Fig 1: The hypermethylated m6A-circRNAs possess potential for translation. A The cumulative fraction of coding probability scores predicted by CPAT for BSJ-distal m6A-circRNAs, BSJ-near m6A-circRNAs and non-m6A-circRNAs. The P value was calculated with the Wilcoxon rank sum test. B Significant higher ratio of binding sites of EIFs and YTHDFs in BSJ-distal m6A-circRNAs than BSJ-near m6A-circRNAs and non-m6A-circRNAs. For each clustered bar, from left to right were binding sites ratios of BSJ-distal m6A-circRNAs, BSJ-near m6A-circRNAs, and non-m6A-circRNAs. P values were calculated by chi-squared test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. C Venn diagram showing the number of m6A-circRNAs with coding potential (circRNAs with ORFs and EIFs binding sites) for non-DE hypermethylated-m6A-circRNA and all hypermethylated-m6A-circRNA. The outer layer indicated total number of corresponding m6A-circRNAs; the inner layer indicated the number of corresponding m6A-circRNAs with coding potential. Fisher’s exact test was used to derive the P value. D GO molecular function (MF) terms enrichment analysis for host genes of hypermethylated m6A-circRNAs with coding potential. E Relative fractions of unbound (free) RNAs, monosome- and polysome-bound RNAs for circZFHX3, circTSHZ1, and circLMTK2 in PDAC cell lines with METTL3 siRNA and control. The HPRT mRNA was used as control. F RIP-qPCR analysis showed EIF3A-, EIF3B-, and EIF3H-bound RNA abundance for circZFHX3, circTSHZ1, and circLMTK2 in PDAC cells with or without METTL3 knockdown. Data in E and F were means ± S.E.M. (n = 3). *, P < 0.05; **, P < 0.01; ***, and P < 0.001 of Student’s t test comparing with each control
Fig 2: eIF3a expression and B cell quantity and function in septic patients. (A, B) Distribution of total (A) and types of B cells (B) in healthy individuals and septic patients. (C) Heat map displaying a differential expression of m6A regulators between sepsis and healthy individuals of three datasets. (D) Spearman correlation between eIF3a expression and levels of different B cell types in three datasets. (E) B cell population in the spleen of LPS treated eIF3afl/– and control mice was determined by staining CD19 and CD220 and detected using flow cytometry. (F) Enrichment analysis of the intersection genes with higher expression in eIF3a-H compared to eIF3a-L across the three datasets. (G) tSNE plot of all patients scRNA-seq data. (H) Patients were classified into eIF3a-H and eIF3a-L groups based on the expression of eIF3a in B cells. (I) Enrichment analysis revealed upregulated and downregulated B cell related pathways in eIF3a-H relative to eIF3a-L. (J, K) Expression of CD70, CCR6, VPREB3, CD127, TBX21 and TP53BP1 in B cells of eIF3a-L and eIF3a-H septic patients. Data are presented as means ± SD (n = 3); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.
Fig 3: eIF3a knockout and severe multi-organ inflammatory responses. (A) Scheme of eIF3a knockout induction via intraperitoneal injection of tamoxifen. (B–F) Body weight change (B), overall survival (C) of eIF3afl/fl and control (NC) mice, and rectal temperatures (D), body posture (E), status of eye, hair, and anus (F) on Day 6. (G, H) Gross anatomy (G) and relative weight (H) of major organs from eIF3afl/fl and control (NC) mice. (I, J) Number of monocytes, neutrophils, and lymphocytes as determined by counting (I) and relative lymphocytes determined by flow cytometry (J) using blood from eIF3afl/fl and control (NC) mice on Day 6. (K, L) Images of HE-stained thymus and spleen (K) and other organ (L) sections. White pulp areas in the spleen were quantified and shown in panel (K). (M) The relative concentration of IL-1, IL-6, IFN-γ in serum of eIF3afl/fl and control (NC) mice on Day 6. Data are presented as means ± SD (n = 5); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
Fig 4: Ectopic eIF3a overexpression protects against LPS-induced inflammation and severe sepsis in the eIF3afl/– mice. (A) Western blot of eIF3a in the eIF3afl/– mice injected with plasmid for ectopic eIF3a overexpression or empty vector. (B) Flow cytometry was performed to detect the proportions of B cells and T cells in the spleen, and the quantification results are shown on the right. (C) Scheme of the eIF3a knockdown, ectopic overexpression and LPS induction of sepsis. (D–H) Status of eye, hair, and anus (D) and clinical scores of sepsis at 24 h (E), overall survival rate (F), body weight (G) and rectal temperature (H) of the mice over 72 h following LPS injection. (I) Gross anatomy of major organs in the OE-eIF3afl/– and Vec-eIF3afl/– mice after sepsis induction. (J, K) Images of HE-stained sections of the spleen (J) and other organs (K) of the OE-eIF3afl/– and Vec-eIF3afl/– mice. A quantified white pulp area in the spleen was presented in the histogram. (L) The relative concentration of IL-1, IL-6, and IFN-γ in serum at 24 h after LPS injection. Data are presented as means ± SD (n = 8); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
Fig 5: eIF3a regulation of B cell production and function. (A, E, I) Scheme of experimental design for quantification of B cells in the spleen (A), hemolytic plaque assay of SRBC using spleen (E), and quantification of IgG levels in the blood (I) from the control and eIF3afl/fl mice. (B, C) B cell population in the spleen as determined by staining CD19 and CD220 and detected using flow cytometry (B) and immunofluorescence microscopy (C). White and orange circles in panel (C) indicate the white pulp (WP) and B cell regions, respectively. (D) Plasma cells in the control and eIF3afl/fl mice as determined by staining CD38 and CD138 and detected using flow cytometry. (F–H) Immunogenic response of the control and eIF3afl/fl mice to injected SRBC. The height of the SRBC precipitate (F) and the amount of hemolytic plaque (G, H) suggest the degree of SRBC hemolysis. Red circles in panel (G) mark the edges of the hemolytic plaques. Panel (H) represents quantification of the relative count of hemolytic plaques from panel (G). (J, K) Expression levels of immunoglobulin heavy and light chains in serum of the control and eIF3afl/fl mice as determined using Mass Spectrometry (J) and intact IgG using ELISA (K). Data are presented as means ± SD (n = 6); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
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