NSUN2-Mediated m⁵C Methylation in Human Disease: Mechanism and Therapeutic Implications

Abstract

The epitranscriptomic landscape defined by reversible post-transcriptional RNA modifications constitutes a sophisticated layer of gene regulatory circuitry that modulates diverse biological processes. As an essential writer enzyme for RNA 5-methylcytosine (m⁵C), NOP2/Sun RNA methyltransferase 2 (NSUN2) catalyzes site-specific m⁵C deposition across an extensive repertoire of cellular transcripts spanning messenger RNAs, transfer RNAs and non-coding RNAs, and orchestrates core post-transcriptional events including transcript stabilization, nucleocytoplasmic trafficking, translational tuning and RNA turnover. Accumulating preclinical and clinical evidence corroborates that perturbed NSUN2 expression rewires the physiological m⁵C epitranscriptomic signature, which functionally contributes to the onset and advancement of numerous human pathological conditions ranging from heterogeneous malignancies and cardiovascular complications to neurodegenerative syndromes, infectious disorders, inflammatory pathologies and systemic metabolic diseases. In this systematic review, we comprehensively consolidate contemporary mechanistic advances underlying NSUN2-dependent m⁵C modification in governing cellular homeostasis and disease pathogenesis, with focused discussion on its multifaceted functions in modulating oncogenic signaling cascades, mitochondrial fitness, neurodevelopmental progression, immune cell polarization and host-virus interaction. We further highlight prospective therapeutic modalities targeting the NSUN2–m⁵C regulatory axis and systematically dissect prevailing translational bottlenecks hindering bench-to-bedside transformation of such targeted interventions. Elucidating the conserved and disease-specific regulatory paradigms of NSUN2 therefore provides profound theoretical implications and practical clinical evidence for developing novel diagnostic biomarkers and precision therapeutic regimens across relevant human disorders.

Introduction

The central dogma of molecular biology has long been extended by the discovery of the epitranscriptome, a dynamic and reversible regulatory layer consisting of widespread post-transcriptional RNA modifications independent of primary DNA sequences.¹ Similar to epigenetic modifications occurring on DNA and histones, chemical modifications of RNA can precisely modulate gene expression in a time- and space-dependent manner. Among all currently characterized RNA modifications, 5-methylcytosine (m⁵C) is highly evolutionarily conserved and ubiquitously expressed. This prevalent epitranscriptomic mark is distributed across nearly all functional RNA subtypes, including tRNAs, mRNAs, rRNAs and diverse non-coding RNAs.^2,3

Generally, epitranscriptomic machineries are classified into three core functional groups: writers (methyltransferases), erasers (demethylases) and readers (effector proteins). Writers are enzymes that catalyze the deposition of chemical modifications on RNAs, erasers reverse such modifications to maintain dynamic epitranscriptomic homeostasis, and readers are effector proteins that specifically bind modified RNA residues and initiate downstream biological events. For m⁵C modification, the methylation is installed by dedicated RNA methyltransferase writers. As one of the most essential m⁵C writers, NOP2/Sun RNA Methyltransferase 2 (NSUN2) predominantly targets mRNAs and partial tRNAs to carry out site-specific methylation.^4,5 Mechanistically, NSUN2 transfers methyl groups from S-adenosylmethionine (SAM) donor to specific cytosine residues of target RNAs. The m⁵C modification is dynamically regulated and can be oxidized by ten–eleven translocation 2 (TET2) or demethylated by AlkB homolog 1 (ALKBH1).^6–8 Furthermore, two canonical reader proteins decode m⁵C-dependent biological signals: ALY/REF export factor (ALYREF) mainly facilitates nuclear export of methylated RNAs, while Y-box binding protein 1 (YBX1) primarily enhances RNA stability and protein translation.^3,5,9

Tight homeostasis of the NSUN2-m⁵C regulatory axis is indispensable for sustaining normal cellular activities. Conversely, aberrant activation or inhibition of this axis disrupts regular RNA metabolism, and such dysregulation has been proven to drive the occurrence and progression of a broad range of human diseases. While existing reviews have either generalized the biological functions of m⁵C epitranscriptomic modification or elaborated the oncogenic role of NSUN2 limited to single malignant diseases, most previous summaries focus on separate biological events or individual disease types. A systematic and cross-disease overview illustrating the conserved and distinct mechanisms of NSUN2 across multiple organ disorders is still lacking. Accordingly, this review comprehensively summarizes current research advances concerning NSUN2-m⁵C signaling in diverse human diseases, and further discusses the translational potential and core challenges of targeted therapies against this axis.

To address this research gap, our updated review systematically elaborates the pathogenic roles of NSUN2-driven m⁵C modification across a broad spectrum of human diseases, including cancers, cardiovascular diseases, neurological disorders, viral infections, inflammatory illnesses and metabolic disorders. Different from previously published reviews, our work builds an integrated research system covering underlying molecular mechanisms, pathological characteristics of different diseases, as well as potential translational therapeutic strategies. Moreover, we incorporate cutting-edge high-impact studies published in recent years to complement updated research findings. This review ultimately intends to clarify the universal and disease-specific regulatory patterns of the NSUN2-m⁵C axis in disease pathogenesis, and offer rational insights for future targeted therapeutic development.

