Search Results (25)

The ability of common carp to withstand both short-term and long-term oxygen deprivation has been well documented; however, the potential genetic mechanisms behind common carp’s hypoxia response remain unclear. Therefore, to understand the possible genetic foundation of their response to hypoxia, comparative genomic analyses were conducted among six common carp varieties: Color, Songpu, European, Yellow, Mirror, and Hebao common carps. We identified 118 single-copy orthologous positively selected genes (PSGs) (dN/dS > 1) in all common carps under study, with GO functions directly related to the cellular responses to hypoxia in Color and European common carp PSGs, such as oxygen transport activity, oxygen binding activity, respiratory burst activity, and superoxide anion production. The Bayes Empirical Bayes (BEB) technique identified possible amino acid substitutions in mitochondrial and hypoxic genes under positive selection. Exonic and intronic structural variations (SVs) were discovered in the CYGB2 hypoxia-related gene of Color and European common carps, as well as in several mitochondrial genes, including MRPL20, MRPL32, NSUN3, GUF1, TMEM17B, PDE12, ACAD6, and COX10 of Color, European, Songpu, Yellow, and Hebao common carps. Moreover, Color common carp and Songpu common carp were found to share the greatest percentage of collinear genes (49.8%), with seven Songpu common carp chromosomes (chr A2, chr A9, chr A13, chr B13, chr B15, chr B2, and chr B12) showing distinct translocation events with the corresponding chromosomes of Color common carp. Additionally, we found 570 translocation sites that contained 3572 translocation-related genes in Color common carp, some of which are directly relevant to mitochondrial and hypoxic GO functions and KEGG pathways. Our results offer strong genome-wide evidence of the possible evolutionary response of Cyprinus carpio to hypoxia, providing important insights into the potential molecular mechanisms that explain their survival in hypoxic environments and guiding future research into carp hypoxia tolerance. Full article

Background: The precise role of RNA modification in post-traumatic stress disorder (PTSD) remains incompletely understood. This study aims to elucidate the effects of five common RNA modifications in PTSD, specifically m⁶A, m⁵C, m¹A, m⁷G, and ψ. Methods: We extracted data from the GEO repository to conduct a series of bioinformatics analyses. These included differential analysis to identify key regulators of five common RNA modifications, model construction using random forest (RF), least absolute shrinkage and selection operator (LASSO), and nomogram techniques, as well as consensus clustering of RNA modification subtypes. Furthermore, GO enrichment analysis was performed on DEGs associated with various RNA modification patterns. Immune cell infiltration was assessed using PCA and ssGSEA. RT-qPCR was performed to validate RNA modification-related genes (RMGs). Results: Twenty-one differentially expressed RMGs were identified. LASSO and RF intersection yielded eight signature genes (YTHDC1, IGFBP1, IGF2BP1, ALKBH5, NSUN4, TET2, TET3, WDR4) that robustly diagnosed PTSD (AUC = 0.804). Furthermore, these feature genes were validated using RT-qPCR, which was basically consistent with the results of bioinformatics analysis. Consensus clustering analysis may reveal two distinguishable subtypes: clusterA marked by high immunoinflammation, and clusterB characterized by high-neuroendocrine dysregulation. Conclusions: RMGs may play a crucial role in the pathogenesis of PTSD. Analyzing RNA modification patterns could offer potential diagnostic markers and help to guide immunotherapeutic approaches or neurotransmitter system interventions for PTSD in the future. Full article

