Oculopharyngeal muscular dystrophy (OPMD) associated alanine expansion impairs the function of the nuclear polyadenosine RNA binding protein PABPN1 as revealed by proximity labeling and comparative proteomics

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disease caused by modest alanine expansion at the amino terminus of the nuclear polyadenosine RNA binding protein PABPN1. PABPN1 is expressed ubiquitously and is involved in multiple steps in RNA processing including optimal cleavage and polyadenylation, polyadenylation signal selection, and export of polyadenylated RNAs from the nucleus. Expanded PABPN1 forms aggregates in a subset of muscle nuclei, but PABPN1 levels are paradoxically low in muscle compared to other tissues. Despite several studies in model systems and patient tissues, it remains unclear whether alanine expansion directly impairs PABPN1 function. The molecular mechanisms leading to OPMD pathology are poorly understood. Here we used a proximity labeling approach to better understand the effect of alanine expansion on PABPN1 function in a cell culture model of skeletal muscle. To avoid the confounding factor of overexpression, PABPN1 constructs containing a carboxy-terminal TurboID tag were expressed in skeletal myotubes at near native levels using an inducible promoter. Although non-expanded PABPN1-TurboID was able to complement RNA export and myoblast differentiation defects caused by deficiency of endogenous PABPN1, alanine expanded PABPN1-TurboID was not. Comparative proteomics revealed increased interaction between expanded PABPN1 and RNA splicing and polyadenylation machinery and follow-up studies identified a dominant negative effect on RNA export in differentiated myotubes. These data indicate that alanine expansion can impair PABPN1 function regardless of the presence of wild type PABPN1 and support a model wherein both loss function and dominant negative effects of expanded PABPN1 contribute to OPMD pathology. Author summaryOculopharyngeal muscular dystrophy (OPMD) is a late onset muscle disease that, unlike other muscular dystrophies, causes progressive weakness primarily in muscles of the face and head. OPMD is most commonly inherited in a dominant fashion and is caused by small expansions in the gene encoding the RNA binding protein PABPN1. Despite the fact that PABPN1 is expressed ubiquitously and contributes to multiple steps in RNA processing, its expansion causes disease almost exclusively in skeletal muscle. The precise molecular events leading to OPMD disease onset have been difficult to characterize as it is challenging to separate functions expanded versus non-expanded PABPN1 in muscle without the confounding factor of overexpressing tagged fusion proteins. Here we fused expanded and non-expanded PABPN1 to the modified biotin ligase TurboID to label proximal proteins. PABPN1-TurboID fusions were expressed in a cell culture model of skeletal muscle at near-native levels. Using these constructs, we discovered that expanded PABPN1 is less functional than non-expanded PABPN1 and causes dominant negative effects on some PABPN1 functions. These findings support a model where both loss of function and dominant negative effects contribute to the molecular pathology of OPMD.