanjing, 210023, China; Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, China; School of Medicine, Southeast University, Nanjing, 210009, China.
Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, China.
Biochem Biophys Res Commun. 2020 Apr 30;525(2):469-476. doi: 10.1016/j.bbrc.2020.02.127. Epub 2020 Feb 24.
The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered.
We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups.
Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1.
These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.
脆性 X 智力低下蛋白（FMRP）是脆性 X 综合征（FXS）的致病基因 FMR1 的产物，它能调节 miRNA 的表达，并参与 miRNA 介导的基因沉默。然而，FMRP 是否反过来受 miRNA 调控，这个问题尚未得到解答。
我们通过原位杂交检测 FMRP 的表达模式。通过种系转化和末端出同源重组分别产生 miR-315 过表达和敲除模型。采用 Western blot 和免疫组织化学检测果蝇 FMRP（dFMRP），并采用 Luciferase 报告基因检测证实 dfmr1mRNA 受 miR-315 调控。通过突触结构定量和电生理方法比较各组的突触功能。
本研究确定了 dFMR1 的转录产物，即 FMR1 的果蝇同源物，是 miR-315 的直接靶标。miR-315 在果蝇的神经系统中主要表达。过表达 miR-315 的果蝇表现出化蛹缺陷和孵化率降低。miR-315 纯合敲除状态在果蝇中是胚胎致死的。这些观察结果表明，miR-315 是果蝇神经系统的关键调节因子。此外，基于计算预测和细胞内 luciferase 及体内试验证明，dfmr1 是 miR-315 的直接靶标。最后，我们以神经肌肉接头作为模型，发现 miR-315 通过靶向 dfmr1 调节突触结构和传递。
这些发现有力地证明了 miR-315 在果蝇神经系统中靶向 dfmr1，作为 FMRP 表达精细调节的调控因子。
