Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Jilin University, China
- Laboratory of Organ Regeneration and Transplantation of The Ministry of Education, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, First Hospital of Jilin University, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Center of Laboratory Animals, China
Figures
Figure 1 with 1 supplement
Development of DRAM system for 5-methylcytosine (m5C) detection.
(A) Schematic diagram of the DRAM assay. DRAM, DRAMmut, and Deaminase system were transfected into HEK293T cells separately. After DRAM transfection, the deaminase was directed by m5C reader to the vicinity of the m5C site and induce C-to-U/A-to-G mutations, whereas transfection of the DRAMmut or Deaminase system failed to effectively induce similar mutations due to the absence of the m5C-recognition-binding domain. (B) The overall design of DRAM, DRAMmut, and Deaminase system.
Figure 1—figure supplement 1
The three-dimensional (3D) structures of ALYREFK171A, YBX1KO W65-N70, ALYREF, and YBX1.
(A, B) Computer simulation of the 3D structure of mutated ALYREF (A) and YBX1 (B) protein. On the left is the full structural model of ALYREFK171A, and on the right the 171st amino acid is colored in yellow and shows the stick structure of mutated A171 (A). The complete structural model of YBX1 after knockdown of W65-N70 (WFNVRN) is shown on the left, and the original L60-K64 and G71-I75 amino acids are colored in yellow on the right (B). (C, D) Computer simulation of the 3D structure of wild-type ALYREF (C) and YBX1 (D). The stick structure of K171 is highlighted in yellow (C). The amino acids L60-K64 and G71-I75 is marked in yellow, while the W65-N70 (WFNVRN) region is highlighted in blue (D). (E–H) The RNA pull-down assay demonstrates the binding of expressed YBX1 (E), YBX1mut (F), ALYREF (G), and ALYREFmut (H) proteins to biotinylated RNA containing C or 5-methylcytosine (m5C). The corresponding quantitative analyses of their binding capacities are shown on the right (n=2).
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Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1E-H, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original western blots for Figure 1—figure supplement 1E-H.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig1-figsupp1-data2-v1.zip
Figure 2 with 3 supplements
DRAM detection system was assayed in an 5-methylcytosine (m5C)-dependent form.
(A, B) Two m5C sites from RPSA (A) and AP5Z1 (B) mRNA detected by deep sequencing of bisulfite-sequencing PCR in HEK293T cells. The m5C sites are highlighted by red color. The m5C fraction of RPSA and AP5Z1 were 75.5% and 27.25% (the number of reads is greater than 1000). (C, D) Sanger sequencing following RT-PCR verified two m5C sites from RPSA (C) and AP5Z1 (D) mRNAs in DRAM-transfected HEK293T cells, respectively. HEK293T cells only expressing DRAMmut or Deaminase were served as negative controls. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3). (E, F) The knockout efficiency of NSUN2 (E) and NSUN6 (F) in HEK293T cell lines verified by western blotting. The protein level of α-Tubulin and GAPDH were served as loading controls, separately. (G, H) DRAM-induced mutations close to m5C sites in AP5Z1 (G) and RPSA (H) mRNAs after NSUN2 and NSUN6 knockout in HEK293T cells. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3).
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Figure 2—source data 1
PDF file containing the original western blots for Figure 2E and F, labeled with relevant bands and processing methods.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig2-data1-v1.zip
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Figure 2—source data 2
Original western blots for Figure 2E and F.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig2-data2-v1.zip
Figure 2—figure supplement 1
Expression of DRAM and DRAMmut in HEK293T cells.
(A, B) Examination of the EGFP-positive cells by fluorescence microscopy. Scale bars, 200 μm. (C) Quantification of the EGFP-positive cells by flow cytometry.
Figure 2—figure supplement 2
Editing of the DRAM system near the 5-methylcytosine (m5C) sites on 28S rRNA and tRNA.
RT-PCR followed by Sanger sequencing verified the presence of m5C modifications at specific sites in DRAM-transfected HEK293T cells, including m5C4447 in 28S rRNA (A), m5C3782 in 28S rRNA (B), m5C48 and m5C49 in tRNAAsp (C), m5C48 in tRNALys (D), and m5C48 and 49 in tRNAVal (E). Mutations were observed in sequences flanking the m5C loci.
Figure 2—figure supplement 3
CRISPR/Cas9-mediated depletion of NSUN2 and NSUN6 in HEK293T.