The Ataxin-2-like (ATXN2L) protein is required to survive embryonic development, as documented in mice with the constitutive absence of the ATXN2L Lsm, LsmAD, and PAM2 domains due to knock-out (KO) of exons 5–8 with a frameshift. Its less abundant paralog, Ataxin-2 (ATXN2), has an extended N-terminus, where a polyglutamine domain is prone to expansions, mediating vulnerability to the polygenic adult motor neuron disease ALS (Amyotrophic Lateral Sclerosis) or causing the monogenic neurodegenerative processes of Spinocerebellar Ataxia Type 2 (SCA2), depending on larger mutation sizes. Here, we elucidated the physiological function of ATXN2L by deleting the LsmAD and PAM2 motifs via loxP-mediated KO of exons 10–17 with a frameshift. Crossing heterozygous floxed mice with constitutive Cre-deleter animals confirmed embryonic lethality among offspring. Crossing with CamK2a-CreERT2 mice and injecting tamoxifen for conditional deletion achieved chimeric ATXN2L absence in CamK2a-positive frontal cortex neurons and reduced spontaneous horizontal movement. Global proteome profiling of frontal cortex homogenate showed ATXN2L levels decreased to 75% and dysregulations enriched in the alternative splicing pathway. Nuclear proteins with Sm domains are critical to performing splicing; therefore, our data suggest that the Like-Sm (Lsm, LsmAD) domains in ATXN2L serve a role in splice regulation, despite their perinuclear location. Full article

Hepatitis B virus (HBV) remains a major global health challenge, with over 296 million people chronically infected worldwide. Despite the availability of antiviral therapies, a functional cure is rarely achieved, highlighting the need for novel therapeutic strategies. RNA 5-methylcytosine (m⁵C) is a pivotal epitranscriptomic mark implicated in RNA stability, transport, and translation. Emerging evidence shows that m⁵C is conserved within HBV RNA and plays critical roles in the viral life cycle. This review provides a comprehensive overview of the molecular mechanisms governing m⁵C deposition and recognition, summarizes recent advances in m⁵C biology, and highlights the emerging role of epitranscriptomic m⁵C regulation in HBV infection. We discuss the identification of HBV-specific m⁵C sites, the functions of key regulatory enzymes, and their interplay in viral RNA stabilization and evasion of innate immune responses. Interplay between m⁵C and other RNA modifications—particularly N6-methyladenosine (m⁶A)—is examined alongside virus-specific m⁵C regulation in EV71, HIV, HCV, EBV, and SARS-CoV-2. Potential links between m⁵C dysregulation and HBV-induced hepatocarcinogenesis are outlined, and emerging therapeutic strategies targeting the m⁵C machinery are highlighted. Together, these insights position the epitranscriptomic landscape as a promising avenue for innovative antiviral strategies. Full article

Sphingosine kinases (SPHKs) are essential enzymes that catalyze the phosphorylation of sphingosine to produce sphingosine-1-phosphate (S1P), which plays pivotal roles in inflammation and immune regulation. In this study, genome-wide association analysis (GWAS) identified the Ydsphk1 gene as closely associated with the resistance of yellow drum (Nibea albiflora) to Vibrio harveyi. Structural prediction showed that YDSPHK1 contains a typical diacylglycerol kinase catalytic (DAGKc) domain (154–291 aa). By constructing and transfecting Ydsphk1 expression plasmids into yellow drum kidney cells, we found that YDSPHK1 is localized in the cytoplasm. Subsequent RNA-Seq analysis of an overexpression plasmid identified 25 differentially expressed genes (DEGs), including 13 upregulated and 12 downregulated. Notably, nsun5 and hsp90aa1 were significantly upregulated, while Nfkbia and hmox1 were downregulated. Promoter analysis indicated that the core regulatory regions of Ydsphk1 are located between −1931~−1679 bp and −419~+92 bp, with two predicted TFAP2A binding sites in the −419~+92 bp region. Further studies demonstrated that varying concentrations of TFAP2A significantly reduced Ydsphk1 promoter activity. These findings underscore the pivotal role of Ydsphk1 in regulating immune responses in yellow drum, particularly through its impact on key immune-related genes and pathways such as NF-κB signaling and ferroptosis. The identification of Ydsphk1 as a mediator of immune regulation provides valuable insights into the molecular mechanisms of immune defense and highlights its potential as a target for enhancing pathogen resistance in aquaculture practices. This study lays a strong foundation for future research aimed at developing innovative strategies for disease management in aquaculture species. Full article

