Extended Data Fig. 7: NSUN2 requires two RNA stems for methylation and binding.
From: Substrate selectivity of the human RNA m5C methyltransferase NSUN2
a,b, EMSA of NSUN2 binding tRNALysTTT, tRNALysTTT(∆Acceptor), tRNALysTTT(∆T-arm), and the Mini substrate. Representative gels among 3 replicates (a), and quantitation (b) are shown. Protein concentrations from left to right are: 0, 16, 32, 65, 130, 260, and 520 nM. For source data, see Supplementary Fig. 1. c, Diagram of Mini (Fig. 5f) and MiniN-LoopMut substrates with mutated nucleotides shown in red and the target cytosine highlighted yellow. d, Methylation activity of NSUN2 with the substrates in c. Ctr, no enzyme control. Data are shown as mean ± s.d (n = 3, two-tailed unpaired t-test). e, In vitro methylation activity of NSUN2 with a series of Mini bulge truncations. Ctr, no enzyme control. Data are shown as mean ± s.d (n = 3, two-tailed unpaired t-test). f, Secondary structure diagrams of minimized oligonucleotide substrates with m5C or U at specific positions. Target cytosines are highlighted in yellow. g, Methylation activity of NSUN2 with the substrates in f at different time points. Data are shown as mean ± s.d (n = 3, two-tailed unpaired t-test). h, Close-up views of NSUN2:tRNA complexes modeled with a pre-existing m5Cyt48 (left) or m5Cyt49 (right). Protein (purple) side chains within 3.8 Å of the modeled RNA base (orange) are shown as sticks. Cytosine residues in the catalytic pocket are colored cyan. i, Secondary structure diagrams of WT (top) and mutant (bottom) RP11 fragments. CNNRR nucleotides are bolded, with motif mutations colored red and complementary mutations colored gray. Target cytosines are highlighted in yellow. j, In vitro methylation activity of NSUN2 with the substrates described in i and RP11CtoU, with the m5C site mutated. Data are shown as mean ± s.d (n = 3, two-tailed unpaired t-test).
