The downregulation of NSUN5 may contribute to preeclampsia.

Preeclampsia (PE) is a complication of pregnancy characterized by the new onset of hypertension after 20 weeks of gestation. The incidence of PE is steadily rising, posing a significant threat to the lives of both the pregnant woman and the fetus. Most studies on PE pathogenesis currently focus on the placenta, but maternal decidualization forms the foundation for placental growth and development. Recent studies have shown that impaired decidualization is also a cause of PE. Decidualization is a process where endometrial stromal cells gradually transform into secretory decidual cells during early pregnancy. While NSUN5 encodes a member of a conserved family of proteins, its role in pregnancy remains unknown. In this study, we conducted experiments and observed a significant downregulation of NSUN5 expression in severe preeclampsia decidual tissues compared to those of normal pregnant women. When inducing decidualization in vitro, we found an increase in NSUN5 expression. However, when we used siRNA to knockdown NSUN5 expression, the process of decidualization was prevented. Moreover, we observed a decrease in ATP content during both cell decidualization and after knockdown of NSUN5. Finally, through immunoprecipitation combined with mass spectrometry, we discovered that the protein ATP5B interacts with NSUN5. Furthermore, after knocking down ATP5B using siRNA, we observed impaired decidualization. Moreover, transfection with siRNA to suppress NSUN5 resulted in a decrease in ATP5B expression. These significant findings provide strong evidence that NSUN5 plays a crucial role in decidualization and is closely associated with the development of PE through its interaction with ATP5B.

Mice with NOP2/sun RNA methyltransferase 5 deficiency die before reaching puberty due to fatal kidney damage.

BACKGROUND: NOP2/Sun RNA methyltransferase 5 (NSUN5) is an RNA methyltransferase that has a broad distribution and plays critical roles in various biological processes. However, our knowledge of the biological functions of NSUN5 in mammals is very limited. Therefore, in this study, we investigate the role of NSUN5 in mice. METHODS: In the present research, we built a mouse model (Nsun5-/-) using the CRISPR/Cas9 system to investigated the specific role of NSUN5. RESULTS: We observed that Nsun5-/- mice had a reduced body weight compared to wild-type mice. Additionally, their survival rate gradually decreased to 20% after postnatal day (PD) 21. Further examination revealed the Nsun5-/- mice had multiple organ damage, with the most severe damage occurring in the kidneys. Moreover, we observed glycogen deposition and fibrosis, along with a notable shorting of the primary foot processes of glomeruli in Nsun5-/- kidneys. Furthermore, we found that the kidneys of Nsun5-/- mice showed increased expression of the apoptotic signal Caspase-3 and accumulated stronger DNA damage at PD 21. CONCLUSIONS: In our study, we found that mice lacking NSUN5 died before puberty due to kidney fatal damage caused by DNA damage and cell apoptosis. These results suggest that NSUN5 plays a vital role in preventing the accumulation of DNA damage and cell apoptosis in the kidney.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

NSUN5-FTH1 Axis Inhibits Ferroptosis to Promote the Growth of Gastric Cancer Cells.

Recent studies revealed that NOP2/Sun RNA methyltransferase 5 (NSUN5) - ferritin heavy chain (FTH1) pathway is associated with ferroptosis in stem cells, whereas its roles in gastric cancer are still unclear. Our study aims to investigate the roles of the NSUN5-FTH1 axis in gastric cancer (GC) and its molecular mechanisms. Stable cell lines were constructed on SGC7901 cells by using shRNAs and pcDNA3.1 expression vectors, respectively. CCK-8 kits were used to determine cell viability. Biochemicals assays were used to detect lipid reactive oxygen species (ROS) and intracellular Fe2+ levels. RNA immunoprecipitation assay, qPCR, and Western blotting were used to determine the changes in biomarkers. GC xenograft mouse model was established to confirm the observation in vivo. An elevation of NSUN5 was observed in GC tumor tissues. NSUN5 inhibited ferroptosis including decreasing cell viability and increasing levels of lipid ROS and Fe2+ in GC cells. Besides, a positive correlation was also observed between NSUN5 and FTH1. Interestingly, NSUN5 regulated the levels of FTH1, instead of FTH1 regulating NSUN5 in GC cells. NSUN5-FTH1 axis regulated erastin-induced ferroptosis in SGC7901 cells. Consistently, silencing NSUN5 or FTH1 inhibited the growth of the SGC7901 tumor in vivo. NSUN5-FTH1 axis promoted the growth of GC cells in part by the regulation of ferroptosis.

Knocking down NSUN5 inhibits the development of clear cell renal cell carcinoma by inhibiting the p53 pathway.