(A) Schematic representation of the construction of NSUN2-/- and NSUN6-/- cell line. HEK293T cells were transfected with plasmids encoding a PX459 editor and guide RNA targeting the NSUN2 or NSUN6 gene. After puro for screening, cells were diluted and seeded into a 96-well plate to allow for clonal selection. Monoclonal cells were then picked and screened for the desired genotype, resulting in the identification of the NSUN2-/- and NSUN6-/- cell line. (B) Mutation detection of NSUN2 and NSUN6 by PCR. M, the marker used is DL2000. (C) Venn diagram showing the overlap of DRAM-edited mRNAs identified across three sets of biological replicates. (D) Motif enrichment analysis based on the sequences spanning 10 nt upstream and downstream of DRAM-edited sites mediated by loci associated with NSUN2 or NSUN6. (E) Stacked Venn diagram showing the overlap of genes detected by Wang et al. and those identified by the DRAM system (Wang et al., 2023).
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Figure 2—figure supplement 3—source data 1
PDF file containing original electrophoresis of the PCR products for Figure 2—figure supplement 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig2-figsupp3-data1-v1.zip
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Figure 2—figure supplement 3—source data 2
Individual raw electrophoresis images of the PCR products for Figure 2—figure supplement 3B.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig2-figsupp3-data2-v1.zip
Figure 3 with 4 supplements
DRAM enables transcriptome-wide analysis of 5-methylcytosine (m5C) methylation.
(A) Schematic of the DRAM-seq method. (B, C) Integrative genomics viewer (IGV) browser traces of DRAM-seq data expressing the indicated constructs in RPSA (B, left panel), TARBP2 (B, right panel), AP5Z1 (C, left panel), and TRAF7 (C, left panel) mRNAs. C-to-U or A-to-G mutations found in at least 10% of reads are indicated by coloring. The previously published RNA bisulfite-sequencing (BS-seq) datasets from two individual studies were displayed as panel ‘Yang et al.’ and ‘Zhang et al.’. (n(DRAM)=3 independent samples, n(Deaminase)=2 independent samples, and n(DRAMmut)=1 independent sample.) (D, E) IGV browser traces of DRAM-seq data in wild-type and methyltransferases knockout cells in AP5Z1 (D) and RPSA (E) mRNAs. C-to-U or A-to-G mutations that were found in at least 10% of reads are indicated by coloring. The previously published RNA BS-seq datasets from two individual studies were displayed as panel ‘Yang et al.’ and ‘Zhang et al.’. n=3 independent samples. (F) Screening process for DRAM-seq assays and principles for screening high-confidence genes. (G) The pie chart shows the distribution of editing sites in different transcript region in cells expressing DRAM (n=3 independent samples). (H) The density map showing the distribution of editing events across the mRNA transcripts detected by DRAM-seq. (I) The frequency plot shows the distribution of the distances of edit events in DRAM-seq relative to the m5C sites from the published BS-seq datasets. The position of each m5C site of BS-seq is. determined as 0, and the relative distance of each site to the nearest edit event in DRAM-seq is calculated and plotted. The plots are presented separately based on the cutoff of upstream and downstream 3000 bp (above) and 80 bp (below) windows. (J, K) Motif analysis discovered within the ±20 nt region around the C-to-U or A-to-G editing site in cells expressing DRAM-CBE (J), APOBEC1(J), DRAM-ABE (K), and TadA-8e (K).
Figure 3—figure supplement 1
DRAM-seq editing in cellular RNAs.
Integrative genomics viewer (IGV) browser traces of DRAM-seq data expressing the indicated constructs in QARS (A), EHD4 (A), AMH (A), MRPS25 (B), TJAP1 (B), and CHD3 (B) mRNAs. C-to-U or A-to-G mutations found in at least 10% of reads are indicated by coloring. The previously published RNA bisulfite-sequencing (BS-seq) datasets from two individual studies were displayed as panel ‘Yang et al.’ and ‘Zhang et al.’.
Figure 3—figure supplement 2
DRAM-seq analysis in NSUN2-depleted and NSUN6-depleted cellular RNAs.
Integrative genomics viewer (IGV) browser traces showing read coverage and C-to-U/A-to-G mutations for eight representative mRNAs in wild-type, NSUN2-depleted cells (A) and NSUN6-depleted cells (B): LLGL2, CEP170B, PHF14, NAPRT1, FAM107B, PCSK6, TIMM50, and FURIN. C-to-U or A-to-G mutations that were found in at least 10% of reads are indicated by coloring. The previously published RNA bisulfite-sequencing (BS-seq) datasets from two individual studies were displayed as panel ‘Yang et al.’ and ‘Zhang et al.’.
Figure 3—figure supplement 3
Analysis of DRAM-seq data repeatability.
