N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

Abstract

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells^1-4. Here we show that N⁶-methyladenosine (m⁶A) modification, contributing to different aspects of messenger RNA metabolism^5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m⁶A-containing R-loops accumulate during G₂/M and are depleted at G₀/G₁ phases of the cell cycle, and that the m⁶A reader promoting mRNA degradation, YTHDF2 (ref. ⁷), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m⁶A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Extended Data Fig. 1. Validation of m⁶A DIP using synthetic RNA and DNA substrates.

Validation of m⁶A DIP using synthetic RNA and DNA substrates. See also Supplementary Note. (a) Schematic demonstrating the sequences of synthetic oligonucleotides and RNA:DNA hybrids used for spike in validation experiments. (b) The relative enrichment of DIP/DRIP performed on 0.1 pmol of spike in synthetic m⁶A-containing RNA:DNA hybrid (81-mer with 46 % GC content) shown in (a) using anti-m⁶A and S9.6 antibodies along with IGG for IP after the indicated time intervals of heat denaturation. (c) S9.6 DRIP exhibits comparable efficiencies in precipitating non-modified and m⁶A-containing RNA:DNA hybrids. Relative enrichment of DRIP performed on the m⁶A-containing RNA:DNA synthetic substrate normalized against that of DRIP done on equivalent amount (0.1 pmol) of non-modified spike in synthetic RNA:DNA hybrid. (d) The results of m⁶A DIP on 0.1 pmol of the indicated spike in synthetic oligonucleotides and RNA:DNA hybrids. Unlike non-modified RNA:DNA hybrid substrate or single stranded m⁶A-containing RNA oligonucleotide, m⁶A-containing RNA:DNA hybrid is efficiently detected by m⁶A DIP technique. Data are means ± SD, n=3 independent experiments.

Extended Data Fig. 2. Detailed schematic illustrating S9.6 DRIP and m⁶A DIP techniques.

Detailed schematic illustrating S9.6 DRIP and m⁶A DIP techniques.

Extended Data Fig. 3. m⁶A is present on the majority of RNA:DNA hybrids in hiPSCs.

m⁶A is present on the majority of RNA:DNA hybrids in hiPSCs. (a) Heatmaps showing the distribution of density of indicated reads across genomic regions containing peaks (3 kb around peak center) of the three categories: m⁶A peaks overlapping with S9.6 peaks (m⁶A/S9.6), m⁶A peaks that do not overlap with S9.6 DRIP peaks (m⁶A only) and S9.6 peaks that do not correspond to m⁶A DIP peaks (S9.6 only). The colour of each line represents the density of reads for a given peak. The width of the heatmaps is normalized by peak length. (b, c) Distribution of the m⁶A/S9.6-, m⁶A only- and S9.6 only peaks at the indicated genomic features (b) and relative to transcription start site (TSS) (c) in hiPSCs.

Extended Data Fig. 4. RNA:DNA hybrids exhibit cell cycle-specific dynamics in hPSCs.

RNA:DNA hybrids exhibit cell cycle-specific dynamics in hPSCs. (a) The diagram illustrating gating of single hiPSCs using PI-Area and PI-Width signals (left panel) and DNA content frequency histogram (right panel) of a representative hiPSCs population used for cell cycle analysis. hPSCs at G₀/G₁, S and G₂/M phases are marked. (b) The coverage plots of m⁶A DIP and S9.6 DRIP densities (CPK) in the intronic regions of the indicated genes. m⁶A and S9.6 peaks are marked with red and blue rectangles.

