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. 2020 Jun 16;52(6):1007-1021.e8.
doi: 10.1016/j.immuni.2020.05.003. Epub 2020 Jun 3.

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development

Yimeng Gao  1 , Radovan Vasic  2 , Yuanbin Song  2 , Rhea Teng  2 , Chengyang Liu  2 , Rana Gbyli  2 , Giulia Biancon  2 , Raman Nelakanti  3 , Kirsten Lobben  2 , Eriko Kudo  4 , Wei Liu  2 , Anastasia Ardasheva  2 , Xiaoying Fu  2 , Xiaman Wang  2 , Poorval Joshi  2 , Veronica Lee  2 , Burak Dura  5 , Gabriella Viero  6 , Akiko Iwasaki  7 , Rong Fan  5 , Andrew Xiao  3 , Richard A Flavell  8 , Hua-Bing Li  9 , Toma Tebaldi  10 , Stephanie Halene  11
Affiliations

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development

Yimeng Gao et al. Immunity. .

Abstract

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Keywords: METTL3; N(6)-methyladenosine; RNA modification; double-stranded RNA; dsRNA; epitranscriptome; hematopoiesis; hematopoietic development; innate immune response; m6A.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Loss of METTL3 results in dysfunctional fetal liver hematopoietic stem cells
(A) Breeding scheme to obtain Vav-Cre-Mettl3fl/fl mice vcMettl3+/+, vcMettl3+/−, and vcMettl3−/− E14.5 fetal livers. (B) Quantitation of Mettl3 expression via Q-RT-PCR, normalized to Gapdh (n=3 biological replicates per group). (C) Quantitation of m6A of mRNA in vcMettl3+/+ and vcMettl3−/− E14.5 fetal livers via ELISA (n=3 biological replicates per group). (D) Fetal liver cell numbers at E14.5 in vcMettl3+/+, vcMettl3+/− and vcMettl3−/− embryos (n=5 biological replicates per group), counted after mechanical dissociation and red blood cell lysis. (E, F) Assessment of colony forming unit (CFU) potential of vcMettl3+/+, vcMettl3+/− and vcMettl3−/− E14.5 fetal liver cells (E) (n=3 biological replicates per group). Demonstration of typical colonies in vcMettl3+/+ and vcMettl3−/− cultures (F). CFU-GEMM, CFU-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM, CFU-granulocyte, macrophage; CFU-M, CFU-macrophage; CFU-G, CFU-granulocyte; BFU-E, burst-forming unit-erythroid. Scale bar, 100 μm. (G) CFUs of E14.5 lineage depleted fetal liver cells transduced with empty, Mettl3 and catalytically dead Mettl3 (Mettl3 CD) expressing retroviral vectors (n=3 biological replicates per group). (H) Serial colony replating assay enumerating colonies/10,000 plated cells (n=3 biological replicates per group). Data are represented as mean ± SEM, and representative of at least two independent experiments; The P values were calculated using two-tailed Student's t test. * p<0.05, ** p<0.01, *** p<0.001. See also Figure S1.
Figure 2.
Figure 2.. Mettl3-deficient fetal liver hematopoietic stem and progenitor cells show aberrant differentiation trajectory and impaired proliferation
(A-D) Determination of LSK and LK cell distribution in vcMettl3+/+, vcMettl3+/− and vcMettl3−/− E14.5 fetal livers by flow cytometry (A). Quantitation relative to single cell population of LSK cells (B) and LK cells (C), and absolute LSK cell numbers per fetal liver (D; n=5 biological replicates per group). (E and F) Determination (E) and quantification (F) of phenotypic hematopoietic stem cell subsets in vcMettl3+/+ and vcMettl3−/− E14.5 fetal livers via flow-cytometric identification of long-term (LT) (Flt3−CD34− LSK) and short-term (ST) (Flt3−CD34+ LSK) hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) (Flt3+CD34+ LSK; n = 3 biological replicates per group). (G and H) Determination (G) and quantification (H) of apoptotic rate in fetal livers via annexin V staining (n = 3 biological replicates per group). (I and J) Assessment (I) and quantification (J) of fetal liver cell proliferation via BrdU uptake and 7AAD staining of DNA content (n = 3 biological replicates per group). Data are represented as mean ± SEM and representative of at least three independent experiments; the p values in (B)–(D) and (H) were calculated using two-tailed Student’s t test. n.s., not statistically significant; **p < 0.01; ***p < 0.001. The p values in (F) and (J) were calculated using two-way ANOVA. **p < 0.01; ***p < 0.001. See also Figures S2 and S3.
Figure 3.
Figure 3.. Loss of METTL3 results in significant upregulation of genes of the innate immune response in LSK cells
(A) Schematics of the experimental design for bulk and single cell RNA-Seq. (B) UMAP (Uniform Manifold Approximation and Projection) representation of vcMettl3+/+ and vcMettl3−/− LSK cells, based on single-cell RNA sequencing (scRNA-seq). Clusters corresponding to entry points for differentiation toward mature cell types are highlighted (n = 2 biological replicates per group). (C) Scatter plot of gene expression average levels (x) and fold changes (y) in vcMettl3−/− versus vcMettl3+/+ LSK cells, examined by bulk RNA-seq. Significantly upregulated and downregulated genes are highlighted in red and green, respectively (FC, fold change; TPM, transcript per million; n = 3 biological replicates per group). (D) Number of upregulated and downregulated genes in vcMettl3−/− LSK cells (n=3 biological replicates per group). (E) Percentage of genes with annotated m6A peaks among genes either upregulated or downregulated upon vcMettl3−/−. The analysis was performed with 52 different published m6A datasets, each corresponding to a dot in the violin plot. Hematopoietic datasets are highlighted. (F) Gene-Concept network visualization of genes upregulated in vcMettl3−/− LSK cells (in red) and their enriched pathways (in grey). The P value in (E) was calculated using two-tailed Wilcoxon rank-sum test. See also Figure S4, Table S1.
