Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic setpoints in physiology. Challenges to osmotic can occur under both physiological and pathophysiological conditions. Increased extracellular osmolarity (hyperosmolarity) causes a rapid decrease in cell volume leading to increased intracellular ionic strength, macromolecular damage, and cell-ECM mechanical strain. To counteract these stressors, cells activate several physiological pathways. One major pathway involves the upregulation of proteins that catalyze the accumulation of solutes called organic osmolytes. Organic osmolytes restore cell volume, reduce cell-ECM mechanical strain, and prevent macromolecular damage. However, the mechanisms by which multicellular animals detect osmotic dyshomeostasis and activate osmosensitive gene expression remain poorly understood. C. elegans responds to hypertonic stress by upregulating the glycerol-3-phosphate dehydrogenase enzyme (GPDH-1) to accumulate the osmolyte glycerol. To understand how the osmotic stress response is coordinated in multicellular animals, we conducted an ENU-based genetic screen with a
gpdh-1::GFP reporter to identify mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants). Through whole genome-resequencing, we discovered that
nio-3 was caused by a missense mutation in the
cpf-2 gene and
nio-7 was caused by a missense mutation in
symk-1. Both
cpf-2 and
symk-1 are interacting components of the highly conserved 3' mRNA cleavage and polyadenylation complex.
cpf-2 and
symk-1 block the hypertonic induction of
gpdh-1 and other osmotically induced mRNAs, suggesting they act at the transcriptional level. Although null mutations in both
cpf-2 and
symk-1 are lethal, the nio alleles are viable under control conditions. However, under hypertonic stress conditions,
nio-3 and
nio-7 are unable to adapt and grow. Both
nio-3 and and
nio-7 suppress phenotypes known to be dependent on 3' mRNA processing and cleavage, suggesting the missense alleles identified in our Nio screen cause partial loss of function in this complex. SYMK-1::GFP and CPF-2::RFP proteins are expressed ubiquitously and co-localize within the nucleus. Interestingly, hypertonic stress causes CPF-2::RFP to rapidly reolocalize into subnuclear puncta. The cell biological properties of SYMK-1::GFP are under investigation. Our data show that
cpf-2 and
symk-1 are essential for physiological activation of the hypertonic stress response through a transcriptional mechanism. We are currently investigating how
cpf-2 and
symk-1 shape the mRNA polyadenylation landscape to control the hypertonic stress response pathway, as well as other stress response pathways, using RNAseq approaches