Three individual DRAM-ABE replicates were analyzed for the number of A-to-G mutations per RNA (A). Three individual DRAM-CBE replicates were analyzed for the number of C-to-U mutations per RNA (B). Pearson correlation coefficient indicated a high degree of overlap between individual replicates.
Figure 3—figure supplement 4
Transcriptome-wide mapping of 5-methylcytosine (m5C) in HEK293T cells.
(A, B) The pie chart shows the distribution of editing sites in different transcript regions in cells expressing DRAM-ABE and DRAM-CBE. (C, D) Gene Ontology (GO) cellular component (C) and molecular function (D) enrichment analysis of genes with DRAM-seq editing events. Statistical analyses were performed using the DAVID tool. p<0.05.
Figure 4
Stable and comprehensive cellular identification of 5-methylcytosine (m5C) loci by DRAM-seq.
(A) Comparison of the overall distribution of genes with m5C modifications detected by DRAM-seq, Yang et al., and Zhang et al. on chromosomes. The mutation sites detected by DRAM-seq on each gene are categorized into dual-colored short lines, with positive strand mutations shown in orange and negative strand mutations in dark green. The line graph and kernel density plot in the inner ring represent the locations and distributions of overlapping genes detected by DRAM-seq (red), Yang et al. (blue), and Zhang et al. (light green). (B) Venn diagram showing the overlap between DRAM-seq and Yang et al.’s edited genes. (D) Venn diagram showing the overlap between DRAM-seq and Zhang et al.’s edited genes. (C, E) Integrative genomics viewer (IGV) browser traces of DRAM-seq data expressing the indicated constructs in the ATG16L1 (B), ARHGEF25 (B), FANCD2 (D), and RPL15 (D) mRNAs. C-to-U/A-to-G mutations found in at least 10% of reads are indicated by coloring, and the m5C site found by bisulfite-sequencing (BS-seq) is also labeled. (F) Genes with DRAM-seq editing events were analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) bioprocess enrichment. (G) Gene Ontology (GO) biological processes enrichment analysis of genes with DRAM-seq editing events. Statistical analyses were performed using the DAVID tool.
Figure 5
Low-input 5-methylcytosine (m5C) detection and transfection efficiency of DRAM system.
(A, B) DRAM analysis of RPSA (A) and AP5Z1 (B) mRNAs with 250 ng, 50 ng, and 10 ng of input RNA. Representative Sanger sequencing plots are shown on the left panel, with mutation sites marked with asterisks. The mutation rates are quantified on the right panel. (C) Flowchart illustrating cell viability analysis by CCK8 reagent after DRAM transfection in HEK293T cells. (D) Quantitative comparison of the relative proliferative capacity of DRAM-expressing and untransfected cells. (E, F) The expression levels of DRAM-CBE (E) and DRAM-ABE (F) systems at different plasmid transfection concentrations were verified by western blotting. (G, H) Editing of RPSA (G) and AP5Z1 (H) mRNA at varying concentrations of DRAM protein expression. The left panels indicate Sanger sequencing results following RT-PCR, while the corresponding quantifications of DRAM-induced mutations are shown in the right panels.
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Figure 5—source data 1
PDF file containing the original western blots for Figure 5E-H, labeled with relevant bands and processing methods.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig5-data1-v1.zip
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Figure 5—source data 2
Original western blots for Figure 5E-H.
- https://cdn.elifesciences.org/articles/98166/elife-98166-fig5-data2-v1.zip
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Additional files
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Supplementary file 1
The detection for 5-methylcytosine (m5C) in RNA.
- https://cdn.elifesciences.org/articles/98166/elife-98166-supp1-v1.docx
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Supplementary file 2
High-confidence editing sites of DRAM-seq.
- https://cdn.elifesciences.org/articles/98166/elife-98166-supp2-v1.xlsx
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Supplementary file 3
Genes with editing sites occurring in DRAM-seq.
(a) DRAM-edited genes. (b) Overlapped genes with Yang et al. (c) Overlapped genes with Zhang et al.
- https://cdn.elifesciences.org/articles/98166/elife-98166-supp3-v1.xlsx
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Supplementary file 4
Primers and sgRNA for molecular cloning, genotyping, and downstream analyses.
(a) The primers for vector construction. (b) The primers used for genotyping. (c) Single guide RNA. (d) The primers for DRAM-Sanger analysis. (e) The primers for qPCR. (f) The primers for bisulfite-sequencing PCR.
- https://cdn.elifesciences.org/articles/98166/elife-98166-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/98166/elife-98166-mdarchecklist1-v1.docx
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Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing
eLife 13:RP98166.
https://doi.org/10.7554/eLife.98166.5