Clear cell renal cell carcinoma (ccRCC) is the most common solid renal tumor. NSUN5, a gene encoding cytosine-5 RNA methyltransferase, has rarely been reported associated with cancer. A bioinformatics analysis revealed that NSUN5 was overexpressed in ccRCC. Gene Ontology and gene set variation analyses showed that NSUN5 was associated with tumor immunity in ccRCC. The effect of immunosuppressive treatment was superior in the low-risk group compared to the high-risk group, and higher stromal score in the high-risk group relative to the low-risk group. A drug sensitivity analysis revealed that the high-risk group was more sensitive to 5-fluorouracil, mitomycin C, methotrexate, and 17-AAG, whereas the low-risk group was more sensitive to crizotinib, sorafenib, foretinib, and ivozanib. NSUN5 knockout decreased ccRCC cell proliferation. The migration speed and number of invasive cells further decreased. The percentage of apoptotic cells increased. In NSUN5-knockout cells, the levels of BAX, caspase-8, caspase-9, and p53 increased significantly, whereas those of Bcl2, CCND1, CCND3, and MMP9 decreased significantly. NSUN5 is highly expressed in ccRCC and inhibits cancer cell invasion, proliferation, and migration while promoting apoptosis by activating the p53 signaling pathway. This study provides insights into the mechanisms of action of NSUN5 in urological tumors and may contribute to improving ccRCC treatment options.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

A novel NSUN5/ENO3 pathway promotes the Warburg effect and cell growth in clear cell renal cell carcinoma by 5-methylcytosine-stabilized ENO3 mRNA.

OBJECTIVES: Clear cell renal cell carcinoma (ccRCC) cells often reprogram their metabolisms. Enolase 3 (ENO3) is closely related to the Warburg effect observed in cells during tumor progression. However, the expression and function of ENO3 in ccRCC cells remain unclear. Therefore, this study investigated the expression and functional significance of ENO3 in the Warburg effect observed in ccRCC cells. METHODS: In this study, B-mode and microflow imaging ultrasound examinations were performed to evaluate patients with ccRCC. The extracellular acidification rate test and glucose uptake and lactate production assays were used to examine the Warburg effect in ccRCC cells. Western blotting, quantitative reverse transcription polymerase chain reaction, and immunochemistry were used to detect the expression of ENO3 and NOP2/Sun RNA methyltransferase 5 (NSUN5). RESULTS: ENO3 upregulation in ccRCC tumor tissues was accompanied by an increase in tumor size. Importantly, ENO3 participated in the Warburg effect observed in ccRCC cells, and high levels of ENO3 indicated a poor prognosis for patients. Loss of ENO3 reduced glucose uptake, lactate production, and extracellular acidification rate as well as inhibited ccRCC cell proliferation. Furthermore, NSUN5 was involved in the ENO3-regulated Warburg effect and ccRCC cell progression. Mechanically, NSUN5 was upregulated in ccRCC tissues, and NSUN5 upregulation mediated 5-methylcytosine modification of messenger RNA (mRNA) in ccRCC cells to promote mRNA stability and ENO3 expression. CONCLUSIONS: Collectively, the destruction of the NSUN5/ENO3 axis prevents ccRCC growth in vivo and in vitro, and targeting this pathway may be an effective strategy against ccRCC progression.

NSUN5 promotes progression and predicts poor prognosis in hepatocellular carcinoma.

The 5-methylcytosine (m5C) RNA methyltransferase NOP2/Sun RNA methyltransferase 5 (NSUN5) has been reported to serve important roles in numerous diseases. However, the functions and clinical significance of NSUN5 in hepatocellular carcinoma (HCC) remain unknown. Clinical information and NSUN5 mRNA sequencing data for 374 patients with HCC were downloaded from The Cancer Genome Atlas (TCGA) database, and NSUN5 mRNA and protein expression levels in 120 patients with HCC (present study cohorts) were assessed using reverse transcription-quantitative PCR, western blotting or immunohistochemistry. The association between NSUN5 mRNA and protein expression levels and the clinical characteristics (or prognosis) of patients with HCC was analyzed using the χ2 or log-rank test. The functions of NSUN5 in HCC were evaluated using in vitro and in vivo experiments, and the mechanism by which NSUN5 affected the progression of HCC was assessed using bioinformatics analysis using LinkedOmics. NSUN5 was significantly upregulated and predicted poor prognosis in HCC according to data from both TCGA database and present study cohorts. NSUN5 significantly promoted HCC proliferation and migration in vitro and significantly induced HCC tumor growth in vivo. Bioinformatics analysis demonstrated that NSUN5 was positively correlated with genes associated with translation in HCC. It was hypothesized that overexpression of NSUN5 strengthened ribosome functions and global protein translation, which may promote the proliferation and migration of HCC. In conclusion, NSUN5 may promote the progression of HCC by enhancing translation, thus making it a potential target for HCC treatment.

