Extended Data Fig. 5: Deletion of TkNAT10 and TkTHUMPD1 in T. kodakarensis.
From: Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping
a, Total RNA from T. kodakarensis was analysed via ac4C-seq. IGV browser traces display representative ac4C sites in rRNA, ncRNA, mRNA and tRNA of T. kodakarensis, visualized as in Fig. 1c. The number in parentheses indicates the number of sites identified for each class of molecules. b, Conserved ___domain architecture of human NAT10 and its homologue in T. kodakarensis, TK0754 (referred to as TkNAT10 in the text). c, Expression of TkNAT10 and TkTHUMPD1 (TK2097) in wild-type T. kodakarensis and the indicated deletion strains was quantified from ac4C-seq data. Shown are mean TMM-normalized values (n = 3 and 2 biological replicates in wild-type and deletion strains, respectively). d, Quantitative LC–MS/MS proteomics analysis of wild-type and ΔTkNAT10 T. kodakarensis. Fold-change in protein abundance was based on comparison of distributed normalized spectral abundance factor for individual proteins. Fold-change for proteins detectable exclusively in the wild-type or the knockout (KO) condition (fold-change = ∞) are graphed at 5.5 and 0.1, respectively, which represents the maximum and minimum of measured values. n = 3 LC–MS/MS runs for each condition. e, Anti-ac4C immuno-northern blot in T. kodakarensis total RNA. Ethidium bromide staining is used to visualize total RNA. Results are representative of two biological replicates. For gel source data, see Supplementary Data 3. f, Relative quantification of ac4C in total RNA isolated from wild-type and ΔTkNAT10 T. kodakarensis strains as measured by LC–MS. Mean of n = 3 technical replicates. n.d., not detectable. g, Scatter-plot depicting misincorporation rate of ac4C sites in wild-type T. kodakarensis is compared with the TkTHUMPD1-deletion strain, showing no effect of the deletion of the gene on the ac4C status. h, Correlation between misincorporation rates in T. kodakarensis compared to P. furiosus and T. sp. AM4 for the different types of ncRNAs identified by ac4C-seq. The Pearson’s correlation coefficient is indicated at the bottom of each plot. n = 4 and 1 independent biological samples for T. kodakarensis and other archaea, respectively. Shading indicates 95% confidence intervals for predictions from a linear model.
