Functional Studies On One Intronic MiR-932 Hosted In Drosophila Neuroligin2

MicroRNA(miRNA)is one class of small(-22 nucleotides),endogenous noncoding RNAs that are involved in post-transcriptional gene regulation of protein coding gene expression across diverse organisms.Despite great progress for two decades in microRNAs(miRNAs),our understanding about the function of miRNAs,especially for intronic miRNA is limited.Nervous system as the most complicated and exquisite network system in human relies on billions of neurons with associated connections between them.Mammal synaptic cell adhesion molecules(CAM)Neuroligins and Neurexins form the trans-pre and post synaptic bridges,with key roles in the growth,differentiation,development and function.We investigate one intronic miR-932 hosted in Drosophila neuroligin 2(dnlg2),since the direct regulation of host gene by intragenic(mostly intronic)miRNA is conceptually plausible but evidence-limited.The Locked Nucleic Acid based in-situ hybridization and microRNA-sensor experiments show that miR-932 is highly expressed in nervous system of fruitfly embryos,larvae,and adult heads.miR-932 and host dnlg2 seem to show a inverse expression pattern during the late embryonic to pupal stages detected by quantitative RT-PCR.Bioinformatics prediction indicates two potential targeting sites by miR-932 in the coding sequence(CDS)rather than 3'UTR of host dnlg2.To support this prediction,taking advantage of Dual-Luciferase Reporter Assays as well as Drosophila S2 cell transfection with dosages of pAc-miR-932 and pAc-dnlg2,we confirm the direct repression of dnlg2 expression by miR-932 at both protein and mRNA level.Transgenic overexpressions of UAS-miR-932 in neuronal or muscle tissues lead to developmental defect to some different extent.In the neuromuscular junction(NMJ)model,miR-932 is involved in the DNlg2 mediated synapse development and function.Consistent with the phenotype of DNlg2KO70 null mutant(decreased bouton numbers and reduced Glutamate Receptors,mainly GluRIIBs);overexpressed miR-932 also causes decreased GluRIIB.Controversially,the presynaptic overexpressed miR-932 leads to increased mEJP amplitude but not frequency,while there are no changes of mEJP in DNlg2KO70,indicating that there must be other potential targets by miR-932.Verified Knock out(miR-932KO)and knockdown flies(UAS-miR-932Sponge)also confirm the direct regulation of intronic miR-932 on host dnlg2.In summary,miR-932 does participate in the regulation of DNlg2 on GluRIIB,synaptic development and neurotransmission.Meanwhile,we also provide evidence that Neurexin-1,the binding partner of DNlg2,can be targeted by its intragenic/exonic miR-4952 through four or five CDS-located target sites.Additionally,by genome-wide computational analysis,we demonstrate that nearly half of intronic miRNAs could regulate the host mRNA via binding to CDS-located regions:that's 44.4%(48/108)in Drosophila melanogaster,60.6%(492/812)in human and 30.2%(19/63)in Caenorhabditis elegans.It suggests that feedback regulation of host gene by intronic miRNA may be a common phenomenon at least in Drosophila and human genome.Our discovery also provides insights into a novel mechanism of tight and efficient control as well as maintenance of CAMs and other key molecules related to synaptic homeostasis and function.