Degenerative retinal diseases can lead to blindness if left untreated. At present, there are no curative therapies for retinal diseases. Therefore, effective treatment strategies for slowing the progression of retinal diseases and thus improving patients’ life standards are urgently needed. The present study aimed to assess the effect of sinusoidal electromagnetic field (EMF) (50 Hz, 1.3 mT) treatment for 15 and 30 min on spontaneously arising retinal pigment epithelial cells (ARPE-19) and retinal ganglion cells (RGC-5) and its short-term post-treatment significance. Our study indicated the beneficial impact of EMF treatment on the proliferative and migratory capacity of the tested cells. ARPE-19 and RGC-5 cells exposed to an EMF exhibited elevated levels of HO-1, increased N6-methyladenosine (m⁶A) and N5-methylcytosine (m⁵C) status mediated by METTL3 and NSUN2, respectively, and changes in levels of DNA damage repair factors, which may contribute to the regenerative properties of ARPE-19 and RGC-5 cells. Overall, this analysis showed that EMF (sinusoidal, 50 Hz, 1.3 mT) treatment may serve as a potential therapeutic strategy for retinal diseases. Full article

Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is a prevalent and aggressive cancer with high rates of metastasis and poor prognosis. Recent research highlights the role of 5-methylcytosine (m⁵C) in cancer progression. NSUN2, an m⁵C methyltransferase, has been implicated in various cancers, but its role in HNSCC remains elusive. Methods: NSUN2 expression and its impact on HNSCC were analyzed by using clinical samples and bioinformatic analysis. m⁵C-Bis-Seq was used to assess changes in mRNA m⁵C modification and identify downstream targets. Both in vitro and vivo studies were performed to evaluate the impact of NSUN2 manipulation on tumor growth and metastasis. Results: Results indicated that NSUN2 was significantly upregulated in HNSCC tissues compared to normal tissues and was associated with poor prognosis. NSUN2 knockdown led to decreased cell proliferation, migration, and invasion in vitro and reduced tumorigenicity and lymph node metastasis in vivo. m⁵C-Bis-Seq revealed altered m⁵C-modification patterns upon NSUN2 knockdown, with LAMC2 identified as a key downstream target. Conclusions: NSUN2-mediated m⁵C-modification enhanced LAMC2 stability, promoting epithelial–mesenchymal transition (EMT) signaling pathways. These findings demonstrate that NSUN2 promotes the initiation and progression of HNSCC by stabilizing the LAMC2 transcript through m⁵C-dependent mechanisms, offering a promising epitranscriptomic-targeted therapeutic approach for HNSCC. Full article

Inherited optic neuropathies (IONs) are rare genetic diseases characterized by progressive visual loss due the atrophy of optic nerves. The standard diagnostic workup involving next-generation sequencing panels has a diagnostic yield of about forty percent. In the other 60% of the patients with a clinical diagnosis of ION, the underlying genetic variants remain unknown. In this case study, we describe a potentially new disease-associated gene, NSUN3, for IONs. The proband was a young woman with consanguineous parents. She presented with bilateral optic atrophy and nystagmus at the age of seven years. Genetic testing revealed the homozygous variant c.349_352dup p.(Ala118Glufs*45) in NSUN3, with a segregation in the family compatible with autosomal recessive inheritance. Additional functional analysis showed decreased NSUN3 mRNA levels, slightly diminished mitochondrial complex IV levels, and decreased cell respiration rates in patient fibroblasts compared to healthy controls. In conclusion, pathogenic variants in NSUN3 can cause optic neuropathy. Trio whole-exome sequencing should be considered as a diagnostic strategy in ION cases where standard diagnostic analysis does not reveal disease-causing variants. Full article

