Identification of regulatory RNA binding proteins that mediate alternative polyadenylation in cardiac myocytes

Abstract

IntroductionCardiovascular disease is the leading cause of death in the US with coronary heart disease (ischemia/reperfusion; I/R injury) accounting for approximately half of these deaths. Recent data from our lab suggests that alternative polyadenylation (APA) plays a role in cardiac gene expression following I/R via modulation of mRNA 3′UTR length, leading to the inclusion or exclusion of regulatory sequence elements. The goal of this work is to identify specific RNA binding proteins (RBPs) that are likely to mediate APA in the myocardium.MethodsThe 3′UTR of the heat shock protein 70.3 (Hsp70.3) mRNA was randomly biotinylated throughout and incubated with cardiac protein extract associated with differential APA of the Hsp70.3 3′UTR. Biotinylated RNA and RBPs were co‐precipitated and bound RBPs were identified using mass spectrometry.ResultsWe identified a total of 45 known RBPs interacting with the Hsp70.3 3′UTR. Of these, 9 unique RBPs were found to bind only under conditions associated with a long 3′UTR, whereas 10 unique RBPs were found to bind only in association with APA truncation of the 3′UTR. Our results identified RBPs previously indicated to play a role in APA and/or mRNA degradation as well as RBPs whose function remains unknown. Work is ongoing to determine the functional role of these RBPs in alternative polyadenylation and cardiac I/R injury.This work was partially funded by a UC CCTST Grant (MT).