Extended Data Fig. 5. m⁶A DIP signal is increased upon RNase H1 knockdown in hPSCs.

m⁶A DIP signal is increased upon RNase H1 knockdown in hPSCs. (a, b) The coverage plots of m⁶A and S9.6 DRIP/DIP densities (CPK) in the regions located downstream of the m⁶A and S9.6 peaks (marked with red rectangles). The location of regions that were used as controls in (d) is designated by blue rectangles. (c) Relative expression of RNase H1 and LINE1 transcripts in hPSCs transfected with control non-targeting (siCTL) and RNase H1 (siRNaseH1) siRNAs. (d) The results of m⁶A DIP qPCR of the indicated repeats and intronic sequences performed on siCTL and siRNaseH1 hiPSCs. The regions without peaks (RANBP17 Downstream and HECW1 Downstream) were used as controls. Generic primers amplifying Alu elements from the indicated families and evolutionarily young L1Hs were used for DRIP qPCRs and qPCR. Data are means ± SD, n=3 independent experiments.

Extended Data Fig. 6. m⁶A is detectable on the RNA components of R-loops.

m⁶A is detectable on the RNA components of R-loops. (a) Schematic representation of the two round (S9.6 followed by m⁶A) DRIP/DIP procedure. (b, c) The results of the two round (S9.6 DRIP followed by m⁶A DIP) DRIP/DIP performed on individual intronic (b) and repetitive (c) m⁶A/S9.6 peak containing sequences. (d) Schematic representation of the m⁶A RIP performed on the RNA isolated from S9.6 IP-ed nucleic acids, followed by RT-qPCR of the candidate sequences. (e) The results of the m⁶A RIP performed on the S9.6 DRIP of individual and repetitive DRIP/m⁶A DIP-peak containing sequences. No RT represents control samples processed without reverse transcription. The regions without peaks (RANBP17 Downstream and HECW1 Downstream) were used as controls in (b and e). Representative results of analysis of one of 3 independent biological samples are presented. Data are means ± SD, n=3 technical repeats.

Extended Data Fig. 7. METTL3 depletion leads to accumulation of RNA:DNA hybrids in hPSCs.

METTL3 depletion leads to accumulation of RNA:DNA hybrids in hPSCs. (a) The results of S9.6 DRIP qPCR of the indicated repeats and intronic sequences performed on siCTL and siMETTL3 hiPSCs sorted at different cell cycle phases. (b) Relative expression of the indicated transcripts in siCTL and siMETTL3 hiPSCs. The difference between the values of Y-axes in (a) and in Fig.3b is due to incorporation of RNase H control samples in the analysis shown in Fig.3b. Data are means ± SD, n=3 independent experiments.

Extended Data Fig. 8. METTL3 depletion leads to an increase in the m⁶A DIP peaks in hPSCs.

METTL3 depletion leads to an increase in the m⁶A DIP peaks in hPSCs. (a) SID-UPLC-MS/MS quantification of m⁶A in the total nucleic acids (left panel), and in ultrafiltrate fractions released upon RNase H treatment of siCTL and siMETTL3 hPSCs (right panel). The data are means of 2 independent experiments. (b) The total numbers of consensus m⁶A peaks in WT and siMETTL3 hiPSCs. (c) Heatmaps showing the distribution of density of indicated reads across peak-containing genomic regions (3 kb around peak center) for S9.6 DRIP in WT and m⁶A DIP in WT and siMETTL3 hPSCs. The colour of each line represents the density of reads for a given peak. The width of the heatmaps is normalized by peak length. (d) Pie chart showing the percentages of transcripts differentially expressed between siCTL and siMETTL3 hPSCs (p<0.01) and without significant changes in expression amongst genes containing siMETTL3-specific m⁶A peaks. P values were calculated using the Standard ballgown parametric F-test. (e) Pie chart demonstrating the percentages of genes differentially expressed between siCTL and siMETTL3 hPSCs containing siMETTL3-specific m⁶A peaks (diff peaks) and without such peaks (No diff peaks). (f) Average profile of m⁶A peak densities for all genes sorted based on levels of their expression in siMETTL3 hPSCs. The colour gradient represents log₁₀ of mean RPKM per bin. (g) Average profile of m⁶A peak densities for all genes sorted based on the fold change of their expression between siCTL and siMETTL3 hPSCs. The colour gradient represents log₁₀ of mean fold change (FC) per bin.