Structure and Basic Functions of NSUN2

NSUN2 is a nuclear-localized RNA methyltransferase. It adopts S-adenosylmethionine (SAM) as the universal methyl donor, and possesses two key functional domains: an RNA recognition motif (RRM) responsible for substrate binding, and a Rossmann fold catalytic core that accommodates SAM cofactors for methylation reactions.¹⁰ NSUN2 mediates m⁵C modification across a broad range of RNA substrates, covering tRNAs, mRNAs and multiple non-coding RNAs including lncRNAs and circRNAs, as summarized in Figure 1.¹¹ Structurally, two conserved catalytic cysteine residues within NSUN2 form a covalent intermediate with cytosine bases of target RNAs during methylation.¹² This interaction activates the electron-rich C5 position of cytosine, which is subsequently subjected to nucleophilic attack by methyl groups donated from SAM, ultimately generating m⁵C marks on target RNAs.¹³ Upon completion of methylation, the covalently linked RNA substrate requires dissociation from NSUN2. A conserved cysteine residue located in motif IV of NSUN2 functions as a proton acceptor to trigger deprotonation of the tetrahedral intermediate, thereby driving the elimination reaction, restoring the unsaturated pyrimidine ring of m⁵C, and releasing modified RNA from the enzyme complex.¹⁰

Figure 1.

Functional roles of NSUN2-mediated m⁵C modification in diverse RNA types.¹¹

NSUN2 acts as a vital post-transcriptional regulator of RNA metabolism through catalyzing m⁵C modification across multiple types of cellular RNAs. For tRNAs, NSUN2-installed m⁵C modifications preserve native tRNA structure and guarantee accurate protein translation. In rRNAs, such epitranscriptomic marks support ribosome assembly and improve overall translational capacity. When targeting mRNAs, NSUN2-mediated m⁵C methylation enhances transcript stability, promotes pre-mRNA splicing and nucleocytoplasmic transport, and further elevates downstream protein translation levels. Beyond canonical coding RNAs, NSUN2 also stabilizes long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). Additionally, this methyltransferase modulates the stability and biological function of other non-coding RNA subgroups, such as enhancer RNAs, vault RNAs and tRNA-derived fragments. Overall, NSUN2 exerts widespread regulatory effects on diverse RNA species, predominantly improving RNA stability and translational performance at the post-transcriptional level.

NSUN2 catalyzes m⁵C modification on target RNAs, and these methylated transcripts further bind to specific reader proteins to execute diverse biological functions.¹⁴ Two well-characterized m⁵C reader proteins exert distinct regulatory roles: ALYREF primarily mediates the nucleocytoplasmic export of modified RNAs, whereas YBX1 is responsible for maintaining RNA stability.¹⁵ Existing evidence reveals that NSUN2 modulates protein synthesis via multiple post-transcriptional pathways, including facilitating mRNA nuclear export, sustaining mRNA stability, modulating miRNA maturation, tuning the expression of both protein-coding genes and long non-coding RNAs, and boosting overall translational activity.^16,17 Functionally, NSUN2 governs cell proliferation by modulating the expression and translation of core cell cycle regulators, as well as maintaining mitotic spindle stability during cell division.^18,19 In addition, NSUN2 acts as a critical cellular sensor for external stress signals, which modulates global protein synthesis and participates in the regulation of cellular senescence.^20,21

Cumulative studies have demonstrated aberrantly elevated NSUN2 expression in multiple solid tumors including breast cancer, gastric cancer and hepatocellular carcinoma (HCC), and its expression level is tightly correlated with patient clinical prognosis.^22–24 Beyond malignancies, pathogenic mutations of NSUN2 are implicated in neurodevelopmental disorders. Moreover, NSUN2-dependent m⁵C modification participates in regulating nervous system development, as well as vascular injury repair and vascular tissue remodeling.^25,26 Given the extensive involvement of NSUN2 in diverse human diseases, this review systematically summarizes the latest research progress regarding the biological functions and underlying mechanisms of NSUN2 across cancers, cardiovascular diseases, neurological disorders, viral infections and inflammatory disorders. The integrated disease regulatory network is summarized in Figure 2.

Figure 2.

Multifaceted regulatory landscape of NSUN2-mediated m⁵C RNA modification in human diseases.

The Role of NSUN2 in Disease Development

NSUN2 and Cancer

NSUN2 is frequently overexpressed in a broad spectrum of human cancers, and exerts potent oncogenic effects by regulating RNA stability, protein translation and cellular metabolic reprogramming through m⁵C RNA modification. Table 1 summarizes core downstream targets, detailed molecular mechanisms and corresponding malignant phenotypes of NSUN2 in different cancer types, facilitating intuitive comparison and systematic integration of existing research evidence across malignancies.

Table 1.

