Journal: The Journal of Biological Chemistry

Article Title: The RNA methyltransferase NSUN2 catalyzes 5-methylcytosine (m 5 C) on IL1B mRNA to promote transcript stability

doi: 10.1016/j.jbc.2026.111290

Figure Lengend Snippet: Nsun2 reduction decelerates the advancement of systemic inflammation in mice. A , the m 5 C methylation level in total RNA of mouse lung tissues administrated with LPS was determined by m 5 C dot blot assay. Methylene blue (MB) staining was set as an internal reference for sample loading. The histogram summarized the ratios of the dot intensities of m 5 C versus methylene blue . Data are means ± SD, n = 6. B and C , published RNA-seq data derived from other researchers was downloaded from the GEO database. We reanalyzed the expression profiles of m 5 C modification-associated genes in lung tissues from mice with systemic inflammation. These systemic inflammation mouse models were conducted by three common methods: LPS treatment ( GSE217695 ), cecal microbiome (CM) treatment ( GSE239388 ), as well as cecal ligation and puncture treatment (CLP) ( GSE179554 ). B , heatmap represents normalized gene expression ( red , high expression; blue , low expression). C , the histogram summarized the relative expression of Nsun2 . Data are means ± SD, n = 3 or 4. D , schema for the strategy used to generate the Nsun2 knockout mice via the CRISPR/Cas9 technology. E , PCR identification of genotypes of C57BL/6N wild-type (WT) mice and Nsun2 mutant (heterozygous) mice. F , determine the protein expression of NSUN2 in lung tissues of WT or Nsun2 +/− mice by IHC. The scale bar represents 100 and 50 μm (high magnification). G , timeline for LPS-induced systemic inflammation as well as body weight measurements and behavioral assessment (15 mice per group). H , weight changes of Nsun2 +/− and control WT mice before injection (0 h) and 16, 18, 20, 22, and 24 h after LPS or PBS injection. The significance of the weight change differences between WT + LPS group and Nsun2 +/− + LPS group was analyzed and plot on the weight change polyline of Nsun2 +/− + LPS group. Weight change (%) = (weight at a specific time-weight at 0 h)/weight at 0 h × 100% (15 mice per group). I , the statistical analysis of the M-CASS scores was measured at 24 h after LPS administration (n = 15). J , representative H&E staining of mouse lung sections from each group. The degree of lung damage was measured via the lung injury scoring system (n = 15). The scale bar represents 100 and 50 μm (high magnification). K , quantitative real-time PCR analysis of the mRNA levels of Il1b , Il6 , Cxcl10 , and Ccl2 in lung tissues. Gapdh served as a loading control. Data are means ± SD, n = 15. Trdmt1 , tRNA aspartic acid methyltransferase 1; Tet , tet methylcytosine dioxygenase; Alkbh , alkB homolog 1. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10; GEO, Gene Expression Omnibus; IHC, immunohistochemical; IL1B, interleukin 1 beta; LPS, lipopolysaccharides; M-CASS, mouse clinical assessment score for sepsis; m5C, 5-methylcytosine; NSUN, NOP2/Sun RNA methyltransferase.

Article Snippet: Nsun2 +/− mice were derived from C57BL/6N mice employing CRISPR/Cas-mediated genome engineering scheme (Mice Nsun2 Gene ID: 28114) in Cyagen Biotechnology Co, Ltd The wild-type and Nsun2 gene trap alleles were distinguished by polymerase chain reaction (PCR) with One Step Mouse Genotyping Kit (Vazyme).

Techniques: Methylation, Dot Blot, Staining, RNA Sequencing, Derivative Assay, Expressing, Modification, Ligation, Gene Expression, Knock-Out, CRISPR, Mutagenesis, Control, Injection, Real-time Polymerase Chain Reaction, Immunohistochemical staining