Mitochondria, the main cellular power stations, are important modulators of redox-sensitive signaling pathways that may determine cell survival and cell death decisions. As mitochondrial function is essential for tumorigenesis and cancer progression, mitochondrial targeting has been proposed as an attractive anticancer strategy. In the present study, three mitochondria-targeted quercetin derivatives (mitQ3, 5, and 7) were synthesized and tested against six breast cancer cell lines with different mutation and receptor status, namely ER-positive MCF-7, HER2-positive SK-BR-3, and four triple-negative (TNBC) cells, i.e., MDA-MB-231, MDA-MB-468, BT-20, and Hs 578T cells. In general, the mito-quercetin response was modulated by the mutation status. In contrast to unmodified quercetin, 1 µM mitQ7 induced apoptosis in breast cancer cells. In MCF-7 cells, mitQ7-mediated apoptosis was potentiated under glucose-depleted conditions and was accompanied by elevated mitochondrial superoxide production, while AMPK activation-based energetic stress was associated with the alkalization of intracellular milieu and increased levels of NSUN4. Mito-quercetin also eliminated doxorubicin-induced senescent breast cancer cells, which was accompanied by the depolarization of mitochondrial transmembrane potential. Limited glucose availability also sensitized doxorubicin-induced senescent breast cancer cells to apoptosis. In conclusion, we show an increased cytotoxicity of mitochondria-targeted quercetin derivatives compared to unmodified quercetin against breast cancer cells with different mutation status that can be potentiated by modulating glucose availability. Full article

Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel–Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2′-O-methylcytidine. The method’s reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials. Full article

RNA modifications implicate pathological and prognosis significance in cancer development and progression, of which, m6A and m5C are representative regulators. These RNA modifications could produce effects on the function of other RNA by regulating gene expression. Thus, in this study, we aimed to explore the correlation between m6A/m5C regulators and early-stage lung adenocarcinoma (LUAD). Only the early-stage LUAD samples were included in this investigation, and the RNA-seq dataset of The Cancer Genome Atlas (TCGA) cohort was utilized to evaluate the expression of 37 m6A/m5C regulated genes. Based on the expression level of these 37 genes, early-stage LUAD patients were divided into 2 clusters, which were performed by consensus clustering, and the m6A/m5C subtypes had significantly different prognostic outcomes (p < 0.001). Cluster1, which has a better prognosis, was characterized by the C3 (inflammatory) immune subtype, low immune infiltration, chemokine expression, major histocompatibility complex (MHC) expression, and immune checkpoint molecule expression. Furthermore, compared with cluster1, cluster2 showed a T cell exhaustion state, characterized by a high expression of immune checkpoint genes, and immune cells, such as T cells, CD8+ T cells, cytotoxic lymphocytes, NK cells, and so on. In addition, patients in cluster2 were with high tumor mutational burden (TMB) and numerous significant mutated oncogene and tumor suppressor genes, such as WNT10B, ERBB4, SMARCA4, TP53, and CDKN2A (p < 0.001). A total of 19 genes were mostly related to the prognosis of LUAD and were upregulated in cluster2 (p < 0.05), showing a positive correlation with the mRNA expression of 37 m6A/m5C regulated genes. The predictive risk model was constructed using Cox and LASSO (least absolute shrinkage and selection operator) regression analysis. Finally, a seven-gene m6A/m5C risk model, comprising of METTL3, NPLOC4, RBM15, YTHDF1, IGF2BP1, NSUN3, and NSUN7, was constructed to stratify the prognosis of early-stage LUAD (p = 0.0049, AUC = 0.791). The high-risk score was associated with a poorer prognosis. This model was also validated using two additional GEO datasets: GSE72094 (p = 0.011, AUC = 0.736) and GSE50081 (p = 0.012, AUC = 0.628). In summary, it was established that the m6A/m5C-regulated genes performed a crosstalk function in the mRNA expression of early-stage LUAD. By interacting with other mRNA genes, m6A/m5C modification disturbs DNA replication and the tumor immune microenvironment (TIME). The seven-gene risk model may be a critical tool for the prognostic assessment of early-stage LUAD. Full article

Targeting RNA methyltransferases with small molecules as inhibitors or tool compounds is an emerging field of interest in epitranscriptomics and medicinal chemistry. For two challenging RNA methyltransferases that introduce the 5-methylcytosine (m⁵C) modification in different tRNAs, namely DNMT2 and NSUN6, an ultra-large commercially available chemical space was virtually screened by physicochemical property filtering, molecular docking, and clustering to identify new ligands for those enzymes. Novel chemotypes binding to DNMT2 and NSUN6 with affinities down to K_D,app = 37 µM and K_D,app = 12 µM, respectively, were identified using a microscale thermophoresis (MST) binding assay. These compounds represent the first molecules with a distinct structure from the cofactor SAM and have the potential to be developed into activity-based probes for these enzymes. Additionally, the challenges and strategies of chemical space docking screens with special emphasis on library focusing and diversification are discussed. Full article