Roles of NSUN2 in Various Human Cancer Types

Cancer Type	NSUN2 Expression	Key Downstream Targets	Mechanism	Functional Outcome	References
Hepatocellular Carcinoma (HCC)	Upregulated	GRB2, RNF115, AATF, PKM2, MALAT1, GDF15, ACSL6	m5C-mediated stabilization of oncogenic mRNAs; activation of Ras/PI3K-AKT signaling; enhancement of glycolysis; ferroptosis suppression; lipid metabolism regulation	Promotes proliferation, metabolic reprogramming, sorafenib resistance, poor prognosis	[23,27–29]
Gastric Cancer (GC)	Upregulated	PGK1, GCLC, ATG9A, YBX1	Stabilizes PGK1 mRNA via m5C/YBX1; activates PI3K/AKT signaling; lactylation-activated NSUN2 stabilizes GCLC; NSUN2/YBX1 axis promotes autophagy	Promotes growth, invasion, metabolic adaptation, 5-FU/doxorubicin resistance	[30–32]
Colorectal Cancer (CRC)	Upregulated	ErbB-STAT3, KSR1 (via LINC02167/YBX1/ILF3), ENO1	m5C-dependent and -independent mechanisms; stabilizes oncogenic mRNAs; activates ERK/MAPK signaling; NSUN2/YBX1/m5C-ENO1 positive feedback loop	Promotes proliferation, invasion, metastasis, poor prognosis	[33–35]
Pancreatic Cancer (PC)	Upregulated	TIAM2	Stabilizes TIAM2 mRNA through m5C modification (YBX1-dependent) and transcriptional upregulation	Promotes proliferation, migration, EMT	[36]
Bladder Cancer (BCa)	Upregulated	HDGF, RABL6/TK1, PRDM11, SOCS3	m5C-mediated stabilization of HDGF mRNA; facilitates splicing/stabilization of RABL6/TK1 mRNA; recruits EZH2 for PRDM11 silencing; methylates SOCS3 mRNA to inhibit stability/nuclear export, activating JAK2/STAT3 pathway	Promotes tumor progression, cisplatin resistance, M2 macrophage polarization	[3,37–39]
Triple-Negative Breast Cancer (TNBC)	Upregulated	tRNA^Val-CAC, ALDH3A2, HK1, PFKM	tRNA m5C enhances codon-dependent translation of glycolytic enzymes	Promotes glycolysis, proliferation, paclitaxel resistance	[40,41]
Lung Cancer (LC)	Upregulated	ME1, GLUT3, CDK2, circFAM190B	Stabilizes metabolic enzyme mRNAs via m5C; circFAM190B m5C modification enhances stability, inhibits autophagy	Promotes metabolism, proliferation, angiogenesis, tumor progression	[42,43]
Acute Myeloid Leukemia (AML)	Upregulated	FSP1, PHGDH, SHMT2	Stabilizes metabolic and ferroptosis-related mRNAs via m5C/YBX1	Enhances proliferation, anti-ferroptosis, survival	[44,45]
Head and Neck Squamous Cell Carcinoma (HNSCC)	Upregulated	LAMC2	m5C-mediated stabilization of LAMC2 mRNA	Promotes proliferation, invasion, lymph node metastasis	[46]
Anaplastic Thyroid Cancer (ATC)	Upregulated	SRSF6, AGX2, ABC transporters	NSUN2-ALYREF complex mediates splicing reprogramming; increases glycosylation of ABC transporters	Promotes chemoresistance	[47]
Prostate Cancer (PRAD)	Upregulated	TRIM28	Stabilizes TRIM28 mRNA via m5C; upregulated by FOXA1	Promotes proliferation and drug resistance	[48]

Key Mechanisms of NSUN2 in Cancer Progression

Proliferation and Tumor Initiation

NSUN2 facilitates tumor initiation and sustained cancer cell proliferation largely via m⁵C-dependent stabilization of oncogenic and cell cycle-related transcripts. The m⁵C modification deposited by NSUN2 protects oncogenic mRNAs including PGK1 and PKM2 from intracellular degradation, thereby elevating their post-transcriptional expression and supporting uncontrolled malignant cell proliferation.^27,30 In HCC, NSUN2-induced m⁵C hypermethylation on GRB2, RNF115 and AATF mRNAs leads to persistent activation of Ras signaling pathway. This cascade accelerates cell cycle progression and ultimately promotes the tumorigenesis and malignant progression of HCC.²³

Invasion and Metastasis

NSUN2 drives cancer cell invasion and metastasis by regulating epithelial-mesenchymal transition (EMT) process and the expression of metastasis-related genes through m⁵C modification. Its key downstream targets vary across different tumor types: TIAM2 in prostate carcinoma (PRAD), UBE2S in osteosarcoma, and LAMC2 in head and neck squamous cell carcinoma (HNSCC).^36,46,49 In colorectal cancer (CRC), lncRNA LINC02167 acts as a molecular scaffold to recruit YBX1 protein to recognize m⁵C-modified KSR1 mRNA. Such binding stabilizes KSR1 transcripts, further activating the ERK/MAPK signaling cascade and eventually promoting colorectal cancer metastasis.³⁴

