Transcriptomic Profiling of NSUN2-KO Murine BMM Cells Post-M1 Polarization
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE295299 | GEO | 2026/06/01
REPOSITORIES: GEO
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Project description:Aims: Mammalian mRNA internal modification m6A has been suggested to be essential in the maintenance of cardiac homeostasis. The role of mRNA cytosine methylation (m5C) in the heart remains unexplored.Methods and results: In contrast with human normal heart, the expression of RNA cytosine methyltransferase NSun2 was found to be significantly elevated in the hypertrophied one. Consistently, NSun2 expression was increased in hypertrophic rat ventricular myocytes induced in vitro, as well as in TAC (transverse aortic constriction) developed hypertrophied murine heart. Cardiomyocyte-specific knockout of NSun2 (Myh6-CreERT2, NSun2 flox+/+) dramatically accelerated heart failure progression in TAC mice. RNA m5C immunoprecipitation followed by sequencing and MeRIP (methylation-specific RNA immunoprecipitation) revealed that NSun2 particularly methylates the mRNA of protein kinase A (PKA) catalytic subunit alpha (PRKACA). Polyribosome and dual luciferase reporter gene analysis further suggested that NSun2 promotes PRKACA translation via RNA methylation. In TAC hypertrophic cardiomyocytes, NSun2 ablation markedly dampened PKA signaling, as evidenced by reduced PKA activity and protein phosphorylation levels of PKA diverse substrates, impaired myocyte contraction and relaxation, and disturbed calcium transients. Cardiac-restricted overexpression of NSun2 via adeno-associated virus 9 (AAV9, NSun2 expression cassette driven by cTnT promoter) sensitized heart hypertrophic response, whereas administration of PKA inhibitor H89 successfully blocked this process, reiterating the importance of NSun2-PRKACA regulation in cardiac hypertrophic program.Conclusions: Our study showed that RNA methyltransferase NSun2 promotes PRKACA translation via RNA methylation, whereby achieving its modulation of PKA signaling within the cardiomyocyte. NSun2-PRKACA regulation is required for cardiac homeostasis maintenance and adaptation to hypertrophic stresses. These findings furnish us with novel understanding of heart function mediated by mRNA cytosine methylation in cardiac homeostasis.
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Project description:NOP2/Sun RNA methyltransferase family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modifications in tRNA, rRNA, and mRNA, playing critical roles in various malignancies. However, its expression and functional relevance in B-acute lymphoblastic leukemia (B-ALL) remain largely undefined. Lineage-negative progenitor cells from Nsun2 knockout (Δ/Δ) and wild-type (fl/fl) mice were used to generate BCR-ABL (P190)-driven murine B-ALL model. Furthermore, RNA sequencing (RNA-seq) was performed to identify NSUN2 targets. Here, we found that NSUN2 expression was significantly higher in bone marrow (BM) cells from B-ALL patients than normal controls (NCs). Knockdown of NSUN2 reduced m5C amounts, induced apoptosis and ferroptosis in B-ALL cells. Moreover, Nsun2 knockout significantly suppressed the leukemic burden and extended the OS in murine B-ALL model in vivo. RNA-seq and subsequent studies identified ferritin heavy chain 1 (FTH1) as a downstream target of NSUN2. Mechanistically, NSUN2 knockdown reduced m5C modifications on 3'UTR of FTH1 mRNA, impairing its stability and reducing FTH1 expression. Overexpression of wild-type (WT) NSUN2, but not its mutants, blocked NSUN2 knockdown-induced cytotoxic effects, indicating that NSUN2 enhances leukemogenesis in an m5C-dependent manner. In addition, NSUN2 facilitated global protein synthesis and ribosome biogenesis by enhancing fibrillarin (FBL) translation efficiency. In contrast, Nsun2 depletion was dispensable for hematopoiesis and exerted minimal impact on ferroptosis and protein synthesis in murine pre-B (B220+) cells, suggesting selective leukemic dependency. Our results demonstrate that NSUN2 facilitates leukemogenesis by increasing FTH1 expression and enhancing FBL-mediated ribosome biogenesis in an m5C-dependent manner. Targeting NSUN2 might provide a promising therapeutic strategy for B-ALL.
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Project description:Autosomal-recessive loss of the NSUN2 gene has been recently identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNA). Whether NSun2 methylates additional RNA species is currently debated. Here, we adapted the individual-nucleotide resolution UV cross-linking and immunoprecipitation method (iCLIP) to identify NSun2-mediated methylation in RNA transcriptome. We confirm site-specific methylation in tRNA and identify messenger and non-coding RNAs as potential methylation targets for NSun2. Using RNA bisulfite sequencing we establish Vault non-coding RNAs as novel substrates for NSun2 and identified six cytosine-5 methylated sites. Furthermore, we show that loss of cytosine-5 methylation in Vault RNAs causes aberrant processing into argonaute-associating small RNA fragments (svRNA). Thus, impaired Vault non-coding RNA processing may be an important contributor to the etiology of NSUN2-deficieny human disorders. mRNA-seq in Embryonic kidney (HEK293) cells transfected with siRNA against Nsun2 vs control
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