Fig 2: NUDT21 regulates Rragd expression via m6A-mediated mRNA degradation.(A and B) RIP-qPCR shows the binding of METTL3 (A) and NUDT21 (B) to the proximal and distal PASs of Rragd mRNA in Jurkat cells (n = 3). (C) WT METTL3 (Mettl3 WT) or catalytic mutant METTL3 (Mettl3-MUT) was overexpressed in Mettl3 knockdown (KD) Jurkat cells, and qRT-PCR was performed to detect the dPAS usage by using two sets of primers designed to target the translated region (common) and distal region (distal) of the Rragd 3′UTR (n = 3). (D) RIP-qPCR shows the binding of NUDT21 to the proximal and distal PASs of Rragd mRNA in control and Mettl3 knockdown Jurkat cells (n = 3). (E) m6A RIP-qPCR assay was applied to detect m6A enrichment in Rragd mRNA in Nudt21 cKO and WT CD4+ T cells (n = 3). (F) Degradation of Rragd mRNA in Nudt21 cKO and WT naïve CD4+ T cells treated with actinomycin D (ActD) for 0, 2, 4, and 8 hours (n = 3). (G) The expression of Rragd was measured by qPCR in Mettl3 cKO and WT naïve CD4+ T cells (n = 3). (H) Immunoblots of protein lysates from Jurkat T cells infected with lentivirus carrying an EV or Mettl3 shRNA (#1 and #2). Data represent one of three independent experiments and are shown as the means ± SEM. *P < 0.05 and **P < 0.01 determined by Student’s t test.

Fig 3: NUDT21 and METTL3 co-regulate the stability and expression of Rragd mRNA.(A) Scatterplot of PDUIs in Mettl3 cKO and WT naïve CD4+ T cells. (B) Scatterplot of PDUIs in Mettl3 cKO versus WT naïve CD4+ T cells overlapped with PDUIs in Nudt21 cKO versus WT naïve CD4+ T cells. (C) Venn diagram depicting the overlap of genes whose 3′UTRs are shortened in Nudt21 cKO and Mettl3 cKO naïve CD4+ T cells. (D) Plot of m6A peak coverage surrounding ±3000 bp of proximal PASs in genes whose 3′UTRs are shortened in Mettl3 cKO or Nudt21 cKO T cells. (E) Scatterplot of PDUIs and expression change of mTOR pathway genes in Nudt21 cKO and WT naïve CD4+ T cells. (F) Reads density of Rragd 3′UTR from RNA-seq and m6A CLIP-seq data. Numbers on the y axis indicate the read coverage. (G) mRNA expression of Rragd by qPCR in Nudt21 cKO and WT naïve CD4+ T cells. (H) Immunoblots of lysates from splenic Nudt21 cKO and WT naïve CD4+ T cells. (I) Representative dot plots show annexin V/7-AAD staining on Jurkat T cells infected with lentivirus carrying an empty vector (EV) or Nudt21 short hairpin RNA (shRNA) and Rragd shRNA. Data represent one of three independent experiments and are shown as the means ± SEM. **P < 0.01 determined by Student’s t test.