Allison Mezzell (University of Cincinnati College of Medicine)| Katherine Vest (University of Cincinnati)
The polyadenylate binding protein nuclear 1 (PABPN1) is an mRNA binding protein that facilitates polyadenylation and alternative polyadenylation, nuclear RNA export, the pioneer round of translation, and RNA decay. The PABPN1 protein contains an N-terminal polyalanine tract of unknown function. Small expansions of the polyalanine tract cause the late-onset disease oculopharyngeal muscular dystrophy (OPMD), which affects the muscles of the eyes, pharynx, and proximal limbs. Individuals with OPMD experience drooping eyelids, trouble swallowing, and loss of mobility. Skeletal muscle is a regenerative tissue that contains a pool of stem cells termed that undergo a well-characterized differentiation program to repair injured or diseased muscle. These stem cells, often termed satellite cells, are affected in OPMD patients and knockdown PABPN1 impairs differentiation of muscle stem cell-derived myoblasts. A deeper understanding of PABPN1 function during muscle stem cell differentiation will provide insight into the pathology of OPMD. Expanded PABPN1 forms insoluble intranuclear aggregates, which have long been implicated in OPMD pathology. However, soluble alanine-expanded PABPN1 interacts with a different subset of proteins than wild type PABPN1, suggesting that alanine expansion impacts PABPN1 protein function. Here, we aim to define PABPN1 protein binding partners in the context of myoblast differentiation and elucidate the impact of alanine expansion. We will use proximity ligation assay coupled with mass spectrometry to label and identify proteins that interact with wild type PABPN1. We generated a fusion construct with mouse PABPN1 and the promiscuous biotin ligase TurboID under the control of a constitutive promoter. However, the resulting PABPN1 overexpression resulted in mislocalization of PABPN1 from the nucleus to the cytoplasm. As an alternative, we generated an inducible lentiviral construct to decrease the expression of the PABPN1-TurboID fusion protein. As a complement to the proximity ligation assay, we are taking a candidate-based approach informed by published studies that define PABPN1 interacting partners in mature murine skeletal muscle. Future studies will focus on comparing wild type PABPN1 to alanine expanded or alanine deleted PABPN1. Our findings will expand the known interactome of PABPN1 and provide additional insight into the mechanisms that cause OPMD utilizing robust methodologies. By identifying PABPN1 interactors we will be able to manipulate them in future studies to further our understanding of OPMD and PABPN1.