Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation

Abstract

Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (m⁵C) and 5-methyluridine (m⁵U) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.

Figure 1.

RNA-mediated activity-based protein profiling (RNABPP) enables the chemoproteomic analysis of RNA modifying enzymes in living cells. (a) Schematic representation of the RNABPP approach. Metabolic labeling with a nucleoside-based activity probe facilitates the incorporation of a chemical “warhead” into cellular RNA. Crosslinked RNA modifying enzymes on mRNA can then be identified through oligo-dT based enrichment and quantitative proteomics analysis. (b) Chemical structure of 5-FCyd and proposed RNA-protein crosslinking between 5-FCyd-labeled RNA and m⁵C RNA methyltransferases. (c) LC-MS/MS analysis of 5-FCyd and 5-FUrd levels in total RNA after metabolic labeling of HEK293T cells for 12 hr with 5-FCyd. 3 independent biological replicates were analyzed. Data represent mean values ± SEM. (d) Western blot analysis of protein-RNA crosslinking in cells after 5-FCyd labeling. NSUN2, NSUN5, NSUN6, and DNMT2 were expressed as 3xFLAG-fusion proteins and cells were treated with 10 μM 5-FCyd (for NSUN2 and NSUN6) and 100 μM 5-FCyd (for DNMT2 and NSUN5) for 12 hr. (e) Oligo-dT based isolation of NSUN2 using RNABPP with 5-FCyd. Cells were fed with 10 μM 5-FCyd, 10 μM 5-azacytidine (5-AzaC), or untreated, and subjected to the RNABPP workflow. For (d) and (e), the experiments were repeated 3 times independently with similar results.

Figure 2.

Proteomic analysis of 5-FCyd-reactive proteins on mRNA using RNABPP. (a) Volcano plot showing enrichment of 5-FCyd-reactive proteins. 3 independent biological replicates were analyzed using TMT-based isobaric tags. Multiple t test (unpaired) was performed to evaluate the statistical significance. P = 0.000065 for NSUN2; p =0.000027 for NSUN5; p = 0.00015 for TRMT2A; p = 0.0015 for TRMT2B; p = 0.0037 for NSUN5C; p = 0.0081 for DUS3L; p = 0.0084 for DPYD; p = 0.025 for DNMT2; p = 0.027 for TOP3A. (b) Western blot validation of proteomics result for m⁵U methyltransferase TRMT2A. (c) Western blot analysis of TRMT2A-RNA crosslinking after metabolic labeling with 10 μM 5-FCyd or 5-FUrd for 12h. For (b) and (c), the experiments were repeated 3 times independently with similar results.

Figure 3.

NSUN2 and TRMT2A are the major RNA m⁵C and m⁵U methyltransferases, respectively. (a) Schematic representation of mRNA isolation protocol for LC-QQQ-MS quantitation of modified nucleotides in HEK293T WT and KO cells. (b) - (e) Quantitation of m⁵C and m⁵U levels in total RNA and mRNA extracted from WT, NSUN2 KO, NSUN5 KO, and TRMT2A KO cell lines. 3 independent biological replicates were analyzed. Data represent mean values ± SEM. Unpaired t test (two-tailed) was performed to evaluate the statistical significance. P = 0.00045 in (b); p = 0.000040 in (c); p = 0.00063 in (c); p = 0.0043 in (d).

Figure 4.

DUS3L installs dihydrouridine on human RNA. (a) Western blot analysis of 5-FCyd-mediated enrichment for DUS3L. After metabolic labeling with 5-FCyd, oligo-dT enriched samples were analyzed by western blot using an anti-DUS3L antibody. (b) Western blot analysis of DUS3L-RNA crosslinking after metabolic labeling with a panel of C5-halogenated pyrimidines. (c) Proposed mechanism of DUS3L-RNA crosslinking mediated by C5-halogenated uridine derivatives. (d) Western blot analysis of protein-RNA crosslinking between WT DUS3L or C396A DUS3L and 5-FUrd or 5-BrUrd labeled RNA. WT or mutant DUS3L transgenes were introduced by transfection of plasmid constructs into DUS3L KO cells. For (a), (b) and (d), the experiments were repeated 3 times independently with similar results. (e) – (g) Quantitation of dihydrouridine (DHU) levels in total RNA, small RNA, and mRNA extracted from WT and DUS3L KO cells. 3 independent biological replicates were analyzed. Data represent mean values ± SEM. Unpaired t test (two-tailed) was performed to evaluate the statistical significance. *: p = 0.012, **: p = 0.0046 in (f); **: p = 0.0072 (#1) and p = 0.0035 (#2) in (g).

Figure 5.

5-FUrd-iCLIP sequencing of DUS3L substrates. (a) Schematic of 5-FUrd-iCLIP workflow. (b) Analysis of RNA-protein crosslinking in DUS3L iCLIP samples. Cells expressing DUS3L or DUS3L C396A were treated with 5-FUrd or left untreated, and covalently linked RNA was detected by anti-biotin western blot after immunoprecipitation and RNase fragmentation. For (b), the experiment was repeated 3 times independently with similar results. (c) Composition of RNA identified by 5-FUrd-iCLIP according to uniquely mapped reads. (d) Abundance of tRNA species as enriched by 5-FUrd-iCLIP; RPM: reads per million. (e) Coverage of all tRNA peaks according to their relative position within the mature tRNA. (f) Consensus motif detected by MEME using all RNA peaks identified by 5-FUrd-iCLIP.

Figure 6.

DUS3L regulates cell proliferation and protein translation efficiency. (a) Fluorescence microscopy analysis of DUS3L localization and OP-puro incorporation by WT and DUS3L KO cells. OP-puro labeling was imaged following CuAAC reaction with Cy3-azide. 2 independent biological replicates were analyzed for each cell line with similar results. (b) Flow cytometry analysis of global protein translation using OP-puro in WT and DUS3L KO cells (c) Plot of OP-puro incorporation as measured by Cy3 fluorescence from (b). The median fluorescence intensity and SEM from 3 independent biological replicates (50,000 cells analyzed per sample, 3 technical replicates per sample) are shown. Unpaired t test (two-tailed) was performed to evaluate the statistical significance. ****: p = 0.00013 (#1) and p = 0.0000094 (#2); ns: p = 0.064. (d) Cell viability of WT and DUS3L KO cells measured by MTS assay. 12 independent biological replicates were analyzed. Data represent mean values ± SEM. Unpaired t test (two-tailed) was performed to evaluate the statistical significance. **: p = 0.0075 (#1) and p = 0.0068 (#2).