Extended Data Fig. 1: Biochemical characterization of NSUN2 interactions with substrate tRNAs and cofactor.
From: Substrate selectivity of the human RNA m5C methyltransferase NSUN2
a, Frequent (> 5%) NSUN2 copy number alterations across various cancer types. Data adapted from cBioPortal. b, SDS-PAGE of purified full-length NSUN2WT and NSUN2C271A. A representative gel among 3 replicates is shown. For source data, see Supplementary Fig. 1. c,d, EMSA for NSUN2 binding tRNALysCTT, tRNAIleTAT, and tRNALysCTT(C48U). Representative gels from 3 replicates (c) and quantitation (d) are shown. For source data, see Supplementary Fig. 1. Estimated KD from each curve is shown under each gel. Protein concentrations for gels from left to right are: 0, 16, 32, 65, 130, 260, and 520 nM. e, Alignment of human NSUN family methyltransferase domain sequences near the catalytic site. Catalytic residues (red, numbered for NSUN2) and conserved motifs (gray) are highlighted. f, Proposed catalytic mechanism of NSUN2 with protein atoms in blue and cofactor atoms in pink. g,h, Cryo-EM structures of NSUN2C271A-tRNALysCTT-SAH complexes in the “D-arm” conformation (g) and “No D-arm” conformation (h). Protein shown in cartoon representation, colored by domain, with RNA shown as an orange ribbon and SAH as pink sticks. i,j, Close-up views of NSUN2:tRNA Anticodon arm interactions in the D-arm (i) and No-D-arm (j) conformations. Sharpened cryo-EM map is overlaid with protein side chains and RNA nucleotides, colored as in g. k, Models of isolated tRNALysTTT (PDB: 1FIR, top) and NSUN2-bound tRNALysCTT (“D-arm” conformation, bottom) displayed as gray and orange ribbons, respectively, with Cyt48 shown as cyan sticks. Solid arrows indicate the distance between Gua19N1 and Cyt56N3. l-r, Close-up views of NSUN2C271A-tRNALysCTT-SAH structure D-arm conformation, near the T-stem (l), T-loop (m), T-arm (n), Acceptor arm (o) 3′ Acceptor end (p), catalytic pocket (q), and m5C48 (r). Sharpened cryo-EM map is overlaid with protein side chains and RNA nucleotides, colored as in g. Only protein sidechains within 3.8 Å of RNA and SAH are labeled. s, Another view of the catalytic pocket shows the RNA chain draping over the occupied cofactor-binding pocket with RNA and cofactor shown as sticks with a transparent surface. Protein is shown as a transparent surface. t, Isothermal titration calorimetry of SAM binding by NSUN2. KD is calculated from two replicates with 95% confidence intervals in brackets. u,v, EMSA of NSUN2 binding WT tRNALysCTT with no cofactor (None), 500 µM Sinefungin (SFG) or SAH. Representative gels among 3 replicates (u), and quantitation (v) are shown. “None” is the same series as “LysCTT” in d. Protein concentrations from left to right are: 0, 16, 32, 65, 130, 260, and 520 nM. For source data, see Supplementary Fig. 1.