Extended Data Fig. 9. YTHDF2 co-localizes with R-loops in vivo and interacts with synthetic m⁶A-marked RNA:DNA hybrids in vitro.

YTHDF2 co-localizes with R-loops in vivo and interacts with synthetic m⁶A-marked RNA:DNA hybrids in vitro. (a) Co-immunostaining of YTHDF2 with R-loops (S9.6) in a representative hiPSCs interphase nucleus. Merged view and individual channels are shown. The area used for S9.6/YTHDF2 signals quantification is indicated. The arrow designates the region used for generation of signal intensity profile shown in (b). (b) The profile showing intensities of YTHDF2, S9.6 and DAPI signals across the nuclear region marked with an arrow in (a). (c) Representative image of hPSCs immunostained for YTHDF2 and R-loops. Merged view and YTHDF2/S9.6 channels are shown. Scale bars are 20 μm The experiments shown in (a, c) were repeated independently 3 times with similar results. (d) Scatter diagram for YTHDF2 and S9.6 fluorescence intensities in an individual hPSCs nucleus. (e) The value of overlapping coefficient of YTHDF2 vs S9.6 intensities quantified for 20 cells immunostained for YTHDF2 and S9.6. The centre value is median, error bars show minimal and maximal values. (f, g) The results of EMSA using recombinant YTHDF2-His fusion (f) or YTHDF2-FLAG (g) and 0.15 pmol of unmodified (non) or m⁶A-containing (meth) RNA oligonucleotides (ssRNA) or corresponding synthetic RNA:DNA hybrids (RNA:DNA). Triangles indicate increasing concentrations of the protein (10, 100, 300 ng). Concentrations of the recombinant protein (g) and NaCl/KCl in the binding buffer are indicated. The RNA and RNA:DNA-protein complexes are arrowed. See also Supplementary Note. The gel images were cropped. The full scans of the gels are shown in Source Data 2.

Extended Data Fig. 10. YTHDF2 interacts with R-loop-containing loci in vivo and its depletion leads to accumulation of RNA:DNA hybrids in hPSCs.

YTHDF2 interacts with R-loop-containing loci in vivo and its depletion leads to accumulation of RNA:DNA hybrids in hPSCs. (a-f) The results of YTHDF2 (a, c, e) and HNRNPA2B1 (b, d, f) ChIP qPCR of the indicated repeats and intronic sequences performed on REBL-PAT hiPSCs (a, b), siCTL and siMETTL3 hPSCs (c, d) as well as on siCNTL and siRNase H1 hPSCs (e, f). (g) Relative expression of YTHDF2 transcript in hiPSCs transfected with siYTHDF2 siRNAs compared with that in non-targeting control siRNA (siCTL) transfected cells. (h, i) The results of S9.6 DRIP qPCR (h) and m⁶A DIP qPCR (i) of the indicated repeats and intronic sequences performed on siCTL and siYTHDF2 hiPSCs. Generic primers amplifying Alu elements from the indicated families and evolutionarily young L1Hs were used for DRIP qPCRs and qPCR. The regions without peaks (RANBP17 Downstream and HECW1 Downstream) were used as controls. Representative results of analysis of one of 3 independent biological samples are presented. Data are means ± SD, n=3 technical repeats.

