A265 - Poster of Distinction
AUTOPHAGY REGULATES EXPRESSION OF THE RISC COMPONENT AGO2 - A CRITICAL REGULATOR OF THE MIRNA SILENCING PATHWAY
M Sibony1, H Mascarenhas1, D Raju1, E Galindo-Mata1, M Silverberg2, D Philpott3, N Jones1
1Cell Biology Program, Research Institute, The Hospital for Sick Children; 2Zane Cohen Centre for Digestive Diseases, IBD group, Mount Sinai Hospital; 3Department of Immunology, University of Toronto, Toronto, ON
Aims: Genome-wide association studies have repeatedly associated a variant of the autophagy dependent gene ATG16L1 with Crohn's disease (CD), thus implicating autophagy, a novel and unexpected pathway, in the development of inflammation. The functional relevance of the ATG16L1-CD variant is unclear, as it was shown to be unnecessary for canonical autophagy, and whether it affects bacterial mediated autophagy remains controversial. We hypothesized that the ATG16L1-CD variant is involved in a different process that contributes to persistent inflammation, the miRNA silencing pathway. miRNA are short non-coding RNA that bind to target mRNA to repress their translation and promote their degradation. Emerging data suggest that altered regulation of pro- and anti-inflammatory genes by miRNA is involved in the pathogenesis of IBD. In order to exert their function, miRNA are loaded onto an RNA induced silencing complex (RISC). AGO2 is a critical component of the RISC complex and the only AGO protein with Slicer activity which results in the generation of unique mature miRNA. RISC formation and turnover occurs on endosomal membranes. Since autophagy and endosomal membranes are closely related, we hypothesized that autophagy and the ATG16L1-CD variant affect RISC components, hence modulating miRNA silencing.
Methods: In order to test our hypothesis we performed immunoblotting assays to compare the expression of RISC components in control cells or cells with increased or disrupted autophagy using bafilomycin, knockdown of ATG dependent genes or treatment with the H. pylori vacuolating cytotoxin (VacA) which disrupts autophagy. We determined the colocalization of autophagic vacuoles with RISC components using confocal microscopy.
Results: Our data show increased AGO2 expression in epithelial cells in which autophagy is disrupted by knocking-down the expression of autophagy proteins atg16, atg5 and atg12, as well as treatment with VacA in comparison with control cells. Increased AGO2 expression was also detected by confocal imaging of cells with disrupted autophagy. In addition, human peripheral blood mononuclear cells also show increased AGO2 when autophagy was disrupted.
Conclusions: Taken together, these results suggest that autophagy is involved in degradation of the critical RISC component AGO2 in both epithelial and myeloid cells. We propose a compelling mechanism by which autophagy may affect miRNA expression and is involved in the etiology of CD.