Muscle-Specific Regulation of PABPN1 Expression

Emory University, Atlanta GA

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Abstract

? DESCRIPTION (provided by applicant): Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant disease that affects muscles of the eyelid, pharynx, and proximal limbs. OPMD results in eyelid drooping, difficulty swallowing leading to aspiration pneumonia, and proximal limb weakness, all of which greatly impact patient quality of life as no pharmacologic treatment is available. OPMD patients have a dominant GCG triplet repeat expansion mutation in the gene polyadenylate-binding protein, nuclear 1 (PABPN1), resulting in an alanine expansion in the PABPN1 protein. PABPN1 regulates critical aspects of pre-mRNA processing, such as polyadenylation and cleavage site selection. Although PABPN1 is ubiquitously expressed, this mutation results in muscle-specific disease. To understand this muscle-specific pathology, we need to determine the unique properties of PABPN1 in muscle. We have found that wild type PABPN1 protein and mRNA levels are low in muscle, with even lower levels in OPMD-affected muscles, relative to unaffected tissues. These low levels may make muscle susceptible to mutations that reduce PABPN1 protein function, while higher PABPN1 levels in non- muscle tissues may compensate for reduced function. Muscle-specific destabilization of murine Pabpn1 mRNA contributes to low protein levels in muscle, and our preliminary data suggest this destabilization is mediated by an AU-rich element (ARE) in the Pabpn1 3'untranslated region (3'UTR). We use murine cells and tissues because PABPN1 is highly conserved from mouse to human. Mutating the Pabpn1 ARE in the context of a luciferase reporter reduces luciferase activity, indicating that the ARE and interacting factors influence transcript stability. RNA binding proteins can bind AREs and influence mRNA stability, and competition between stabilizing and destabilizing factors, as well as the levels of these factors across tissues, determines mRNA stability. We have shown that HuR, a canonically stabilizing RNA binding protein that binds AREs, interacts with the Pabpn1 3'UTR and is present at low levels in muscle. These data suggest that low levels of HuR may interact with the ARE to partially stabilize the transcript in muscle, but muscle-specific interactions with destabilizing factors lead to overall Pabpn1 destabilization. We hypothesize that a muscle-specific complement of RNA binding proteins interacts with regulatory elements in the PABPN1 3'UTR leading to low PABPN1 levels in muscle relative to other tissues. In Aim 1, we will determine the role of the Pabpn1 ARE and HuR in modulating muscle-specific Pabpn1 mRNA stability. In Aim 2, we will use both candidate- and discovery-based approaches to identify interactions between RNA binding proteins and the Pabpn1 3'UTR that are altered in muscle relative to kidney, an unaffected tissue in OPMD. Our long-term objective is to understand the molecular mechanisms resulting in muscle-specific susceptibility to OPMD pathogenesis, which may identify novel therapeutic targets to modulate PABPN1 levels in OPMD patient muscles.