Fig. 4: TET2 restrains urea cycle through mRNA oxidation.

a, b Tet2 KO increases mRNA stability of urea cycle enzymes. Primary liver cells (a) and primary lung cells (b) from WT and Tet2 KO mice were treated with 5 μg/mL actinomycin D for different time points as indicated. mRNA decay of urea cycle enzymes was quantified by qPCR. n = 3 biologically independent animals per group. c Tet2 KO increases mRNA levels of urea cycle enzymes in primary lung cells. n = 3 biologically independent animals per group. d Catalytic activity of TET2 is required to induce mRNA decay of urea cycle enzymes. WT and catalytic mutant (R1896S) TET2 were transfected into HepG2 cells. mRNA levels of urea cycle enzymes were quantified by qPCR. n = 3 biologically independent samples per group. e LC-MS quantifying 5mC and its oxide 5hmC levels in methylated RNA in the absence or presence of TET2 catalytic domain (CD) in vitro. 5mC at 5.3 min, 5hmC at 5.6 min. n = 2–3 biologically independent samples per group. f TET2 associates with endogenous 3′ UTR and CDS of urea cycle enzyme mRNAs. TET2 binding sites were mapped in the mRNA of urea cycle enzymes by qPCR of TET2 RIP product. RIP, RNA immunoprecipitation assay. n = 2–3 biologically independent samples per group. g Conserved motifs in 3′ UTR of five urea cycle enzyme mRNAs. h TET2 KO increases mRNA 5mC levels of urea cycle enzymes. mRNA of urea cycle enzymes was immune-precipitated from control and TET2 KO HepG2 cells with anti-5mC antibody and quantified by qPCR. n = 3 biologically independent samples per group. i TET2 KO reduces mRNA 5hmC levels of urea cycle enzymes. mRNA of urea cycle enzymes was immune-precipitated from control and TET2 KO HepG2 cells with anti-5hmC antibody and quantified by qPCR. n = 2–3 biologically independent samples per group. j TET2 promotes mRNA decay via mediating its oxidation from 5mC to 5hmC.