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TET2 chemically modifies tRNAs and regulates tRNA fragment levels

Abstract

The ten-eleven translocation 2 (TET2) protein, which oxidizes 5-methylcytosine in DNA, can also bind RNA; however, the targets and function of TET2–RNA interactions in vivo are not fully understood. Using stringent affinity tags introduced at the Tet2 locus, we purified and sequenced TET2-crosslinked RNAs from mouse embryonic stem cells (mESCs) and found a high enrichment for tRNAs. RNA immunoprecipitation with an antibody against 5-hydroxymethylcytosine (hm5C) recovered tRNAs that overlapped with those bound to TET2 in cells. Mass spectrometry (MS) analyses revealed that TET2 is necessary and sufficient for the deposition of the hm5C modification on tRNA. Tet2 knockout in mESCs affected the levels of several small noncoding RNAs originating from TET2-bound tRNAs that were enriched by hm5C immunoprecipitation. Thus, our results suggest a new function of TET2 in promoting the conversion of 5-methylcytosine to hm5C on tRNA and regulating the processing or stability of different classes of tRNA fragments.

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Fig. 1: Validation of RNA binding by TET2 in mESCs.
Fig. 2: The TET2-bound transcriptome.
Fig. 3: Distribution of hm5C and TET2 binding overlap within tRNAs.
Fig. 4: hm5C is enriched on tRNAs and depleted in the absence of TETs.
Fig. 5: Loss of TET2 affects the balance between classes of tRFs.

Data availability

RNA sequencing data generated for this study have been deposited in the NCBI GEO with accession number GSE133472. Raw mass spectrometry data are available on figshare (https://doi.org/10.6084/m9.figshare.c.5133581).

Code availability

Software utilized for each analysis is detailed in the relevant Methods section. Scripts and R markdown documents to generate figures are available from the corresponding author upon request.

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Acknowledgements

We thank R. Martienssen for support, encouragement, and discussions; M. Liu and R. Kohli for the kind gift of recombinant TET2 protein; T. Christopher for technical help; G. Xu for his generous gifts of Tet1/2/3-tKO cells; K. Ingvarsdottir and R. Warneford-Thomson for tissue culture help; and S. Erhardt for helpful discussion. R.B. acknowledges support from the NIH (R01GM127408). C.H. was supported in part by the National Natural Science Foundation of China (32070613, 31800687), the Natural Science Foundation of Hunan Province of China (2020JJ4179) and the Fundamental Research Funds for the Central Universities of China (531107051157). B.A.G. was supported in part by the NIH (R01GM110174, R01AI118891 and P01CA196539). A.J.S. acknowledges assistance from the Cold Spring Harbor Laboratory Shared Resources, which are funded in part by the Cancer Center Support Grant (5PP30CA045508). J.E.W. is a Rita Allen Foundation Scholar and is supported by NIH grant R35-GM119735.

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Contributions

C.H. and R.B. conceived the project and designed the experiments. C.H. generated the ESC lines and performed most of the experiments. J.B. performed rescue experiments and small RNA sequencing. K.A.J. performed all MS under the supervision of B.A.G. A.J.S. sequenced and analyzed small RNAs. J.E.W. helped with RNA biochemistry. C.H. and R.B. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Chongsheng He or Roberto Bonasio.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Generation of epitope-tagged Tet2 alleles and CLIP quantification.

a, Genotype validation for 6xHis–HA knock-in at the Tet2 locus by Sanger sequencing. The targeted allele scheme (top), expected protein and DNA sequence (middle), and sequencing traces (bottom) for the two clones used in subsequent experiments are shown. b, Quantification of Fig. 1D; fluorescence signal (crosslinked RNA) was normalized to WB signal (protein). Bars represent the mean + s.e.m. P-value is from a Student’s t-test.

Extended Data Fig. 2 Additional analyses on TET2 CLIP-seq.

a, Fluorescence image of two CLIP replicates from two cell lines (#65 and #75) used for CLIP-seq library construction. The dashed red boxes indicate the position of the excised bands. Bottom panel, Western blot for HA was used as a loading control (bottom). Uncropped blot images are shown in Supplementary Fig. 1. b, Percentage of transcripts from the indicated classes enriched > 2-fold in TET2 CLIP compared to input (black bars). The non-enriched portion of each class is shown in gray. Only transcripts detected (> 1 read) in at least one replicate were considered. snRNA, small nuclear RNAs; rRNA, ribosomal RNAs; miRNA, micro RNAs; lncRNA, long noncoding RNAs; misc, RNAs not included in the other displayed categories; pseudo, pseudogene-derived RNAs; snoRNA, small nucleolar RNAs; scaRNA, small Cajal body RNAs; mRNA, protein-coding messenger RNAs. c, Same as (B) but considering as enriched only RNAs containing a peak (see text for details) with FDR-corrected P-value < 10−5.

Extended Data Fig. 3 Replicate consistency for the hm5C RIP experiment.

Clustered heatmap showing the Pearson’s correlation coefficients between RPKMs calculated on all annotated genes in input and hm5C RIP for two biological replicates each.

