Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Abstract

Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m⁵C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m⁵C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m⁵C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f⁵C sites on select tRNA. Finally, we show that f⁵C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m⁵C dioxygenases and mapping oxidative m⁵C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

Fig. 1. Probing m⁵C oxidation in RNA with 5-ethynylcytidine (5-EC).

a Structure of reported m⁵C oxidation products in RNA. b Proposed capture of m⁵C dioxygenases by metabolic labeling with 5-EC. 5-EC mimics m⁵C in RNA and upon enzymatic oxidation generates an electrophilic ketene that can covalently trap nearby nucleophiles. Source data are provided as a Source Data file. c Strategy for metabolic labeling with 5-EC. The nucleoside is fed to mammalian cells and incorporated into cellular RNA. Incorporation can be measured by quantitative mass spectrometry or click chemistry imaging. d LC-MS/MS analysis of 5-EC, m⁵C, and 5-EU in total RNA of HEK293T cells after treatment with 1 mM 5-EC or DMSO for 16 h. Three independent biological replicates were analyzed. Data represent mean values ± s.d. e Fluorescence microscopy analysis of 5-EC cellular incorporation. HEK293T were treated with 1 mM 5-EC or DMSO for 16 h, fixed and labeled with Cy3-N₃ before imaging by fluorescence microscopy. The experiments were repeated three times independently with similar results. Scale bar = 10 µM.

Fig. 2. Proteomic analysis of m⁵C dioxygenases using 5-EC RNABPP.

a 5-EC RNABPP workflow. Cells are metabolically labeled with 5-EC and crosslinked RNA-protein complexes are isolated by oligo(dT)-based enrichment. Enriched RNA-protein complexes are digested with RNase and then analyzed by mass spectrometry-based proteomics. b Volcano plot showing the enrichment of 5-EC reactive proteins. Experiment was performed independently in triplicate and protein abundance was quantified by spectral counting. Multiple student’s unpaired two-sided t-tests were used to assess statistical significance. c Western blot validation of ALKBH1, TET2, and MMP8. 293 Flp-In cells overexpressing the respective FLAG-tagged protein were treated with 1 mM 5-EC or DMSO and subjected to the RNABPP workflow before western blot analysis. Experiments were performed in triplicate with similar results. Source data are provided as a Source Data file.

Fig. 3. ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in mRNA and small RNA.

a Strategy for mRNA isolation and LC-QQQ-MS quantification of modified nucleosides in HEK 293T wild-type (WT) and KO cells. Total RNA is isolated and subjected to two rounds of poly(A) enrichment, followed by rRNA and small RNA depletion. The mRNA is then digested to nucleosides and analyzed by LC-QQQ-MS. b Quantification of hm⁵C in total RNA from WT and ALKBH KO cell lines. p-values: WT vs. KO #1, p = 0.0000106; WT vs. KO #2, p = 0.0000103. c Quantification of f⁵C in total and mRNA from WT and ALKBH1 KO cell lines. p-values: WT vs. KO #1 in total RNA, 0.000004; in small RNA, 0.000001; WT vs. KO #2 in total RNA, 0.000007; in small RNA, 0.000036. d Quantification of f⁵C in small RNA from WT and ALKBH1 KO cells. p-values: WT vs KO in total RNA, p = 0.000109; in small RNA, p = 0.00000013. e Quantification of f⁵C in total and mRNA from WT and TET2 KO cell lines. p-values: WT vs. KO #1 in total RNA, 0.003594; in small RNA, 0.001040; WT vs. KO #2 in total RNA, 0.002192; in small RNA, 0.049386. f Quantification of f⁵C in total, mRNA, and small RNA from WT and NSUN2 KO cells. p-values: WT vs KO in total RNA, 0.285820; in mRNA, 0.00088; in small RNA; 0.000017. g Quantification of f⁵C in total, mRNA, and small RNA from WT and NSUN3 KO cells. p-values: WT vs KO in total RNA, 0.000007; in mRNA, 0.001198; in small RNA; 0.291189. Three independent biological replicates were analyzed, except in f and g, where n = 6 in total RNA measurements. Data represent mean values ± s.d. An unpaired t test (two-tailed) was used to measure the statistical significance *p  <  0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 4. 5-EC-iCLIP sequencing of ALKBH1 m⁵C RNA substrates.

a Strategy for 5-EC-iCLIP workflow. Cells overexpressing ALKBH1 are metabolically labeled with 5-EC and the RNA-crosslinked protein is immunoprecipitated (IP) and digested. RNA is reverse transcribed (RT) and prepared for Illumina sequencing. b Analysis of RNA-ALKBH1 crosslinking in immunoprecipitated 5-EC-iCLIP samples after biotin labeling of RNA. Experiment was repeated two times with similar results. c Distribution of uniquely mapped reads identified by 5-EC-iCLIP; lncRNA, long non-coding RNA. d Abundance of tRNA species enriched by 5-EC-iCLIP. cDNA scores from reads were normalized to reads per million (RPM). e Heatmap showing enrichment (in RPM) of all crosslink peaks in tRNA targets according to their relative position in the mature tRNAs. Peaks below 5000 RPM are not visible on color scale. f Distribution of all peaks in tRNA Leu-CAA according to their relative position in the mature tRNA. g Distribution of crosslink peaks in all tRNA targets identified by 5-EC-iCLIP according to their relative position in the mature tRNAs. h tRNA consensus motif identified by MEME from all tRNA peaks identified by 5-EC-iCLIP. i non-tRNA consensus motif identified by MEME from iCLIP non-tRNA peaks.

