The molecular evolution of DEDDh exonucleases reveals novel functions in innate immune signalling

Abstract

The Promyelocytic Leukemia (PML) protein is the major constituent of PML nuclear bodies (NBs), a subnuclear compartment involved in regulating a diverse array of cellular processes. PML NBs have tumour suppressive functions through their regulation of post-translational modifications, cell cycle progression, senescence, maintenance of genome stability, and apoptosis. However, there are many remaining questions about the functions of the PML-I isoform and its role in the cytoplasm. Work presented in this thesis examines the molecular evolution of PML and identifies a new role for PML-I and a novel family of related genes, PML-like exon 9 (Plex9), in repressing LINE-1 retroelements and regulating cGAS-STING signalling in jawed vertebrates. Duplication of DNA encoding the PML C-terminus gave rise to Plex9 genes in Teleostei, which lack homologs of both PML and the TREX1 DEDDh exonuclease (a regulatory of cGAS-STING). In an example of convergent evolution, zebrafish (Danio rerio) Plex9.1 functionally replaces TREX1 to suppress LINE-1. We then characterize an ortholog of PML encoded in the genome of the spotted gar (Lepisosteus oculatus). Gar Pml is a cytoplasmic exonuclease belonging to the DEDDh family that also suppresses LINE-1 retrotransposition. The PML protein lost exonuclease activity but evolved SUMOylation sites to form NBs in amniote species. However, human PML-I retained the ability to restrict LINE-1 by shuttling in a CRM1-dependent manner to the cytoplasm. Thus, we shed new light on the molecular evolution and functional divergence of PML protein over the course of vertebrate evolution. cGAS-STING signalling is involved in mammalian wound healing and age-related senescence, but it has not been examined in other vertebrate species. We examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in the hearts of zebrafish. During the early stages of limb-fin regeneration and age-related senescence, Pml, Trex1 and Plex9.1 exonucleases are downregulated, presumably to allow cGAS-STING activation. Therefore, in jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by the collective actions of Trex1, Pml and Plex9 DEDDh exonucleases.