Figure 4.
Figure 4.. OAS family genes are highly upregulated via epigenetic regulation in response to Mettl3 loss in HSPCs
(A) Oas family gene expression in vcMettl3+/+ and vcMettl3−/− E14.5 fetal liver LSK cells as measured by RNA-seq (n=3 biological replicates per group). (B) Percentage of genes with annotated m6A peaks in Oas genes compared to the remaining genes upregulated in vcMettl3−/− LSK. The meta-analysis was performed with 52 different published m6A datasets. (C) Number of genes with significantly increased (red) versus decreased (green) H3K4me3 occupancy around the transcription start site (TSS) in vcMettl3−/− versus vcMettl3+/+ LSK cells (n=2 biological replicates per group). (D) Scatterplot comparing gene expression changes (y-axis) versus H3K4me3 occupancy changes (x-axis) between vcMettl3−/− and vcMettl3+/+ LSK cells. Genes with consistent and significant epigenetic and gene expression changes are highlighted (red=up, green=down). (E) H3K4me3 occupancy profiles of Oas1g, Oas2 and Rag1 (3k bp around the TSS) in vcMettl3+/+ and vcMettl3−/− LSK cells. The average normalized signal and standard error from 2 replicates is displayed. The P value in (B) was calculated using two-tailed Wilcoxon rank-sum test. See also Table S1.
Figure 5.
Figure 5.. Loss of METTL3 leads to aberrant dsRNA formation upon depletion of m6A
(A) Detection of dsRNA via immunocytochemistry in vcMettl3+/+ and vcMettl3−/− fetal liver cells using the dsRNA specific antibody J2. Scale bar, 50 μm. Data are representative of four independent experiments. (B) Quantification of J2 intensity of each cell normalized to its DAPI signal (vcMettl3+/+ n=69, vcMettl3−/− n=66). Data are representative of four independent experiments. (C) Schematics of the criteria used to identify the 94 genes significantly enriched in J2 RIP of vcMettl3−/− versus vcMettl3+/+ fetal liver cells. (D) J2 RIP-Seq signals from six representative J2 vcMettl3−/− enriched genes, compared with Actb and Gapdh as negative controls (n=2 biological replicates per group). (E) Percentage of genes with annotated m6A peaks among the 94 genes enriched in vcMettl3−/− J2 RIP compared with invariant genes. The meta-analysis was performed considering 52 different published m6A datasets. (F) Distributions of the transcript length of vcMettl3−/− J2 RIP enriched genes compared with invariant genes. (G) Distributions of the predicted folding energy of vcMettl3−/− J2 RIP enriched genes compared with invariant genes. The P values in (E,F,G) were calculated using two-tailed Wilcoxon rank-sum test. See also Figure S5, Table S2.
Figure 6.
Figure 6.. Loss of METTL3 induces an aberrant dsRNA induced innate immune response
(A) Scheme depicting the dsRNA induced innate immune response pathways. (B) Q-RT-PCR determination of expression of dsRNA sensor genes Ifih1 (MDA5) and Ddx58 (RIG-I) in E14.5 fetal livers, normalized to Gapdh (n=3 biological replicates per group). (C-D) Detection of PKR phosphorylation and eIF2α phosphorylation in fetal liver in response to Mettl3 deletion via Western blot (C); bands from three representative blots were quantified via Image J, p-PKR and p-eIF2a protein expressions were normalized to PKR and eIF2a expression, respectively (D) (n=3 biological replicates per group). (E) RtcB Q-RT-PCR analysis of tRNA specific cleavage by RNase L, normalized to U6 (n=3 biological replicates per group). (F) Q-RT-PCR determination of expression of immune response genes downstream of interferon activation in E14.5 fetal livers, normalized to Gapdh (n=3 biological replicates per group). (G) Q-RT-PCR determination of IFN III (Ifnl3) and I (Ifna4, Ifnb1) expression in E14.5 fetal livers, normalized to Gapdh (n=3 biological replicates per group). Data are represented as mean ± SEM, and representative of at least three independent experiments; The P values were calculated using two-tailed Student's t test. * p<0.05, ** p<0.01. See also Figure S6.
Figure 7.
Figure 7.. Inhibition of the innate immune response partially rescues hematopoietic failure secondary to Mettl3 deletion
(A) Determination of the effect of CRISPR/Cas9 mediated Mavs deletion on colony formation in vcMettl3−/− compared to vcMettl3+/+ Lin− fetal liver cells (n=3 biological replicates per group). (B) qRT-PCR measurement of Oas and interferon response gene expression secondary to control versus targeted sgRNA-mediated deletion of Mavs in vcMettl3−/− compared to vcMettl3+/+ fetal liver cells, normalized to Gapdh (n = 3 biological replicates per group). (C and D) Determination (C) and quantification (D) of the engraftment of vcMettl3−/− cells with CRISPR-Cas9-mediated deletion of Mavs or overexpression of Mettl3 6 weeks post-transplantation via flow cytometric detection of CD45.2 fetal liver cells versus CD45.1 control cells (n = 4 separate mice per group). One independent experiment is shown. (E) Model of the Mettl3 deletion-induced dsRNA-mediated innate immune response. Data are represented as mean ± SEM, and representative of at least two independent experiments unless stated otherwise; The P values were calculated using two-tailed Student's t test. n.s. not statistically significant, * p<0.05, ** p<0.01, *** p<0.001. See also Figure S7.
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