Fig. 1

m⁶A marks the RNA components of RNA:DNA hybrids in hPSCs. (a) m⁶A and 5-methyldeoxycytosine (5mC) co-immunostaining of KaryoMAX-treated hiPSCs without RNases and after RNase A treatment. Merged images are shown. Mitotic cells are arrowed. (b) The ratios of the indicated deoxynucleotides obtained from the quantification of LC-MS/MS peaks in KaryoMAX-treated and untreated hiPSCs/hESCs DNA. Data are means ± SD, n=2 MS experiments. (c) Immunostaining of hiPSCs using anti-m⁶A and anti-phospho-Histone H3 antibodies without RNases and after RNase A or combined RNases A/H treatments. Merged views are presented. (d) Box plots showing quantification of m⁶A signal intensity in the interphase and mitotic hiPSCs at indicated immunostaining conditions. The elements of the box plots are: center line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum of all the data; n=20 nuclei for each condition. Significance was determined by unpaired two-tailed Student’s t-test. No adjustments were made for multiple comparisons. (e) Schematic illustrating design of the experiment on SID-UPLC-MS/MS analysis of hPSCs-derived nucleic acids released and retained upon RNase H treatment. (f) SID-UPLC-MS/MS quantification of m⁶A and ribo-m⁵C in the fractions of hESCs- and hiPSCs-derived nucleic acids released upon RNase H treatment. Data are shown as means ± SD, n=4/n=3 MS experiments for m⁶A/ribo-m⁵C quantification. Scale bars are 10 μm in (a) and 5 μm in (c). KaryoMAX treatment was used to enrich hPSCs for mitotic cells in (a, b). The experiments shown in (a, c) were repeated independently 6 times with similar results.

Fig. 2

m⁶A is present on the majority of the RNA:DNA hybrids in hPSCs. (a) Schematic illustrating the m⁶A DIP technique. See also Extended Data Fig. 2. (b) The coverage plots of m⁶A DIP and S9.6 DRIP densities (CPK) in the intron of CAMTA1-201 gene. m⁶A DIP and S9.6 DRIP peaks are marked with red and blue rectangles. (c) Distribution of m⁶A and S9.6 peaks at the indicated genomic features in hiPSCs. (d) Venn diagram illustrating an overlap between m⁶A DIP and S9.6 DRIP consensus peaks in REBL-PAT hiPSCs. (e) Heatmaps showing the distribution of density of m⁶A DIP and S9.6 DRIP reads across genomic regions containing the peaks (3 kb around peak center) of the three categories: m⁶A peaks overlapping with S9.6 peaks (m⁶A/S9.6), m⁶A peaks that do not overlap with S9.6 DRIP peaks (m⁶A only) and S9.6 peaks that do not correspond to m⁶A peaks (S9.6 only). The color of each line represents the density of reads for a given peak. The width of the heatmaps is normalized by peak length. Median numbers of reads per normalized region within each of the peak subsets are plotted over the top of the heatmaps. As the exact mode of genomic distribution of m⁶A-containing RNA:DNA hybrids was initially unknown, we performed detection of both narrow and broad peaks in the datasets. The results shown were obtained from analyses of the narrow m⁶A and S9.6 peaks.

Fig. 3

RNA:DNA hybrids exhibit cell cycle-specific dynamics in hPSCs. (a) The m⁶A DIP and S9.6 DRIP consensus narrow peak counts of the indicated repetitive elements in hiPSCs. (b) The results of m⁶A DIP and S9.6 DRIP qPCR of the indicated repeats performed on hiPSCs sorted at different cell cycle phases. Generic primers amplifying evolutionarily young L1Hs were used. (c, d) The results of S9.6 DRIP (c) and m⁶A DIP qPCR (d) of the RNA:DNA peaks localized in the introns of the indicated genes (See Extended Data Fig. 9b) performed on hiPSCs sorted at different phases of the cell cycle. Data are means ± SD, n=3 independent experiments in (b-d).

