Fig 2: Arg2 TSB modulates surface markers and metabolic parameters in primary BMDM(A–D) Surface markers expression. Pro-inflammatory markers CD80 (A) and CD86 (B) and anti-inflammatory markers CD71 (C) and CD206 (D) surface levels in Arg2 vs NC TSB transfected BMDM (in presence and absence of LPS) were compared by flow cytometry (n = 3, in single). Histograms depict the results obtained in one representative experiment. The bar graphs represent the means of mean fluorescence intensity ± SEM of positive cells in three biological replicates. Data were compared by one-way ANOVA (Sidak’s multiple comparisons test, *p = 0.05, **p < 0.01, ****p < 0.0001). (E) Representative Seahorse metabolic flux trace of BMDM transfected with Arg2 vs NC TSB in absence (left) or presence (right) of LPS (10 ng/mL) (n = 5, 4–7 technical replicates). (F) Quantitative oxidative parameters changes in BMDM transfected with Arg2 vs NC TSB in absence or presence of LPS (n = 5, 4–7 technical replicates). Data were compared by two-way ANOVA (Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: 6-Phosphogluconate dehydrogenase (6PGD) blockade results in loss of suppressive function in regulatory T cells (Tregs).(A–B) Frequency of CD4+Foxp3+ cells in spleen of 20-day-old Pgd+/+Foxp3Cre (wild type [WT]) was evaluated and demonstrated (A) in total cells or (B) in gated CD4+ T cells. Bar graphs show respective statistical differences for percentage of Foxp3+ cells and Foxp3 gMFI. (C) Expression of CD98, CD71, and CD40L on Tregs from WT and Pgdfl/flFoxp3Cre mice. (D–E) YFP+ cells were isolated from 20-day-old WT and Pgdfl/flFoxp3Cre mice. (D) Purified YFP+ cells are shown and (E) Foxp3 mRNA levels were evaluated by real-time PCR. (F) Isolated Tregs (YFP+) were cultured in vitro in presence of IL-2 (700 IU/ml) and anti-CD3/anti-CD28 coaled beads (Treg:beads ratio 1:3) and cells number was assessed at 24 and 48 hr time points. Results are representative of three independent experiments with N = 4. (G) Cytokine release in media as cultured in above culture condition. (H–I) Isolated Tregs (YFP+) from Pgdfl/flFoxp3Cre mice showed lower suppressive activity in suppression assay, as described in Materials and methods. Representative histogram of CFSE dilution pattern (Treg:Teff = 1:1 ratio) (H) and bar graph of T cell proliferation as a function of serial Treg:Teff ratios (I) are shown. Results are from three independent experiments with N = 4. (J) Tregs (YFP+) from WT and Pgdfl/flFoxp3Cre mice and T effector (CD4+CD45RBhigh) cells were isolated, mixed, and transferred to Rag1-/- mice as inflammatory bowel disease (IBD) model. Colons were evaluated 45 days after cells injection. (K) Weight change in IBD mouse model. (L) On day 45 post IBD induction Rag1-/- mice colon were measured for length and thickness. (M) Representative hematoxylin and eosin staining of Rag1-/- mice colon on day 45 after IBD induction. Results are from two independent experiments with N = 8 per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: A genome-wide pooled CRISPR screen identifies novel RNA-binding proteins which regulate TfR1 expression.a, b Workflow (a) and cell sorting strategy (b) of the genome-wide CRISPR screen for TfR1 regulators. c Gene ontology analysis of the TfR1 CRISPR screen hits. Statistical analysis was performed using Immuno-Navigator83. d Volcano plot of TfR1 phenotype (casTLE Effect, the most likely effect size as determined by casTLE) and the confidence in that effect size (casTLE Score) from the CRISPR screen. The screen was performed in duplicate. e–g Validation of the screen hits. Two gRNAs per gene were chosen for retests. K562-Cas9 cells were transduced with lentiviral vectors encoding individual gRNAs together with mCherry. Then cells were stained with anti-TfR1 antibody and analyzed using flow cytometry (e). Change in TfR1 expression levels between untransduced (mCherry-, gray) and transduced (mCherry+, red) were calculated and summarized (f, g). Each symbol represents the results of individual gRNAs. Data were pooled from two independent experiments. h, i Effects of wild-type or catalytic-dead mutant METTL16 (PP185/186AA, F187G) on surface TfR1 expression in the presence of DFO. Similar expression levels of wild-type or mutant METTL16 protein were confirmed (h). Surface TfR1 expression levels of cells expressing indicated gRNAs and proteins were examined by flow cytometry (i). Similar results were obtained in two independent experiments. Source data are provided as a Source Data file.

Fig 5: METTL16 regulates the deposition of m6A modification on DNA-damage response-associated mRNAs.a Outline of the in vitro erythroid culture experiments (Methods). b mRNA expression levels in cultured erythroblasts from E13.5 control and Mettl16fl/flCreERT2+ mice (n = 3 biological replicates). Data are expressed as mean ± SD. The p-values were calculated using two-tailed Student’s t-test. c–f MeRIP-seq analyses using poly(A)+ RNAs in cultured erythroblasts from control and Mettl16fl/flCreERT2+ mice. Significantly differentially methylated sites were identified using RADAR (n = 3 independent experiments). c Heatmap showing the relative methylation levels at these sites. d The fractions within the 5' UTR, CDS, 3' UTR, and ncRNA. e The metagene distribution of the differentially methylated sites in mRNA. f Sequence motif logos identified from these sites. g, h GO analysis of downregulated (g) and upregulated (h) genes with hypomethylation in erythroblasts from Mettl16fl/flCreERT2+ mice. i Venn diagram comparing genes downregulated with hypomethylation in erythroblasts from Mettl16fl/flCreERT2+ mice and genes downregulated in CD71+Ter119+ erythroblasts from Mettl16fl/flEpor-Cre+ mice. The GO analysis revealed that DNA-repair-related genes were enriched in the intersect. Statistical analysis was performed using Immuno-Navigator83 (g–i). Source data are provided as a Source Data file.