Fig. 5. Transcriptome-wide profiling of RNA f⁵C using Aldehyde-reactive probe (ARP)-sequencing.

a Workflow for ARP sequencing. Fragmented total RNA extracted from WT and ALKBH1 KO cells is incubated with ARP, and labeled RNA is enriched through streptavidin pulldown. Libraries are generated through RT, cDNA circulation, and PCR amplification. b Distribution of enriched ALKBH1-dependent peaks (cDNA value > 10) identified by ARP sequencing. c IGV tracks showing the reads mapped to mt-tRNA-Met in input and IP samples of WT and ALKBH1 KO cells. d Top ALKBH1-dependent f⁵C-containing tRNA substrates enriched by ARP pulldown. Only peaks with cDNA score >100 and present in the WT sample and absent from the ALKBH1 KO control were considered.

Fig. 6. Validation of 5-EC-iCLIP tRNA hits.

a Strategy for LC-QQQ-MS quantification of modified nucleosides in individual tRNAs isolated from HEK 293T wild-type (WT) and ALKBH1 KO cells. Individual tRNAs are enriched by antisense pulldown, digested to mononucleosides and analyzed by LC-QQQ-MS. b–g Quantification of oxidized m⁵C products in b mt-tRNA-Met, p-values: WT vs KO for m⁵C, 0.026079; for f⁵C, 0.000132; for hm⁵C, 0.000244, c tRNA-Leu-CAA, p-values: WT vs KO for m⁵C, 0.156295; for f⁵C, 0.000170; for hm⁵C, 0.000002, d tRNA-Glu-CTC, p-values: WT vs KO for m⁵C, 0.975934; for f⁵C, 0.002584, e tRNA-Gly-CCC, p-values: WT vs KO for m⁵C, 0.161660; for f⁵C, 0.001039, f tRNA-Gln-CTG, p-values: WT vs KO for m⁵C, 0.365015; for f⁵C, 0.171454, and g tRNA-Val-CAC, p-values: WT vs KO for m⁵C, 0.152377; for f⁵C, 0.124395; for hm⁵C, 0.366551. h Schematic workflow for pyridine borane f⁵C sequencing. Pyridine borane converts f⁵C residues to dihydrouridine, which is read as uridine, thus generating a C-to-T signature that can be identified in sequencing (i) Validation of presence and ALKBH1-dependence of f⁵C at the wobble base of mt-tRNA-Met by pyridine borane sequencing. j Validation of presence and ALKBH1-dependence of f⁵C at the wobble base of tRNA-Leu-CAA. k Presence and ALKBH1-dependence of f⁵C at various positions of tRNA-Val-CAC and tRNA Glu-CTC. For (b)–(g), three independent biological replicates were analyzed. Data represent mean values ± s.d. An unpaired t-test (two-tailed) was used to measure the statistical significance *p  <  0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For (h)–(j), two independent biological replicates were performed and analyzed. Source data are provided as a Source Data file.

Fig. 7. In vitro activity of ALKBH1 on structured and linear substrates.

a Workflow to assess activity of ALKBH1 in vitro. Purified ALKBH1 is incubated with RNA substrate and cofactors at 37 °C for 30 min. The reaction is quenched, and RNA is purified, digested, and analyzed by LC-QQQ-MS. Activity was measured by quantifying the decrease of m⁵C and the generation of oxidized products hm⁵C and f⁵C. b Sequences of anticodon stem loops (ASLs) tested in the assay. All substrates have an m⁵C residue at the wobble base. c Conversion of m⁵C and generation of hm⁵C and f⁵C in various ASL substrates. d Sequences of linear substrates tested in the assay. e Conversion of m⁵C and generation of hm⁵C and f⁵C in linear substrates. For (c) and e, to account for loading differences, values were first quantified as ratios over A and then normalized to the maximum possible ratio for that substrate. Three independent replicates were performed, and data represent mean values ± s.d. An unpaired t-test (two-tailed) was used to measure the statistical significance *p  <  0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For (c), p-values (+ALKBH1 versus No enzyme) for substrate 3, 0.000006; for substrate 8, 0.0141996; for mt-Met, 0.0009131. For (e), P-values (f⁵C versus hm⁵c) for substrate 3, 0.0000927; substrate 4, 0.009919; for substrate 5, 0.0000889; for substrate 8, 0.000914; for mt-Met, 0.0004327. Source data are provided as a Source Data file.