Fig. 4

m⁶A reader proteins interact with RNA:DNA hybrids. (a) Western blot of RNA:DNA hybrids protein co-IP probed with indicated antibodies. Top1 and Lamin B1 serve as positive and negative controls for R-loop IP, respectively. The experiments were repeated independently 2 times for METTL3 and 3 times for other proteins with similar results. The blots were cropped. The full scans of the blots are shown in Source Data 1. (b, c) Immunostaining of hiPSCs using anti-YTHDF1 and anti-phospho-Histone H3 antibodies imaged at two different magnifications. (d, e) Immunostaining of hiPSCs for HNRNPA2B1 and m⁶A imaged at two different magnifications. (f, g) Immunostaining of hiPSCs for YTHDF2 and phospho-Histone H3 imaged at two different magnifications. Merged views and YTHDF1/HNRNPA2B1/YTHDF2 channels (b, d, f) or merged views and individual channels (c, e, g) are shown. The locations of the views shown in (c, e, g) are marked with dotted rectangles in (b, d, f). Scale bars are 10 μm. The experiments shown in (b-g) were repeated independently 4 times with similar results. (h) Microscale thermophoresis binding curves for YTHDF2 interaction with m⁶A-containing/non-modified RNA:DNA hybrid and m⁶A-marked/non-modified ssRNA synthetic substrates The binding is shown as fraction protein bound as a function of substrate concentration. Binding curves are fitted to the data points from experiments for m⁶A-containing (filled circles/triangles) and unmodified (open circles/triangles) substrates. Dissociation constant values are shown for each of the interactions. Error bars show SD, the centre values are means, n=6 independent series of experiments.

Fig. 5

YTHDF2 depletion leads to accumulation of R-loops, increased accretion of m⁶A on RNA:DNA hybrids and cell growth retardation. (a) Immunostaining of WT and YTHDF2 KO HAP1 for YTHDF2 and phospho-Histone H3. The experiments were repeated independently 3 times with similar results. (b) Immunostaining of WT and YTHDF2 KO HAP1 for R-loops alongside the quantification of S9.6 nuclear signal. (c) DRIP qPCR of the indicated sequences performed on WT and YTHDF2 KO HAP1. RANBP17 and HECW1 downstream regions lacking DRIP peaks were used as controls. (d) SID-UPLC-MS/MS quantification of m⁶A and ribo-m⁵C in S9.6-IPs performed on WT and YTHDF2 KO HAP1 and normalized for dA or rA. RNase H-pre-treated samples were used as controls. Data are means ± SD, n=5 (left) and n=7 (right panel) measurements of 4 independent samples. (e) The growth curves of WT and YTHDF2 KO HAP1. (f) Immunostaining of WT/Ythdf2 KO mNSCs for R-loops alongside the quantification of S9.6 nuclear signal. (g) DRIP and m⁶A DIP qPCR of mouse LINE-1 ORF1 performed on WT and Ythdf2 KO mNSCs. Individual channels (a) or S9.6 channel (b, f) with merged views are shown. Scale bars are 10 μm. Data are means ± SD, n=3 independent experiments in (c, e, g). The elements of the box plots (b, f) are: centre line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum of all the data; n, sample size, ***p < 0.0001. Significance was determined by unpaired two-tailed Student’s (b, f) or unpaired two-tailed Welch's (e) t-test.

Fig. 6

YTHDF2 depletion leads to elevated levels of H2AX phosphorylation in human and mouse cells. (a) Representative images of WT and YTHDF2 KO HAP1 cells immunostained for γH2AX, and quantification of γH2AX signal intensity in these cells. (b) The results of γH2AX ChIP qPCR of the indicated sequences performed on WT and YTHDF2 KO HAP1. Generic primers amplifying Alu elements from the indicated families and evolutionarily young L1Hs were used. Data are means ± SD, n=3 independent experiments. (c) Representative images of WT/Ythdf2 KO embryonic brain cortex and mNSCs immunostained for γH2AX alongside quantification of γH2AX signal intensity in these tissues/cells. (d) Immunostaining of siCTL, siMETTL and siHNRNPA2B1 hPSCs for γH2AX and quantification of γH2AX signal intensity in these cells. (e) Representative images of YTHDF2 KO HAP1 cells transfected with GFP-RNase H1 and GFP-only expression constructs immunostained for γH2AX, alongside the quantification of γH2AX signal intensity in the GFP-positive cells. P value is indicated. Examples of the nuclei used for signal quantification are marked with dotted shapes. Merged images and S9.6 channel views are shown in (a, c-e). Scale bars are 10 μm. The elements of the box plots shown in (a, c-e) are: centre line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum of all the data, n, sample size, ***p < 0.0001. Significance was determined by unpaired two-tailed Student’s t-test, no adjustments were made for multiple comparisons.