Abstract
MiR-124 is a highly expressed miRNA in the brain and regulates genes involved in neuronal function. We report that miR-124 post-transcriptionally regulates PARP-1. We have identified a highly conserved binding site of miR-124 in the 3′-untranslated region (3′UTR) of Parp-1 mRNA. We demonstrate that miR-124 directly binds to the Parp-1 3′UTR and mutations in the seed sequences abrogate binding between the two RNA molecules. Luciferase reporter assay revealed that miR-124 post-transcriptionally regulates Parp-1 3′UTR activity in a dopaminergic neuronal cell model. Interestingly, the binding region of miR-124 in Parp-1 3′UTR overlapped with the target sequence of miR-125b, another post-transcriptional regulator of Parp-1. Our results from titration and pull-down studies revealed that miR-124 binds to Parp-1 3′UTR with greater affinity and confers a dominant post-transcriptional inhibition compared to miR-125b. Interestingly, acute or chronic cocaine exposure downregulated miR-124 levels concomitant with upregulation of PARP-1 protein in dopaminergic-like neuronal cells in culture. Levels of miR-124 were also downregulated upon acute or chronic cocaine exposure in the mouse nucleus accumbens (NAc)-a key reward region of brain. Time-course studies revealed that cocaine treatment persistently downregulated miR-124 in NAc. Consistent with this finding, miR-124 expression was also significantly reduced in the NAc of animals conditioned for cocaine place preference. Collectively, these studies identify Parp-1 as a direct target of miR-124 in neuronal cells, establish miR-124 as a cocaine-regulated miRNA in the mouse NAc, and highlight a novel pathway underlying the molecular effects of cocaine.
Highlights
MicroRNAs are a class of noncoding RNAs that post-transcriptionally regulate gene expression[1]
MiR-183 levels were downregulated in the striatum upon cocaine treatment[14]. Consistent with these findings, a microarray study revealed that cocaine exposure resulted in the upregulation of ~ 25 miRNAs and downregulation of ~ 9 miRNAs in the hippocampus[10]. In addition to these miRNAs, miR-124 is emerging as a key cellular miRNA regulated by cocaine exposure in the central nervous system (CNS)12,15–17. miR124 is highly expressed across the b rain[18] and is involved in neurogenesis, synaptic signal transmission, neurodegeneration, neuroadaptation, synapse morphology, long-term potentiation, and neuronal homeostasis[19,20,21]
The miR-124 target site was located at the nucleotide position 312–331 of Parp-1 3′-untranslated region (3′UTR) (Fig. 1C) and was highly conserved in several mammalian species (Fig. 1C), another critical feature of miRNA-mediated gene regulation[2]
Summary
MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression[1]. Decreased miR-124 levels have been reported in several central nervous system (CNS) disorders including Parkinson’s disease, dementia, multiple sclerosis, and substance use disorders[25,26,27] Since these studies establish the importance of miR-124 in the brain, understanding the role of this miR will provide key insights into the molecular actions of cocaine. PARP-1 and PARylation have been shown to be involved in the molecular effects of cocaine in the N Ac42–44 Given these important roles of PARP-1, we tested the functional significance of our in silico prediction studies in both a dopaminergic neuronal cell model and rodent brain. MiR-124 levels were significantly reduced in the NAc of animals after induction of conditional place preference (CPP) for cocaine These studies identify Parp-1 as a direct target of miR-124 in neuronal cells and unravel a novel regulatory mechanism underlying the molecular effects of cocaine exposure
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