Cellular stress responses play essential roles in cell and organismal survival and contribute to a wide range of physiological processes and diseases in humans. The molecular architecture of most stress response pathways are well defined. A striking exception to this is osmotic stress response, where the relevant stress sensors and signaling mechanisms in animals are poorly understood. Most studies of the osmotic stress response use cultured cells, where in vivo complexities, i.e. the extracellular matrix, tissue mechanical properties, etc., are not replicated. To better mimic these conditions, we study the osmotic stress response in a live animal, the nematode C. elegans. Like humans, C. elegans responds to osmotic stress by metabolizing glucose to produce organic osmolytes, such as glycerol. We performed an unbiased forward genetic screen to identify mutants that exhibit no induction of osmolyte biosynthesis genes (Nio genes) and discovered multiple alleles of nio-2, which encodes the sole C. elegans homolog of the O-GlcNAc transferase (ogt-1; OGT in humans). OGT post-translationally O-GlcNAcylates Ser/Thr residues of cytosolic and nuclear proteins but also exhibits important GlcNAcylation independent functions. Mammalian cells lacking OGT do not survive, but C. elegans lacking ogt- 1 are viable and fertile, providing a unique opportunity to study the role of ogt-1 in cellular physiology. ogt-1 mutants are unable to adapt and grow in hypertonic environments and exhibit reduced organic osmolyte levels and no induction of the osmolyte biosynthesis protein GPDH-1. However, osmotic induction of osmolyte biosynthesis gene mRNAs is normal, suggesting that ogt-1 functions post-transcriptionally. These defects can be rescued by expression of wild type or catalytically inactive human OGT, showing that non-canonical functions of OGT in the osmotic stress response are conserved from C. elegans to humans. We also discovered mutations in interacting components of a conserved 3? mRNA processing complex that phenocopy ogt-1. We hypothesize that non-canonical functions of ogt-1 facilitate upregulation of stress-induced mRNA translation via interactions with 3? RNA processing complex proteins during osmotic stress. To test this hypothesis, we will determine the temporal, functional, and regulatory requirements for ogt-1 in the osmotic stress response (Aim 1), identify the specific gene expression mechanism(s) that are affected by ogt-1 (Aim 2), and determine if ogt-1 regulates the osmotic stress response via interactions with 3? mRNA cleavage and polyadenylation components also identified in our Nio screen.
(Aim 3). Our studies will delineate a novel paradigm in stress signaling and reveal new mechanisms by which OGT impacts cell physiology.
Cellular responses to environmental fluctuations and stress play key roles in human health and disease. In an unbiased genetic screen in C. elegans, we discovered that an enzyme called OGT, which mediates an unusual type of protein modification, and genes involved in the maturation of mRNA specifically regulate the translation of mRNAs upregulated in response to cellular dehydration, which is a commonly encountered stress in several human tissues. These enzymes are essential for cellular viability in mammals; therefore our studies in C. elegans present a unique opportunity to define the role of these highly conserved but poorly understood proteins in cellular responses to the environment.
