Fig. 1: Neuronal Nsun2 expression and activity modulates tRNA cytosine methylation in the adult cortex.
a Left, schematic representation of conditional Nsun2 ablation with loxP sites surrounding Nsun2 exon 6 and representative RNA-seq profiles from the cortex of adult CamK-Cre+,Nsun2lox/2lox (KO) and WT control mice. Right, immunoblot (top) for Nsun2 protein expression in cortical tissue homogenate (Histone H3 was used as a loading control; two-tailed, t(5) = 3.381, *P = 0.0196; n = 3 WT, 4 KO). Uncropped western blots depicted in Supplementary Fig 1b. Nsun2 mRNA levels (bottom) were measured in forebrain tissue homogenate using qPCR with Gapdh as a housekeeping gene (n = 9 WT, 8 KO; two-tailed, Mann–Whitney U = 0, ***P < 0.0001). Data are presented as mean values +/− SEM. b Top left, schematic representation of viral vector containing hSyn1 promoter to drive neuronal expression, mouse Nsun2 transcript, and GFP fusion protein (top). Brain atlas depiction of discrete PFC bilateral microinjections that were confirmed via GFP illumination (left) and immunostaining of Nsun2GFP (right; top scale bar = 100 μm, bottom scale bar = 20 μm). Note nucleolar staining pattern of Nsun2GFP. Bottom left, a significant increase in Nsun2 mRNA was detected by qPCR in PFC tissue after AAV8hSyn1-Nsun2GFP injection compared to AAV8hSyn1-GFP controls using Gapdh as a housekeeping gene (n = 7–8/condition; two-tailed, Mann–Whitney U = 0, ***P = 0.0003). Data are presented as mean values +/− SEM. c Left, schematic of tRNAGlyGCC, depicting 11 cytosines that were queried with tRNA bisulfite sequencing. Yellow circles represent unmethylated cytosines, blue circles represent cytosines methylated by Nsun2, and green circles represent cytosines methylated by Dnmt2. Right, representative tRNA bisulfite sequencing methylation maps for WT & Nsun2 KO cortex and AAVNsun2 PFC. tRNA bisulfite methylation maps include one cytosine per column and reads are represented as horizontal lines. d Methylation maps for differential methylation levels of Nsun2 and Dnmt2 cytosine targets compared to appropriate control for tRNAGlyGCC, tRNAGluCTC, and tRNAAspGTC (n = 3–5 biological replicates per condition (denoted as 1–5), two-tailed t test, ***FDR adj. P < 0.001; *FDR adj. P = 0.038 vs. same-batch control for each group; statistics for each cytosine shown in Supplementary Table 2). Source data are available as a Source Data file.
