Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning.

O- GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O- GlcNAc binding activity derived from Clostridium perfringens OGA ( Cp OGA) with TurboID proximity labeling in Drosophila , we developed an O- GlcNAcylation profiling tool that translates O- GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O- GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O- GlcNAcylated in the mushroom body of Drosophila brain. Hypo- O- GlcNAcylation induced by ectopic expression of active Cp OGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O- GlcNAcylation in Drosophila , and suggests a possibility that O- GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.