Drug Resistance

NSUN2 mediates chemotherapy resistance across multiple cancers through modulating three core biological processes: DNA damage repair, cellular ferroptosis and drug efflux. In bladder cancer, NSUN2 maintains the stability of R-loop structures to reduce cisplatin-induced DNA damage, thereby strengthening tumor cell resistance to cisplatin treatment.³⁸ In gastric cancer, lactylation modification activates NSUN2 enzymatic activity. Functional NSUN2 further stabilizes GCLC mRNA, elevates intracellular GSH content and blocks doxorubicin-triggered ferroptosis, which ultimately develops chemotherapy resistance.³¹ In anaplastic thyroid cancer, NSUN2 triggers alternative splicing reprogramming via m⁵C modification of SRSF6 mRNA. This regulatory axis enhances the glycosylation level of ABC drug transporters and facilitates drug excretion, resulting in widespread multidrug resistance.⁴⁷

Metabolic Reprogramming

NSUN2 facilitates tumor metabolic reprogramming across diverse malignancies, which fuels cancer progression via upregulating glycolysis in triple-negative breast cancer (TNBC) and lung cancer (LC), promoting lipid synthesis in HCC, and modulating serine metabolism in acute myeloid leukemia (AML).^40,42,45 In HCC, NSUN2 stabilizes glycolysis-related transcripts including GLUT1 and HK2 to accelerate cellular glucose uptake. Such metabolic alteration helps tumor cells gain survival superiority against infiltrating CD8⁺ T cells within the tumor microenvironment.⁵⁰ In AML, NSUN2 mediates m⁵C modification on FSP1 mRNA. The reader protein YBX1 further binds to these methylated transcripts to improve mRNA stability, which suppresses intracellular lipid peroxidation and protects leukemic cells from ferroptotic cell death.⁴⁴

NSUN2 and Cardiovascular Diseases

NSUN2 exerts context-dependent dual effects in the maintenance of cardiovascular physiological homeostasis and the regulation of pathological progression, and plays an indispensable regulatory role in cardiovascular health and lesion development by modulating endothelial biological function, myocardial injury response and vascular senescence through m⁵C RNA modification. Table 2 summarizes the core downstream targets, detailed molecular mechanisms and corresponding functional outcomes of NSUN2 in different cardiovascular diseases, facilitating the intuitive comparison and systematic integration of existing research evidence in the cardiovascular field.

Table 2.

Roles of NSUN2 in Major Cardiovascular Diseases

Cardiovascular Disease	NSUN2 Expression	Key Downstream Targets	Mechanism	Functional Outcome	References
Atherosclerosis	Upregulated	ICAM-1, SHC (p66SHC/p52SHC/p46SHC)	Upregulates ICAM-1 mRNA via m5C modification to induce endothelial inflammation; activates SHC/ROS/p38MAPK cascade	Promotes endothelial inflammation, leukocyte adhesion, vascular senescence and atherogenesis	[20,21]
Doxorubicin-Induced Myocardial Injury	Upregulated	Nrf2 (HO-1, NQO1)	Stabilizes Nrf2 mRNA via m5C methylation to prolong its half-life; upregulates antioxidant targets HO-1, NQO1	Relieves DOX-induced myocardial injury via anti-apoptosis and cardiac function preservation	[51]
Abdominal Aortic Aneurysm (AAA)	Upregulated	Autotaxin (ATX)	Upregulates endothelial autotaxin (ATX) expression and secretion via m5C modification; recruit T lymphocytes to aortic vessel wall	Induces aortic inflammation, medial damage and vascular dilatation to accelerate AAA progression	[52]

Atherosclerosis

Endothelial dysfunction, inflammatory activation and vascular senescence are core pathological drivers of atherosclerosis, and NSUN2 participates in the occurrence and progression of atherosclerotic lesions via dual regulation of endothelial inflammation and vascular oxidative senescence relying on its m⁵C methyltransferase activity. Mechanistically, NSUN2 catalyzes m⁵C methylation modification across the 5’ untranslated region (5’ UTR), coding sequence (CDS) and 3’ untranslated region (3’ UTR) of ICAM-1 mRNA, which markedly elevates the expression level of adhesion molecule ICAM-1 at the post-transcriptional level. This molecular cascade further aggravates endothelial inflammatory injury triggered by TNF-α or homocysteine stimulation, and ultimately promotes excessive leukocyte adhesion to vascular endothelial cells, a key initial event of atherosclerotic plaque formation.²⁰ Beyond inflammatory regulation, NSUN2 also modulates high glucose-induced vascular endothelial senescence via oxidative stress-dependent pathways in human umbilical vein endothelial cells (HUVECs). NSUN2-mediated m⁵C methylation of SHC mRNA facilitates the translational activation of three SHC protein isoforms, namely p66SHC, p52SHC and p46SHC. The upregulated SHC isoforms further boost intracellular reactive oxygen species (ROS) accumulation and trigger the phosphorylation and activation of downstream p38MAPK signaling pathway, thereby exacerbating endothelial oxidative stress and accelerating vascular senescence in a hyperglycemic microenvironment.²¹

