The Post-Transcriptional Regulation of Peripheral Myelin Protein 22 by MicroRNAs

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DESCRIPTION (provided by applicant): The process of myelination requires the precise and timely expression of several proteins which are necessary for the proper esheatment of axons. The mechanisms by which the myelinating cells of the central and peripheral nervous systems coordinate this elaborate process still remain largely unknown. Peripheral myelin protein 22 (PMP22) is an integral membrane protein that is primarily expressed in myelinforming Schwann cells of the peripheral nervous system. The misexpression of PMP22 is implicated in a host of hereditary demyelinating peripheral neuropathies including Charcot-Marie-Tooth disease type 1A which has an estimated prevalence of approximately 1 in every 2500 live births. Both duplication and gene deletion result in neuropathic phenotypes indicating the necessity for precise control over protein expression. PMP22 also appears to serve a function in cell cycle regulation, where it was first discovered as a gene upregulated during growth arrest. Aberrant expression of PMP22 is also described in several cancers, including osteosarcoma, breast cancer, and some gliomas. Interestingly, PMP22 mRNA is widely detected in the body, but protein expression is highly restricted to the myelinating Schwann cells, epithelial cells and select motor neurons, suggesting post-transcriptional regulation. The 5' and 3'-UTRs of the PMP22 gene have been shown to influence the expression of the mRNA. The 5'-UTR contains three known promoter regions resulting in three distinct RNA transcripts, but same protein. The mechanism by which the 3'-UTR of PMP22 appears to reduce protein translation remains unknown. MicroRNAs (miRNAs) are small, endogenous regulatory RNA molecules that exert their action post-transcriptionally by binding to the 3'-UTR of RNA and preventing translation through several possible mechanisms. It is my overall hypothesis that PMP22 expression is regulated by specific miRNAs in Schwann cells. To test this hypothesis, I will demonstrate that steady-state PMP22 RNA and protein levels can be influenced by inhibition of the miRNA biogenesis pathway (Aim 1). I will also map out specific functional miRNA binging sites within the 3'-UTR of PMP22 using PMP22 3'-UTR-luciferase reporter constructs and demonstrate that the binding of these specific PMP22 targeting miRNAs regulate its expression (Aim 2). Finally, it will be demonstrated that under- and overexpression of specific PMP22 targeting miRNAs will modify the steady-state levels of PMP22 mRNA and protein in cultured Schwann cells (Aim 3). At the conclusion of these experiments, I hope to reveal novel mechanisms governing the regulation of PMP22 that may provide new therapeutic targets for associated disease states.