GoKawiilCloning, protein expression and purification
The full-length human NSUN2 gene was cloned into pET-21(+) (Novagen) with an N-terminal hexahistidine tag and a TEV cleavage site. Protein was expressed in Escherichia coli Rosetta DE3 cells (Sigma) grown in ZYM-5052 auto-induction media at 18 °C for 18 h (ref. 45). Cells were lysed by sonication, and the clarified lysate was subjected to immobilized metal affinity chromatography using Ni-NTA resin (Thermo Fisher). The nickel-affinity chromatography eluate was further purified by ion-exchange chromatography using a linear gradient from 100 mM to 1 M NaCl. The protein was further polished by size-exclusion chromatography with a buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 5 mM DTT. All NSUN2 constructs were purified similarly, and the representative gel-filtration profiles and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels of the purified proteins are shown (Supplementary Figs. 1 and 2). The human METTL1–WDR4 complex was expressed and purified using previously reported methods38,39.
RNA transcription and purification
DNA templates containing human tRNA genes for in vitro transcription were assembled using primer extension polymerase chain reaction (PCR) with a 5′ extended T7 promoter sequence (GCGAAATTAATACGACTCACTATA-[tRNA gene]). The PCR products were purified using a GeneJET PCR purification kit (Thermo Fisher). DNA templates were transcribed in vitro using 0.1 mg ml−1 recombinant T7 RNA polymerase produced in-house in a final buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM spermidine, 0.01% TritonX-100, 5 mM DTT, 2 mM rNTPs, 18–22 mM MgCl 2 and 20 ng µl−1 of DNA template. RNA transcripts were extracted using acid phenol:chloroform pH 4.5 (with IAA, 125:24:1) and precipitated in 1.2 volumes of isopropanol supplemented with sodium acetate pH 5.2 at −20 °C. Precipitated RNA was resuspended in a buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 2 mM MgCl 2 , then further purified using electrophoresis with a 10% urea-PAGE gel. Target RNAs were eluted with a buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 2 mM MgCl 2 for 16 h to extract RNA by passive diffusion. Extracted RNA was precipitated again with sodium acetate and isopropanol at −20 °C, and resuspended in a final RNA dissolving buffer of 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 2 mM MgCl 2 . Purified RNAs were analysed by native PAGE gels for evaluation (Supplementary Fig. 2). All RNAs in this study were annealed by incubating at 75 °C for 5 min, followed by slow cooling to 25 °C over 30 min before use. A list of DNA primers used for PCR is provided in Supplementary Table 1. Wild-type tRNA sequences and secondary structures were obtained from gtRNAdb46. The mitochondrial tRNASerGCT sequence and its secondary structure are adapted from ref. 47. Secondary structure diagrams of non-tRNA substrates were adapted from RNAfold calculations at 37 °C using the Andronescu 2007 energy model with coaxially stacked dangling ends unless stated otherwise48. For lncRNA RP11, the fragment was designed around the reported NSUN2-dependent m5C site Hg19, Chr1:854196.
In vitro methylation assay
In vitro methylation reactions with NSUN2 were performed with 50 nM enzyme and 100 nM RNA in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 5% glycerol, 5 mM DTT and a 200 nM [3H]-SAM:SAM mixture at a molar ratio of 1:4 (Figs. 1, 4–6 and Extended Data Figs. 6 and 8) or 1:3 (Figs. 2 and 3). Reactions with METTL1–WDR4 were performed under the same conditions for Extended Data Fig. 6d, and with 100 nM enzyme and 150 nM tRNA in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 5% glycerol, 5 mM DTT and a 1:3 molar ratio of [3H]-SAM:SAM mixture at a total concentration of 300 nM for Fig. 5e. Reactions were incubated at 37 °C for 1 h and blotted onto Hybond-N+ hybridization membranes. Membranes were washed with a buffer containing 20 mM Tris-HCl, pH 7.5, and 0.01% Triton X-100 before being placed in scintillation vials and suspended in 4 ml of a scintillation cocktail (RPI) for counting using an AccuFLEX LSC-8000 scintillation counter (Hitachi). A list of RNA sequences used is available in Supplementary Table 2.
NSUN2 in vitro crosslinking assay
In vitro crosslinking assays were performed with 500 nM NSUN2C271A, 1 µM RNA and 50 µM SAM in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 500 µM MgCl 2 , 5% glycerol and 5 mM DTT. Reactions were incubated at 37 °C for 1 h, then quenched with an SDS–PAGE gel loading buffer containing 300 mM Tris-HCl, pH 6.8, 12% (w/v) SDS and 40% glycerol supplemented with 10 mM DTT and heated at 95 °C for 5 min. About 350 ng of protein from each sample was loaded and separated by SDS–PAGE, then stained with Coomassie Blue.
EMSA
Dephosphorylated RNAs were 5′ radiolabelled using 0.4 U µl−1 T4 polynucleotide kinase (PNK) in 1X PNK buffer (New England BioLabs) with 133 nM [γ-32P]ATP (Revvity). Radiolabelled RNAs were purified over Bio-Gel P-6 SEC beads (BioRad) equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 2 mM MgCl 2 . RNA–protein complexes were formed by mixing the indicated concentrations of protein with 1 nM radiolabelled RNA substrate in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3 mM MgCl 2 , 10% glycerol, 5 mM DTT and 10 µM of non-specific DNA oligonucleotides at 25 °C for 1 h, then resolved with a native 8% Tris-borate polyacrylamide gel. Gels were dried and imaged using a Typhoon FLA 9500 phosphorimager (GE Healthcare). Band quantitation was performed with ImageLab v.6.1 (BioRad) using a background subtraction disk size of 8.0 mm. K D values were determined by fitting fractional binding data for three replicates to a Hill slope model with a maximum saturation value constrained to 1.0 using Prism 10.
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