Myocardial Injury

NSUN2 also serves as a critical protective regulator in chemotherapy-related myocardial damage and physiological cardiac structural maintenance. Upon doxorubicin (DOX) exposure, NSUN2 expression is significantly upregulated in cardiomyocytes as an endogenous compensatory mechanism against myocardial oxidative damage. Functionally, NSUN2 catalyzes m⁵C methylation modification on Nrf2 mRNA, which prolongs the mRNA half-life and stabilizes Nrf2 transcripts at the post-transcriptional level. The elevated Nrf2 protein abundance further activates downstream antioxidant effector molecules including HO-1 and NQO1, subsequently mitigating intracellular ROS accumulation and suppressing ROS-mediated cardiomyocyte apoptosis to alleviate DOX-induced myocardial injury.⁵¹ Apart from its antioxidative cardioprotective role, NSUN2 is essential for maintaining normal cardiac morphological structure and systolic function in vivo. Genetic or pharmacological inhibition of NSUN2 in mice triggers obvious pathological cardiac remodeling, manifested as ventricular wall thickening, enlarged left ventricular volume and impaired left ventricular systolic function, confirming the indispensable role of NSUN2 in sustaining cardiac physiological homeostasis.⁵¹

Abdominal Aortic Aneurysm (AAA)

AAA is a life-threatening vascular degenerative disease characterized by progressive aortic wall dilation, immune cell infiltration and extracellular matrix degradation. NSUN2 participates in AAA pathological progression via modulating endothelial-derived inflammatory signaling and immune cell recruitment. Mechanistically, NSUN2 upregulates the expression and extracellular secretion of endothelial autotaxin (ATX), a key pro-inflammatory mediator that modulates vascular immune microenvironment. Increased ATX secretion further facilitates the directional migration of T lymphocytes into aortic vessel walls. Excessive infiltration of T cells exacerbates local vascular inflammation, accelerates aortic medial layer destruction, and ultimately drives the initiation and progression of abdominal aortic aneurysm.⁵²

NSUN2 and Neurological Disorders

Compromised NSUN2 function is tightly correlated with the occurrence and progression of multiple neurodevelopmental defects and neurodegenerative diseases. Mechanistically, NSUN2 is indispensable for maintaining normal neuronal physiological functions, given its central role in governing neuronal RNA methylation homeostasis, RNA metabolic turnover and subsequent protein biosynthesis. The loss-of-function or aberrant expression of NSUN2 disturbs physiological RNA processing and translational machinery in neurons, which further triggers neuronal dysfunction and irreversible neurological lesions.

Intellectual Disability (ID)

Pathogenic variants of NSUN2 are well-established genetic drivers of autosomal recessive intellectual disability type 5 (MRT5), a rare congenital neurodevelopmental syndrome with highly consistent clinical manifestations across affected patients. Typical phenotypic features of MRT5 include global intellectual disability, primary microcephaly, proportional short stature, severe language developmental delay and distinctive craniofacial dysmorphism.^53,54 Of note, a novel homozygous frameshift mutation of NSUN2 (c.1171_1175delACCAT, p.Thr391fs18) has recently been documented in a Chinese pedigree harbouring two affected offspring, both of whom presented with moderate intellectual disability, microcephaly and prominent language impairment without additional atypical complications.⁵⁴ At the molecular level, NSUN2 deficiency abolishes physiological tRNA m⁵C modification, which disrupts tissue-specific translational profiling and destabilizes mitotic spindle assembly during cell division. Such molecular defects ultimately interfere with normal neural progenitor cell proliferation and division, laying a fundamental pathological basis for neurodevelopmental abnormalities in NSUN2-deficient individuals.^19,55

Neurodegenerative Diseases

Beyond developmental neurological disorders, NSUN2 dysfunction also contributes to the onset and progression of multiple neurodegenerative disorders via disturbing non-coding RNA modification, synaptic function and neuronal translational homeostasis. In Alzheimer’s disease (AD), neuronal NSUN2 depletion triggers a marked reduction in the m⁶A modification level of miR-125b, which further induces excessive tau hyperphosphorylation, a hallmark pathological change of AD. Conversely, exogenous NSUN2 overexpression is capable of alleviating tau-induced neurotoxicity and reversing relevant pathological damages partially.²⁶ In addition, NSUN2 insufficiency in mice disrupts physiological synaptic plasticity and further induces typical autism-like behavioural abnormalities, which is attributed to dysregulated protein synthesis dependent on aberrant RNA methylation landscape.⁵⁶ Mechanistically, NSUN2 loss facilitates angiogenin-dependent endonuclear tRNA cleavage. The resultant fragmented tRNA suppresses global cellular protein translation, activates multiple intracellular stress response cascades, and eventually triggers neuronal apoptosis and microcephalic phenotypes in vivo.⁵⁷

NSUN2 and Viral Infections

As a vital m⁵C RNA methyltransferase, NSUN2 participates in the modulation of host-virus interaction through its catalytic activity. Specifically, NSUN2-dependent m⁵C RNA methylation acts as a pivotal regulatory node governing both viral replication kinetics and host innate antiviral immune defence responses, which determines the final outcome of viral infection.

