Downregulation of Ahcy with and without blue light exposure significantly alters global gene expression in the Drosophila eye
One-carbon metabolism influences the response to oxidative stress through antioxidant biosynthesis and the transcriptional status of the cell via epigenetic mechanisms. One-carbon metabolism is responsible for the production of the universal methyl donor, S-adenosylmethionine (SAM), which generates the potent methyltransferase inhibitor, S-adenosylhomocysteine (SAH). The essential metabolic enzyme, S-adenosylhomocysteinase (AHCY), functions within one-carbon metabolism and is the sole enzyme to catabolize the metabolite S-adenosylhomocysteine (SAH). Under oxidative stress conditions, AHCY becomes oxidized at a critical residue for its activity correlating with increased SAH abundance in the Drosophila head. Here, we profiled the photoreceptor transcriptome at a single time point after 3-hour blue light exposure relative to untreated controls in the Drosophila eye. We used RNAi expressed in all cells of the eye under longGMR-Gal4 to knockdown Ahcy and examined photoreceptor cells enriched using nuclei-immunoenrichment. As a control, we expressed non-specific RNAi against mCherry. Overall design: To knockdown Ahcy in the Drosophila eye, we crossed longGMR-Gal4, Rh1-KASHGFP flies to UAS-RNAi flies targeting either Ahcy (y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC03222}attP40/CyO, Bloomington Stock #51477) or mCherry (control) in a cnbw background. We then collected male progeny, which were raised in 12-hour light: 12-hour dark conditions at 25C until 5 days post-eclosion. 150 male flies were collected per sample (3 replicates). We affinity enriched photoreceptor nuclei for RNA isolation as previously described (Jauregui-Lozano et al., 2021, Genetics). Nuclear RNA was purified using ZYMO Direct-zol RNA Microprep Kits (#R2061). RNA library were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760S). rRNA was depleted using NEBNext® RNA Depletion Core Reagent Set with RNA Sample Purification Beads (E7870L). Libraries were sequenced using NovaSeq X Plus.