Poster number 94 submitted by Alfa Dhakal
Role of ADARs in the pathogen response of C. elegans
Alfa Dhakal (School of Medicine), Chinnu Salim (Department of Biology), Heather Hundley (Department of Biology)
All organisms must be able to fight infection from pathogen to survive. To be prepared to fight infections, cells have equipped machinery to activate signaling pathways and secrete effector molecules. My project focuses on the role of ADARs in the pathogen response of C. elegans. ADARs are RNA binding proteins that regulate gene expression by either binding or editing (modifying). Since these RBPs act on double-stranded RNA which mimics the viral genome, most ADAR studies have focused on understanding the mechanism of viral infection and how a host responds. However, the role of ADARs in bacterial infection and host-defense mechanisms have not been studied.
There are two ADARs in C. elegans ADR-1 and ADR-2 with double stranded RNA binding domains and a deaminase domain. Using a standard slow killing assay with Pseudomonas aeruginosa as a pathogen, we observed that both adr-1(-) and adr-2(-) single mutants and adr-1(-); adr-2(-) double mutant animals are susceptible. Interestingly, an ADR-1 mutant that is unable to bind to dsRNA also showed a sensitive phenotype suggesting that ADARs are regulating certain transcripts by binding. On doing a pilot RNA sequencing, with WT, adr-1(-) and the ADR-1 dsRNA binding mutant worms after pathogen exposure, we observed that many genes that were downregulated in adr-1(-) and ADR-1 dsRBM1 mutant were also down in normal food suggesting that ADR-1 is regulating these genes basally. Further, RNA sequencing and differential gene expression analysis of WT, adr-1(-), adr-2(-) and adr-1(-); adr-2(-) worms grown on normal food was done. Among the genes overlapping between double mutant and single mutant worms compared to WT, we found “collagen” genes to be significantly down. Collagens are involved in epithelial tissue integrity and form a first line of defense against pathogen. This led to our hypothesis that ADARs are regulating the collagen genes to provide organismal survival to pathogen.
Ongoing studies are focused on finding the cuticular structure changes on ADAR mutant worms using SEM and finding the tissue specific contributions and molecular mechanisms of ADARs in pathogen response.
Keywords: RNA binding protein, pathogen, gene expression