Hepatitis B Virus (HBV)

Chronic hepatitis B virus infection is a major risk factor for viral hepatitis, liver cirrhosis and HCC. NSUN2 exerts a pro-viral effect on HBV life cycle via direct m⁵C modification of viral transcripts. Mechanistically,NSUN2 targets and mediates m⁵C methylation at two conserved loci of HBV RNA, namely C131 and C2017. This epitranscriptomic modification substantially improves the stability of full-length HBV RNA, thereby facilitating robust viral replication. Intriguingly, HBV itself can hijack host epitranscriptomic machinery to amplify viral replication: the HBV core protein is capable of upregulating endogenous NSUN2 expression in host hepatocytes, which in turn further boosts HBV RNA methylation and replication, ultimately forming a positive feedback amplification loop between NSUN2 and HBV replication.⁵⁸ Consistent with these regulatory effects, genetic ablation of NSUN2 or site-directed mutagenesis of the two functional m⁵C modification sites markedly destabilizes HBV transcripts and suppresses viral replication across multiple validated experimental models, including HepG2-NTCP cell lines, primary human hepatocytes (PHHs), and heterozygous Nsun2 knockout C57BL/6JGpt mice.⁵⁸

Other Viruses and Protozoan Pathogens

Beyond HBV, NSUN2 also broadly modulates host innate antiviral immunity against diverse viral pathogens, and additionally regulates the developmental cycle of protozoan parasites through conserved m⁵C methylation machinery. In host antiviral immune cascades, NSUN2 targets interferon regulatory factor 3 (IRF3) mRNA for m⁵C modification. Such epigenetic modification accelerates the degradation of IRF3 transcripts, reduces IRF3 protein abundance and subsequently inhibits downstream interferon-β (IFN-β) production, ultimately dampening host type I interferon-mediated antiviral defence and favouring viral immune evasion.⁵⁹ In line with this immunosuppressive role, NSUN2 knockout activates robust host innate immune responses. Specifically, NSUN2 deletion elevates the level of non-coding RNAs transcribed by RNA polymerase III; these accumulated endogenous non-coding RNAs are further sensed by the pattern recognition receptor RIG-I, which potentiates type I interferon signalling and confers broad-spectrum inhibitory effects on the replication of both RNA viruses and DNA viruses.⁶⁰ Apart from its regulatory function in virus-host crosstalk, NSUN2 also participates in the life cycle regulation of Plasmodium. NSUN2-catalysed m⁵C methylation stabilizes Plasmodium mRNA at the post-transcriptional level, which is indispensable for sustaining normal gametocyte development of this malaria-causing parasite.⁶¹

NSUN2 and Inflammatory Diseases

As a key epitranscriptomic regulator bridging RNA modification and immune response, NSUN2 is critically involved in the initiation and amplification of systemic and local inflammatory responses. Accumulating evidence has demonstrated that NSUN2 exerts a predominant pro-inflammatory effect across multiple inflammatory diseases. It drives sustained inflammatory cascades mainly via modulating the activation and functional polarization of innate and adaptive immune cells, as well as modulating the transcription and secretion of pro-inflammatory cytokines, thereby exacerbating inflammatory tissue damage and disease progression.

Th17 Cell-Mediated Colitis

Aberrant expansion and hyperactivation of Th17 cells represent a core immunopathological hallmark of inflammatory bowel disease, particularly ulcerative colitis. NSUN2 aggravates intestinal inflammatory injury by facilitating Th17 cell differentiation and effector function via epitranscriptomic regulation. Mechanistically, NSUN2 forms a functional complex with retinoic acid receptor-related orphan receptor gamma t (RORγt), the master transcription factor governing Th17 cell lineage specification. This protein interaction enables targeted m⁵C methylation modification on mRNAs encoding Th17 signature pro-inflammatory cytokines, namely IL-17A and IL-17F. Such site-specific RNA methylation markedly increases the stability of these cytokine transcripts, augments their protein secretion, and further amplifies intestinal mucosal inflammatory responses. Collectively, the NSUN2-RORγt regulatory axis potentiates Th17-mediated immune inflammation and ultimately exacerbates the progression of experimental colitis.⁶²

Bladder Cancer-Associated Inflammation

Chronic inflammation within the tumor microenvironment serves as a critical driver of bladder malignant progression, in which tumor-associated macrophage polarization dominates immune landscape remodeling. NSUN2 remodels immunosuppressive microenvironment and accelerates bladder cancer development through modulating macrophage polarization via m⁵C-dependent post-transcriptional regulation. Mechanistically, NSUN2 mediates m⁵C methylation on suppressor of cytokine signaling 3 (SOCS3) mRNA. This epitranscriptomic modification impairs both the transcript stability and nucleocytoplasmic export of SOCS3, resulting in remarkable downregulation of endogenous SOCS3 expression. Given that SOCS3 acts as a canonical negative regulator of JAK2/STAT3 signaling, its depletion triggers persistent activation of the oncogenic JAK2/STAT3 pathway. The hyperactivated signaling cascade further promotes M2-type macrophage polarization. Functionally, M2 macrophages are potent immunosuppressive cells that restrain anti-tumor immune responses, remodel the tumor immune microenvironment into an immunosuppressive state, and consequently fuel the proliferation and malignant progression of bladder cancer.³⁹

NSUN2 and Other Diseases

Metabolic Diseases

Emerging evidence reveals that NSUN2-mediated m⁵C epitranscriptomic modification is also indispensable for maintaining systemic metabolic homeostasis, whose dysregulation contributes to glucose intolerance, insulin resistance and ectopic lipid deposition. NSUN2 governs adipogenesis, myogenesis and hepatic glucose-lipid metabolism through distinct downstream mRNA targets and reader proteins, exerting protective effects against metabolic disorders via two well-characterized regulatory axes.