Extended Data Fig. 4 RNA fractionation and mass spectrometry.

a, Representative gel (6% polyacrylamide, 7 M urea) showing the three RNA fractions analyzed in Fig. 4: total RNA (no fractionation), small RNAs < 200 nts (column-based size selection), and tRNAs (gel purification). The band corresponding to tRNAs (~70 nts) is indicated by the arrow. b, Mass spectrometry chromatograms of nucleosides fragmented into nucleobases. Data were acquired by isolating a precursor ion (nucleoside), fragmenting the precursor ion, and then isolating and detecting a known fragment ion (nucleobase). The top two chromatograms show C (244.093 → 112.050 m/z) and a spiked-in heavy C standard. The bottom two chromatograms show hm5C (256.103 → 142.061 m/z) and a spiked-in heavy hm5C standard (277.103 → 145.061 m/z). The representative chromatograms shown were obtained from the same run on tRNAs from Tet1/2/3 tKO cells transiently transfected with TET2 WT (Fig. 4D). Only one known nucleoside, 5-aminomethyluridine (nm5U), is isobaric with hm5C, and can be easily distinguished from hm5C by retention time. c, Western blot for TET2 comparing WT (+/+) and presumptive KO (–/–) clones as determined by PCR screening and Sanger sequencing. Tubulin is shown as loading control. d, Mass spectrometry quantification of hm5C in size-selected small RNAs < 200 nts from WT (left) Tet2 single KO (middle) and Tet1/2/3 triple KO (right) mESCs. Bars represent mean + s.e.m. ***, P < 0.001. P-values are from one-way ANOVA followed by Holm-Sidak test. Uncropped gel and blot images for a and c are shown in Supplementary Fig. 1.

Extended Data Fig. 5 Additional small RNA sequencing dataset comparing Tet2−/− with E14 ESCs.

a, Coverage of tRNA genes by non-CCA (left) and CCA-containing (right) reads in small RNAs purified from control (E14) or Tet2/ cells. Plots show the average RPMs. Position of the three types of tRFs discussed in the text are indidated (tRF5, tRF3a, and tRF3b). b, Quantification of (a) but only considering size-filtered reads; 28–35 for tRF5, 17–19 (inclusive of CCA) for tRF3a, and 22 (inclusive of CCA) for tRF3b. c, Differential expression analysis for individual tRFs in E14 and Tet2/ cells. Estimated (DESeq2) fold changes are plotted on the x axis and the log-converted P-value on the y axis. Blue and red dots highlight individual tRFs that pass an adjusted P-value cutoff of 0.1 and are downregulated or upregulated in the KO, respectively. d, Overlap of TET2-bound tRNAs as determined by CLIP (Fig. 2), and the tRF3a significantly upregulated in Tet2/ cells as determined in (c). The TET2-bound tRNAs were grouped according to the predicted sequence of the tRF3 produced from them. The P-value was calculated based on the hypergeometric distribution. e, Same as (d) but showing the overlap with tRNAs enriched by hm5C RIP (Fig. 3). f, Comparison of estimated log2(fold-changes) for all tRF3a significantly enriched in Tet2/ cells in two independent experiments (exp 1, Fig. 5; and exp 2, Extended Data Fig. 5). Replicates are from three independent cell cultures and RNA purifications per genotype.

Extended Data Fig. 6 Examples of tRNAs methylated by NSUN2 and regulated by TET2.

a, Heatmap for the % of unconverted BS-seq reads on tRNAs as reported by Legrand et al.10 (GEO series GSE81825). b, Overlap of tRF3a (top) or tRF3b (bottom) detected as significantly upregulated in Tet2-/ cells compared to WT in Fig. 5 with highly methylated targets of NSUN2 (>75% m5C at NSUN2 sites). P-values are from Fisher’s test comparing overlaps with methylated and unmethylated tRNAs. c, Levels (RPMs) for a tRF3a from LeuCAA tRNAs in WT and Tet2/ cells. The two plots show data from two independent experimental replicates, corresponding to Fig. 5 (left) and Extended Fig. 5 (right). Mean ± s.e.m. are shown. d, Genomic browser snapshot for CLIP and hm5C at the chr11.tRNA1911-LeuCAA locus. Matching inputs are shown. The y axis represents RPMs. e, Schematic depiction of methylation patterns on chr11.tRNA1911-LeuCAA as determined by BS-seq in Legrand et al.10. The position of m5C in the anticodon and after the variable loop (VL) is indicated by thicker circles and the % of uncovered reads is shown using the same color scale used in (a). f–h, Same as (c–e) but for chr13.tRNA988-SerGCT. i–k, Same as (c–e) but for chr13.tRNA112-SerTGA.

Supplementary information

Supplementary Information

Supplementary Figure 1 (uncropped blots).

Reporting Summary

Supplementary Table 1

Read numbers for the CLIP experiment.

Supplementary Table 2

Read numbers for the RIP experiment.

Supplementary Table 3

List of tRNAs recovered by TET2 CLIP (Fig. 2) and hm5C RIP (Fig. 3).

Supplementary Table 4

Synthetic nucleotide sequences.

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He, C., Bozler, J., Janssen, K.A. et al. TET2 chemically modifies tRNAs and regulates tRNA fragment levels. Nat Struct Mol Biol 28, 62–70 (2021). https://doi.org/10.1038/s41594-020-00526-w

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