Figure 5.

The C-terminal KA-1 of CeMETT10 facilitates m⁶A modification of RNAs. (A) Alignment of amino acid sequences around the arginine-rich region (RRR) in the C-terminal KA-1 domains from various organisms (Supplementary Figures 2 and 3), including invertebrates (C. elegans, O. vulgaris, T. urticae, A. queenslandica and A. tenebosa), a fission yeast (S. pombe), and vertebrates (H. sapiens, G. gallus, C. picta, X. laevis and D. rerio). (B) Schematic diagram of CeMETT10-FL and its variants used in the methylation assays in (D) and (E). MTD, KA-1a and KA-1b are colored cyan, magenta, and green, respectively. RRR (arginine-rich region: RARKRAK) is colored blue, and its mutant is red (RRR/7E). (C) Secondary structures of sams-hp-ls and U6 snRNA. (D, E) Steady-state kinetics of the methylation of sams-hp-ls (D) and U6 snRNA (E) by METT10-FLΔL, -MTD, and -FLΔL_RRR/7E. Various concentrations of RNA [0–8 μM for sams-hp-ls (D) and 1–4 μM for U6 snRNA (E)] were incubated with 0.4 μM METT10- FLΔL, -MTD or -FLΔL _RRR/7E in the presence of 1 mM SAM, at 37°C for 4 min, and the initial reaction velocities were calculated. The bars in the graph are the SD of three independent experiments. (F) Gel-shifts of sams-hp-ls by wild-type METT10-FLΔL (0–1.0 μM), -MTD (0–100 μM) or -FLΔL _RRR/7E (0–20 μM). The graphs on the left in (F) and (G) are magnified views of the graphs on the right at the lower protein concentration ranges. (G) Gel-shifts of U6 snRNA by wild-type METT10-FLΔL (0–1.0 μM), -MTD (0–100 μM) or -FLΔL _RRR/7E (0–20 μM). The bars in the graphs (F, G) are the SD of three independent experiments.