NSUN2 modulates the balance between lipogenesis and myogenesis through m⁵C-dependent mRNA nuclear export regulation. Specifically, NSUN2 catalyzes m⁵C methylation on YBX2 and SMO mRNAs. The nuclear m⁵C reader protein ALYREF specifically identifies these methylated transcripts and promotes their efficient nucleocytoplasmic translocation. This post-transcriptional regulatory axis suppresses excessive fat biosynthesis and facilitates skeletal muscle generation, thereby maintaining normal muscle-adipose tissue homeostasis.⁶³ In liver metabolic regulation, NSUN2 exhibits beneficial effects on ameliorating metabolic dysfunction. Knockdown of NSUN2 markedly ameliorates impaired glucose tolerance and insulin resistance, and alleviates excessive hepatic lipid accumulation. From the molecular perspective, NSUN2-mediated m⁵C modification stabilizes ACSL6 mRNA to sustain its physiological expression level, which is essential for restraining hepatic steatosis and correcting systemic glucose metabolic disorders.²⁹

Corneal Diseases

Corneal epithelial wound healing is a highly coordinated biological process relying on normal proliferation and migration of human corneal epithelial cells (HCECs), and impaired epithelial repair contributes to persistent corneal epithelial defects and refractory ocular surface diseases. NSUN2 participates in corneal epithelial regeneration and wound repair via m⁵C-dependent translational regulation. Mechanistically, NSUN2 catalyzes m⁵C methylation on UHRF1 mRNA. The m⁵C reader protein ALYREF further recognizes this modified transcript and forms a regulatory complex with NSUN2, which jointly enhances the translational efficiency of UHRF1 mRNA. Functionally, genetic deletion of NSUN2 restrains the proliferative and migratory capacities of HCECs, thereby retarding the repair process of corneal epithelial wounds in vitro and in relevant preclinical models.⁶⁴

Therapeutic Implications and Future Directions

Collectively, NSUN2-mediated m⁵C RNA methylation serves as a pivotal epitranscriptomic mediator. It modulates disease initiation and progression across multiple systems, including cancers, cardiovascular diseases, neurological disorders, infectious diseases, inflammatory disorders, metabolic disorders and corneal diseases. Given its broad yet disease-specific regulatory functions, NSUN2 has emerged as a promising therapeutic target for human diseases. Current research efforts have focused on developing targeted interventions against the NSUN2-m⁵C regulatory axis, among which small-molecule enzymatic inhibitors, RNA interference-based gene silencing strategies, and rational combination therapeutic regimens represent three predominant and promising research directions for future clinical translation.

Small-Molecule Inhibitors

Cutting-edge progress in chemical probe design enables the development of highly selective small-molecule inhibitors targeting NSUN2 catalytic activity. Using cysteine-directed activity-based protein profiling (ABPP), researchers have identified azetidine-acrylamides as a novel class of NSUN2 inhibitors. These compounds function as stereoselective, irreversible covalent binders that target the conserved catalytic cysteine residue within NSUN2 active domain.⁶⁵ Functional assays demonstrate that such inhibitors effectively suppress NSUN2 methyltransferase activity and reduce global tRNA m⁵C modification levels, while exerting minimal cross-reactivity against other NSUN family homologues.⁶⁵ Apart from phenotypic screening, rational structure-guided drug design has also produced active-site-targeted NSUN2 inhibitors. Preclinical data reveal that this inhibitor suppresses NSUN2 enzymatic function, reduces the expression of its downstream target genes, and reverses multidrug resistance in anaplastic thyroid cancer.⁴⁷ Nevertheless, all currently available NSUN2 inhibitors remain confined to preclinical laboratory probes. Further structural modification to optimize pharmacokinetic properties and comprehensive in vivo efficacy and safety evaluation are urgently needed to facilitate their subsequent clinical translation.

Combination Therapies

Given the limited efficacy of single-agent NSUN2 inhibition in solid tumors, multiple preclinical studies have validated the therapeutic potential of NSUN2 blockade combined with cancer immunotherapy and conventional chemotherapy.