Mutations in RNA Methyltransferase Gene NSUN5 Confer High Risk of Outflow Tract Malformation.

NSUN5, encoding a cytosine-5 RNA methyltransferase and located in the 7q11.23 locus, is a candidate gene for tetralogy of Fallot (TOF). Deletion of the 7q11.23 locus in humans is linked to cardiac outflow tract (OFT) disorders including TOF. We identified four potential pathogenic mutations in the coding region of NSUN5 and which were enriched in TOF patients by an association study of 132 TOF patients and 2,000 in-house controls (P = 1.44 × 10-5). We then generated a Nsun5 null (Nsun5 -/-) mouse model to validate the human findings by defining the functions of Nsun5 in OFT morphogenesis. The OFT did not develop properly in the Nsun5 deletion embryonic heart. We found a misalignment of the aorta and septum defects caused by the delayed fusion of the membraneous ventricular spetum as an OFT development delay. This caused OFT development delay in 27 of 64 (42.2%) Nsun5 -/- mice. Moreover, we also found OFT development delay in 8 of 51 (15.7%) Nsun5 +/- mice. Further functional experiments showed that the loss of Nsun5 function impaired the 5-methylcytosine (m5C) modification and translation efficiency of essential cardiac genes. Nsun5 is required for normal OFT morphogenesis and it regulates the m5C modification of essential cardiac genes. Our findings suggest the involvement of NSUN5 in the pathogenesis of TOF.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans.

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.

Insights into the Regulatory Role of m6A Epitranscriptome in Glioblastoma.

N6-methyladenosine (m6A) is one of the most widespread and abundant internal messenger RNA modifications found in eukaryotes. Emerging evidence suggests that this modification is strongly linked to the activation and inhibition of cancer pathways and is associated with prognostically significant tumour subtypes. The present review describes the dynamic nature of m6A regulator enzymes, as methyltransferases, demethylases and m6A binding proteins, and points out thevalue of the balance among these proteins in regulating gene expression, cell metabolism and cancer development. The main focus of this review is on the roles of m6A modification in glioblastoma, the most aggressive and invariably lethal brain tumour. Although the study of m6A in glioblastoma is a young one, and papers in this field can yield divergent conclusions, the results collected so far clearly demonstrate that modulation of mRNA m6A levels impacts multiple aspects of this tumour, including growth, glioma stem cells self-renewal, and tumorigenesis, suggesting that mRNA m6A modification may serve as a promising target for glioblastoma therapy. We also present recent data about another type of epitranscriptomic modification, the methylation of cytosine at a specific site of 28S rRNA, as it was recently shown to affect the biology of glioma cells, with high potential of clinical implications.

Expression of the RNA methyltransferase Nsun5 is essential for developing cerebral cortex.

Nsun5 gene, encoding a cytosine-5 RNA methyltransferase, is deleted in about 95% patients with Williams-Beuren syndrome (WBS). WBS is a neurodevelopmental disorder and characterized by cognitive disorder. We generated single-gene Nsun5 knockout (Nsun5-KO) mice and reported that the Nsun5 deletion leads to deficit in spatial cognition. This study focused on investigating the influence of Nsun5 deficiency in the development of cerebral cortex. In comparison with wild-type littermates, the cortical thickness in postnatal day 10 Nsun5-KO mice was obviously reduced with an abnormal laminar organization, and the processes of pyramidal cells were shorter and finer. Nsun5 was selectively expressed in radial glial cells (RGCs) of cerebral cortex from embryonic day (E) 12.5 to E16.5, but not in intermediate progenitor cells (IPCs) or neocortical neurons. The Nsun5 deletion did not alter proliferation of RGCs or differentiation of RGCs into IPCs. Notably, the ablation of Nsun5 disrupted the growth of radial glial scaffolds, thus numerous basal processes of RGCs failed to reach pial basement membrane. Level of cell polarity regulator Cdc42 protein in radial glial scaffolds of E14.5 Nsun5-KO mice was reduced, but the level of Cdc42 mRNA was unchanged. The dysfunction of glial scaffolds impeded the radial migration of upper-layer and deeper-layer neurons to cause their subcortical accumulation and apoptosis, resulting in an obvious thinness of the cortical plate in E18.5 Nsun5-KO mice. These findings establish a critical role of Nsun5 in development of cerebral cortex through regulating radial glial scaffolds of RGCs to control migration of neocortical neurons.