Leber’s hereditary optic neuropathy (LHON) is a maternally transmitted disease caused by mitochondria DNA (mtDNA) mutation. It is characterized by acute and subacute visual loss predominantly affecting young men. The mtDNA mutation is transmitted to all maternal lineages. However, only approximately 50% of men and 10% of women harboring a pathogenic mtDNA mutation develop optic neuropathy, reflecting both the incomplete penetrance and its unexplained male prevalence, where over 80% of patients are male. Nuclear modifier genes have been presumed to affect the penetrance of LHON. With conventional genetic methods, prior studies have failed to solve the underlying pathogenesis. Whole exome sequencing (WES) is a new molecular technique for sequencing the protein-coding region of all genes in a whole genome. We performed WES from five families with 17 members. These samples were divided into the proband group (probands with acute onset of LHON, n = 7) and control group (carriers including mother and relative carriers with mtDNSA 11778 mutation, without clinical manifestation of LHON, n = 10). Through whole exome analysis, we found that many mitochondria related (MT-related) nuclear genes have high percentage of variants in either the proband group or control group. The MT genes with a difference over 0.3 of mutation percentage between the proband and control groups include AK4, NSUN4, RDH13, COQ3, and FAHD1. In addition, the pathway analysis revealed that these genes were associated with cofactor metabolism pathways. Family-based analysis showed that several candidate MT genes including METAP1D (c.41G > T), ACACB (c.1029del), ME3 (c.972G > C), NIPSNAP3B (c.280G > C, c.476C > G), and NSUN4 (c.4A > G) were involved in the penetrance of LHON. A GWAS (genome wide association study) was performed, which found that ADGRG5 (Chr16:575620A:G), POLE4 (Chr2:7495872T:G), ERMAP (Chr1:4283044A:G), PIGR (Chr1:2069357C:T;2069358G:A), CDC42BPB (Chr14:102949A:G), PROK1 (Chr1:1104562A:G), BCAN (Chr 1:1566582C:T), and NES (Chr1:1566698A:G,1566705T:C, 1566707T:C) may be involved. The incomplete penetrance and male prevalence are still the major unexplained issues in LHON. Through whole exome analysis, we found several MT genes with a high percentage of variants were involved in a family-based analysis. Pathway analysis suggested a difference in the mutation burden of MT genes underlining the biosynthesis and metabolism pathways. In addition, the GWAS analysis also revealed several candidate nuclear modifier genes. The new technology of WES contributes to provide a highly efficient candidate gene screening function in molecular genetics. Full article

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues. Full article

The 5-methylcytosine (m5C) modification on an mRNA molecule is deposited by Nsun2 and its paralog Nsun6. While the physiological functions of Nsun2 have been carefully studied using gene knockout (KO) mice, the physiological functions of Nsun6 remain elusive. In this study, we generated an Nsun6-KO mouse strain, which exhibited no apparent phenotype in both the development and adult stages as compared to wild-type mice. Taking advantage of this mouse strain, we identified 80 high-confident Nsun6-dependent m5C sites by mRNA bisulfite sequencing in five different tissues and systematically analyzed the transcriptomic phenotypes of Nsun6-KO tissues by mRNA sequencing. Our data indicated that Nsun6 is not required for the homeostasis of these organs under laboratory housing conditions, but its loss may affect immune response in the spleen and oxidoreductive reaction in the liver under certain conditions. Additionally, we further investigated T-cell-dependent B cell activation in KO mice and found that Nsun6 is not essential for the germinal center B cell formation but is associated with the formation of antibody-secreting plasma cells. Finally, we found that Nsun6-mediated m5C modification does not have any evident influence on the stability of Nsun6 target mRNAs, suggesting that Nsun6-KO-induced phenotypes may be associated with other functions of the m5C modification or Nsun6 protein. Full article