Combination with Cancer Immunotherapy

NSUN2 blockade exhibits prominent synergistic effects with immune checkpoint inhibitors (ICIs) by remodeling the tumor immune microenvironment. Pharmacological or genetic inhibition of NSUN2 downregulates programmed death-ligand 1 hepatocellular carcinoma (PD-L1) expression and promotes intratumoral infiltration of cytotoxic CD8⁺ T cells, thereby amplifying the anti-tumor immune response and enhancing the therapeutic efficacy of ICIs.^66,67 In HCC, concurrent inhibition of the GLUT1/NSUN2 metabolic axis using WZB117 plus PD-L1 checkpoint blockade produces synergistic anticancer effects. This combined treatment limits tumor growth and abolishes intratumoral immunosuppression.⁵⁰

Combination with Conventional Chemotherapy

NSUN2 upregulation is closely correlated with broad-spectrum chemoresistance across multiple malignancies. Targeted inhibition of NSUN2 reverses established chemoresistant phenotypes triggered by aberrant DNA damage repair, ferroptosis suppression and metabolic reprogramming, and restores tumor cell sensitivity to first-line chemotherapeutics including cisplatin, 5-fluorouracil and paclitaxel.^31,38,40 Accordingly, combinatorial administration of NSUN2 inhibitors and conventional chemotherapy holds great promise for improving clinical outcomes, particularly for malignant tumors characterized by high NSUN2 basal expression and intrinsic chemotherapy resistance.

Challenges and Future Perspectives

Although accumulating preclinical findings support NSUN2 as a viable therapeutic target, several unresolved bottlenecks still hinder the clinical translation of NSUN2-targeted therapies. Firstly, poor target specificity constitutes a major obstacle. The high conservation of catalytic domains among NSUN family homologues complicates the development of selective NSUN2 inhibitors, which inevitably raises the risk of off-target interference with other NSUN paralogs during clinical intervention.¹⁷ Secondly, universal NSUN2 inhibition may trigger systemic toxicities to normal tissues. Physiologically, NSUN2 is indispensable for maintaining normal cellular homeostasis, including stem cell stemness preservation and systemic metabolic modulation. Global pharmacological suppression of NSUN2 activity therefore disrupts essential physiological processes and leads to adverse effects.¹⁰ Thirdly, the context-dependent functional pattern of NSUN2 further limits its clinical application. Both the expression level and biological functions of NSUN2 exhibit obvious heterogeneity across different tumor types and individual patient cohorts. Such variability highlights the necessity of precise patient stratification guided by reliable predictive biomarkers before NSUN2-targeted treatment.⁶⁸

To address the above limitations and advance the clinical translation of NSUN2-targeted strategies, future research should focus on four core directions. Firstly, comprehensive profiling of disease-specific m⁵C epitranscriptomic landscapes is required to uncover tissue- and disease-specific downstream targets of NSUN2, which helps clarify its divergent roles across distinct pathological backgrounds. Secondly, further structural optimization is urgently needed to develop high-potency and highly selective NSUN2 inhibitors with improved pharmacokinetic properties, minimizing off-target effects and systemic toxicities to normal tissues. Thirdly, optimized multi-agent combination regimens deserve in-depth exploration. Rational triple-combination therapies integrating NSUN2 inhibitors, immune checkpoint inhibitors and conventional chemotherapy may achieve synergistic anti-tumor efficacy and overcome acquired therapeutic resistance. Lastly, large-scale prospective clinical trials should be conducted to validate the diagnostic and prognostic value of NSUN2 expression in multiple diseases, laying solid clinical evidence for biomarker-guided individualized treatment regimens.

Conclusions

NSUN2-mediated m⁵C methylation is a conserved post-transcriptional mechanism that regulates RNA stability, translation and non-coding RNA metabolism, thereby modulating cell cycle progression, immune homeostasis and cell apoptosis. This review summarizes the versatile roles of dysregulated NSUN2-m⁵C signalling across multiple human diseases, including cancers, cardiovascular, neurological, infectious, inflammatory and metabolic disorders. Distinct from passive pathological alterations, abnormal NSUN2 activity actively drives disease initiation, progression and therapeutic resistance across different tissue contexts. Although preclinical studies have uncovered key NSUN2-associated signalling pathways, several critical limitations still restrict the development of this field. Current research relies heavily on bulk transcriptomic analysis, lacking high-resolution cell-specific and dynamic m⁵C profiling data. Furthermore, the crosstalk between m⁵C and other common RNA epigenetic modifications remains poorly defined, and relevant clinical cohort evidence is still inadequate to support translational applications. Preclinical NSUN2-targeted therapies including small-molecule inhibitors and RNA interference have shown promising anti-disease effects, yet low drug specificity, systemic toxicity and context-dependent NSUN2 functions hinder further clinical translation. Future studies should integrate single-cell epitranscriptomic sequencing, optimized drug design and large-scale clinical validation to fill these research gaps. Elucidating the NSUN2-m⁵C regulatory network will advance epitranscriptomic mechanistic research and facilitate the development of novel diagnostic biomarkers and precision therapies.

Funding Statement

Natural Science Foundation for Higher Education Institutions in Jiangsu Province (grant 23KJB310004). Scientific Research Project of Jiangsu Provincial Health Commission (grant MQ2024046).

Author Contributions

XZ, SZ, and HG wrote the first manuscript. XZ, HG, PJ and LZ contributed to the literature search. PJ and LZ contributed to the design of the article structure, the exploration of innovative points, the revision and correction of drafts and funding. All authors made a significant contribution to the work reported whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas. All authors took part in drafting, revising or critically reviewing the article. All authors gave final approval of the version to be published. All authors have agreed to submit this article to the International Journal of General Medicine. All authors agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest in this work.