PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans

Abstract

MicroRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential “prion-like” domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59’s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation. Bistability plays a central role in many gene regulatory networks (GRNs) that control developmental processes where distinct and mutually exclusive cell fates are generated in a defined order. While genetic analysis has identified a number of gene types that promote these transitions, we know little regarding the mechanisms and players that ensure these decisions are robust and, in many cases, irreversible. We leveraged the powerful genetics and phenotypes associated with temporal patterning mutants of C. elegans to identify genes whose depletion restores normal development in animals that express miRNA alleles that do not sufficiently down-regulate their targets. These efforts identified pqn-59, the C. elegans ortholog of the human UBAP2L gene. Like UBAP2L, PQN-59 forms a hub for a number of RNA/RNA-binding protein mediated processes in cells including the formation of stress granules in adverse environmental conditions and translational activation during normal development. We demonstrate that pqn-59 depletion impacts protein translation and leads to the stabilization of several mature miRNAs; further connecting aspects of mRNA translation and miRNA-mediated gene regulation to this new family of RNA-binding proteins.