Fig. 1: The KA-1 domain of METTL16 facilitates m6A modification of U6 snRNA and splicing.
From: Structures and mechanisms of U6 snRNA m6A modification by METTL16
a Schematic diagram of S. pombe METTL16 (spMETTL16) compared with human METTL16 (hsMETTL16) and C. elegans METT10 (ceMETT10). The N-terminal methyltransferase domain (spMTD, magenta) and the C-terminal KA-1 domain (spKA-1, cyan) of S. pombe, used in the experiments, are depicted. b In vitro methylation of the S. pombe U6 snRNA (spU6 snRNA) transcript by spMETTL16 and spMTD under standard conditions. spU6 snRNA (0.5 μM) was incubated with 200 nM spMETTL16 or spMTD in the presence of 1 mM SAM for 4 min at 37 °C. Error bars represent the standard deviation (SD) of three independent experiments (N = 3), and the center of the error bands indicates the mean of the measured values. c Steady-state kinetics of methylation of the spU6 snRNA transcript by spMETTL16 and spMTD. spU6 snRNA (0–10 μM) was incubated with 200 nM spMETTL16 or spMTD in the presence of 1 mM SAM at 37 °C. All experiments were independently repeated three times with similar results. d, e Semi-quantitative RT-PCR analyses of two introns: d SPAC18B11.09 C and e ckn1. The nucleotide sequences of the respective 5′ splice sites (−3 to +4) are shown. The upper and lower bands on the gel represent retained and spliced introns, respectively. Intron retention in the Δmtl16 strain was rescued by ectopic expression of plasmid-encoded full-length METTL16 (spMETTL16), but not by the methyltransferase domain (spMTD) alone. All experiments were independently repeated three times with similar results (Supplementary Fig. 2a, b). f Nucleotide sequence of S. pombe U6 snRNA (spU6 snRNA, left). The nucleotide sequence of the spISL used for crystallization of the spKA-1–spISL complex (spISL, right). g Gel retardation assay of spISL by spKA-1. spISL RNAs were incubated with various concentrations of spKA-1 (0–10 μM). The fractions of the shifted RNA in the gel (left) were quantified (right). The experiments were independently repeated two times with similar results. Source data for b, c, g are provided as a Source data file.