Agenesis and Hypomyelination of Corpus Callosum in Mice Lacking Nsun5, an RNA Methyltransferase.

Williams-Beuren syndrome (WBS) is caused by microdeletions of 28 genes and is characterized by cognitive disorder and hypotrophic corpus callosum (CC). Nsun5 gene, which encodes cytosine-5 RNA methyltransferase, is located in the deletion loci of WBS. We have reported that single-gene knockout of Nsun5 (Nsun5-KO) in mice impairs spatial cognition. Herein, we report that postnatal day (PND) 60 Nsun5-KO mice showed the volumetric reduction of CC with a decline in the number of myelinated axons and loose myelin sheath. Nsun5 was highly expressed in callosal oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) from PND7 to PND28. The numbers of OPCs and OLs in CC of PND7-28 Nsun5-KO mice were significantly reduced compared to wild-type littermates. Immunohistochemistry and Western blot analyses of myelin basic protein (MBP) showed the hypomyelination in the CC of PND28 Nsun5-KO mice. The Nsun5 deletion suppressed the proliferation of OPCs but did not affect transition of radial glial cells into OPCs or cell cycle exit of OPCs. The protein levels, rather than transcriptional levels, of CDK1, CDK2 and Cdc42 in the CC of PND7 and PND14 Nsun5-KO mice were reduced. These findings point to the involvement of Nsun5 deletion in agenesis of CC observed in WBS.

Cognitive deficits in mice lacking Nsun5, a cytosine-5 RNA methyltransferase, with impairment of oligodendrocyte precursor cells.

Williams-Beuren syndrome (WBS) is a microdeletion disorder with cognitive phenotype. NSUN5 gene, which encodes a cytosine-5 RNA methyltransferase, is located in WBS deletion locus. To investigate the influence of NSUN5 deletion on cognitive behaviors, we produced single-gene Nsun5 knockout (Nsun5-KO) mice. Here, we report that adult Nsun5-KO mice showed spatial cognitive deficits. Size of the brain and hippocampal structures and the number of CA1 or CA3 pyramidal cells in Nsun5-KO mice did not differ from WT mice. Basal properties of Schaffer collateral-CA1 synaptic transmission in Nsun5-KO mice were unchanged, but NMDA receptor (NMDAr)-dependent long-term potentiation (LTP) was not induced. The NMDA-evoked current in CA1 pyramidal cells was reduced in Nsun5-KO mice without the changes in expression and phosphorylation of NMDAr subunits NR2A and NR2B. Although the protein level of AMPA receptor subunit GluR2 was attenuated in Nsun5-KO mice, the AMPA-evoked current was not altered. Hippocampal immuno-staining showed the selective expression of Nsun5 in NG2 or PDGFRα labeled oligodendrocyte precursor cells (OPCs), but not in pyramidal cells or astrocytes. Analysis of RT-PCR determined the Nsun5 expression in purified populations of OPCs rather than neurons or astrocytes. The Nsun5 deficiency led to decreases in the number and neurite outgrowth of OPCs in the hippocampal CA1 and DG, with the decline in NG2 expression and OPCs proliferation. These findings indicate that the Nsun5 deletion suppresses NMDAr activity in neuronal cells probably through the disrupted development and function of OPCs, leading to deficits in NMDAr-dependent LTP and spatial cognitive abilities.

Gene structure variation in segmental duplication block C of human chromosome 7q 11.23 during primate evolution.

Segmental duplication, or low-copy repeat (LCR) event, occurs during primate evolution and is an important source of genomic diversity, including gain or loss of gene function. The human chromosome 7q 11.23 is related to the William-Beuren syndrome and contains large region-specific LCRs composed of blocks A, B, and C that have different copy numbers in humans and different primates. We analyzed the structure of POM121, NSUN5, FKBP6, and TRIM50 genes in the LCRs of block C. Based on computational analysis, POM121B created by a segmental duplication acquired a new exonic region, whereas NSUN5B (NSUN5C) showed structural variation by integration of HERV-K LTR after duplication from the original NSUN5 gene. The TRIM50 gene originally consists of seven exons, whereas the duplicated TRIM73 and TRIM74 genes present five exons because of homologous recombination-mediated deletion. In addition, independent duplication events of the FKBP6 gene generated two pseudogenes at different genomic locations. In summary, these clustered genes are created by segmental duplication, indicating that they show dynamic evolutionary events, leading to structure variation in the primate genome.