UC Riverside

RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3′ untranslated regions (UTRs) to regulate mRNA stability and translation. In addition to individual effects of RBPs, there are also examples of co-regulatory interactions between multiple RBPs occupying the same 3’UTR. Two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2), are known to bind overlapping sets of transcripts and individual examples of cooperative interactions between the proteins have been described. To further assess the extent of co-regulation between these proteins, transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-sequencing. The presence of PUM binding on the same 3′ UTR corresponded with both cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3′ UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. We also characterized a new role of both PUM1 and PUM2 in regulating cell adhesion and migration. PUM double knockout (DKO) T-REx-293 cells grew in clumps, which arose from an inability to escape cell-cell contacts. In addition, defects in collective cell migration and actin morphology were also observed. RNA-sequencing further validated defects in gene categories related to adhesion and migration. In total, this work highlights the importance of further study into the biological roles of RBPs as well as the complex interactions between them.