Abstract
Mutations of the neurofibromin 1 gene cause neurofibromatosis type 1, a disease in which learning and behavioral abnormalities are common. The disease is completely penetrant but shows variable phenotypic expression in patients. The repertoire of regulatory interactions utilized by neurons to control neurofibromin 1 expression is poorly understood. Here, we examined the contribution of microRNAs into this regulatory network. Using reporter assays, we provided evidence that miR-128 and to a lesser extent miR-137 and miR-103 reduced neurofibromin 1 reporter levels through specific binding to Nf1 3′-UTR. Mutations in all three predicted binding sites eliminated the reporter response. MiR-128 and miR-137, unlike miR-103 that showed a more ubiquitous expression, were predominantly expressed in brain with a distribution that resembled neurofibromin 1 expression in different tissues as well as during the course of neuronal development. In the nervous system, all three microRNAs showed highest expression in neurons and least in Schwann cells and astrocytes. Overexpression of miR-128 alone or with miR-103 and miR-137 significantly reduced endogenous neurofibromin 1 protein levels, while antisense inhibition of these microRNAs enhanced translation of endogenous neurofibromin 1 and reporter in primary cultures of hippocampal neurons. These findings revealed a significant additional mechanism by which neurofibromin 1 is regulated in neurons and implicated new candidates for the treatment of multifarious neurofibromatosis type 1 cognitive symptoms.
Highlights
Neurofibromatosis type 1 (NF-1) is a common autosomal dominant genetic disorder that affects 1 in 3500 individuals worldwide
An average decrease of 16.1% (P,0.01), 33.1% (P,0.05), and 30% (P,0.05) in endogenous Neurofibromin 1 (NF1) protein levels was measured with miR-128, miR-128/103, and miR-128/137 plasmid overexpression, respectively (Figs. 6A and B). These findings indicated that a) miR-103, miR-137, and miR128 function synergistically to regulate endogenous NF1 protein but not mRNA levels in neurons, and b) endogenous miR-103, miR-137, and miR-128 expression levels are not saturated for NF1 regulation of expression in neurons, allowing a controlled amount of Nf1 mRNA to be translated in accordance with the tuning hypothesis
This study has revealed a previously unknown mechanism by which NF1 levels are regulated in the nervous system
Summary
Neurofibromatosis type 1 (NF-1) is a common autosomal dominant genetic disorder that affects 1 in 3500 individuals worldwide. The disease is characterised by a spectrum of somatic and cognitive symptoms that include cafeau lait macules, Lisch nodules, neurofibromas and learning disabilities [1]. Individuals affected by NF-1 are heterozygous for the Nf1 gene mutation, as homozygous mutations appear to be lethal [2]. Neurofibromin 1 (NF1), the Nf1 gene product, is a large protein that contains a central Ras-GTPase-activating (RasGAP) domain thought to function as a negative regulator of proto-oncogene RAS and downstream effectors [3,4,5]. Despite the fact that NF-1 is a single-gene disease, it is presented by a variable expressivity of symptoms even within families, indicating that other modifier genes are involved in the disease process. Studies have far recognized ubiquitination through ETEA [6] and alternative splicing mediated by RNA binding proteins HuR and CELF as possible contributing factors [6,7]
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