To date, the m⁵C methylation of RNA, including mRNA, rRNA and tRNA, is believed to be mainly mediated by two groups of RNA methyltransferases, DNMT2 and the NSUN protein family.

DNMT2, also known as TRDMT1 (tRNA methyltransferase 1), generally influences tRNA methylation (25). The m⁵C site is located on cytosine 38 in the anticodon loop of tRNA^Asp-GUC, tRNA^Gly-GCC, tRNA^Val-AAC (39, 40), and receives a methyl group from the cofactor S-adenosyl-methionine (SAM) (115). Studies showed that simultaneous knockout of DNMT2 and NSUN2 led to deficient tRNA methylation, protein synthesis and cellular differentiation, causing the death of experimental mice, although deficiency of either DNMT2 or NSUN2 alone did not show detectable effects. The results suggested that DNMT2 plays a role in tRNA methylation and cell survival (41). In addition, the role of DNMT2 in mRNA methylation and expression modulation was also reported. DNMT2 deficiency is associated with alterations in mRNA expression and methylation profiles and the inhibition of cell proliferation and migration (42).

The NSUN family also utilizes SAM as a methyl donor (116). The NSUN family consists of seven members, NSUN1-7, and each target different types of RNAs. The RNA targeting specificity of the NSUN family was reviewed in 2019 by Katherine E. Bohnsack et al., with NSUN1, 4, and 5 responsible for rRNA methylation, NSUN2, 3, and 6 responsible for tRNA methylation (NSUN2 also promotes mRNA methylation), and NUSN7 responsible for enhancer RNA (eRNA) methylation (25). However, within the last three years, studies have provided new insights in this regard. Among the seven RNA methyltransferases, NSUN2 was the first discovered and most widely studied. The role of NSUN2 in promoting tRNA methylation is wellknown, and NSUN2 deficiency directly causes a decrease in tRNA m⁵C levels (47). Notably, NSUN2 is predominantly distributed in the nucleus and catalyzes methylation of cytoplasmic tRNA; it is also capable of introducing m⁵C to mitochondrial tRNA (48, 49). However, NSUN2 silencing did not significantly affect mitochondrial tRNA stability (49), which suggested that NSUN2 may not be necessary for tRNA methylation within the mitochondria. NSUN2 is also responsible for biological processes, including cell proliferation (50) and carcinogenesis. Enhanced levels of NSUN2 and NSUN2-mediated m⁵C are observed in patients with gastric cancer (GC) (30, 34, 51), esophageal squamous cell carcinoma (ESCC) (52, 53), hepatocellular carcinoma (HCC) (35, 54), hypopharyngeal squamous cell carcinoma (HPSCC) (55), prostate cancer (56), cervical cancer (57), nasopharyngeal carcinoma (58) and uveal melanoma (59). NSUN2 also affects immune cells, as hyperexpression of NSUN2 in T cells promotes IL-17A secretion by methylating IL-17A mRNA at cytosine C466 both in vitro and in vivo, which stimulates its translation (60). In addition, under conditions of oxidative stress-induced cellular senescence, NSUN2-mediated m⁵C, together with METTL3/METTL14-mediated m⁶A, synergistically upregulates the expression of p21 (61). Note that NSUN2 is also involved in the functioning of m⁵C readers, including ALYREF, whose nuclear-cytoplasm transportation is partly modulated by NSUN2 (62). Other members of the NSUN family are also active in catalyzing m⁵C RNA modifications. NSUN1, or NOP2 (nucleolar protein 2), catalyzes rRNA m⁵C modifications, thus affecting biological processes including ribosome biogenesis (43), cell proliferation (44), healthspan modulation (45) and HIV-1 viral latency (46). NSUN3, a putative tRNA methyltransferase, plays a role in tumor progression (27, 63, 64), immune cell infiltration (64, 66) and multisystem mitochondrial diseases (67). Mechanistically, m⁵C modification of tRNA occurs at C34 in the anticodon loop (65, 117). Current studies have revealed that NSUN3 expression is upregulated in patients with low-grade glioma (63) and head and neck squamous cell carcinoma (HNSCC) (64), and NSUN3-mediated m⁵C modification of tRNA enhances metastasis by stimulating the translation of mitochondrial mRNA (27). NSUN3-associated immune cell infiltration mainly includes CD8+ T cells (66) and M2 macrophages (64). Deficiency of NSUN3 also leads to severe dysfunction within the mitochondria, such as combined oxidative phosphorylation deficiency, which may lead to early-onset encephalomyopathy and seizures (67). NSUN4 facilitates mitoribosomal assembly by methylating C911 in 12S rRNA and interacting with MTERF4 (mitochondrial transcription termination factor 4) (68). In addition to rRNA methylating activity, NSUN4 also acts as a tRNA (69) and mRNA (70) methyltransferase, and NSUN4-mediated m⁵C modification in the 3’-UTR (3’-untranslated region) of SOX9 (SRY-box transcription factor 9) mRNA is necessary for adaptation to higher temperatures (69) and chondrogenic differentiation regulated by SOX9 (70). NSUN4 also promotes HCC generation (71, 72) and neutrophil infiltration (66). NSUN5 participates in rRNA methylation, introducing m⁵C3782 into human and m⁵C3438 into mouse 28S rRNA (73, 74). Overexpression of NSUN5 is associated with tumorigenesis in HCC (75) and colorectal cancer (CRC) patients (76), while NSUN5 deficiency causes a reduction in total protein synthesis, thus impairing cell proliferation (73). In patients with tetralogy of Fallot (TOF) (118) and William’s-Beuren syndrome (WBS) (119), NSUN5 is drastically downregulated. Previous studies regarded NSUN6 as at RNA methyltransferase, which identifies C72 at the 3′ end of the tRNA acceptor stem and targets tRNA^Cys and tRNA^Thr (77, 78), but recent investigations have discovered that NSUN6 exhibits mRNA methylating bioactivity (79, 82, 120, 121). mRNA methylated by NSUN6, which primarily targets the 3’-UTR at the consensus sequence motif CTCCA, increased in transcript and protein levels (79). The role of NSUN6 in cancer development remains unclear, but studies have shown that NSUN6 acts as a protective factor against triple-negative breast cancer (TNBC) (36), pancreatic cancer (31), testis cancer (79), thyroid cancer (79) and ovary cancer (79) but is a risk factor for CRC (80, 81). One possible explanation of the controversial role of NSUN6 in different types of cancers is that NSUN6 expression level in different immune cells within the TME differs based on the tumor context. For instance, NSUN6 is mainly expressed in Tregs in TNBC (36), but in exhausted CD8+ T cells, proliferating T cells and myofibroblasts in CRC (80). In addition, NSUN6 is also related to the promotion of the cell cycle (82), infiltration of B cells, CD4+ T cells and CD8+ T cells (36, 80), and formation of antibody-secreting plasma cells (83). NSUN7 methylates eRNA, a noncoding RNA associated with transcription modulation, and enhances the expression of mRNAs coding for Pfkl, Sirt5, Idh3b and Hmox2 in a peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α)-dependent manner. These effects are likely to facilitate adaptive metabolic alterations under starvation (84).

Readers, or binding proteins of m⁵C sites, include ALYREF and YBX1. ALYREF is reported to be an important oncogenic factor and is associated with poor prognosis in patients with various types of cancer, including HCC (72, 85, 86), glioblastoma (87, 88), glioma (63), neuroblastoma (89), lung adenocarcinoma (90, 91), HNSCC (92, 93), bladder cancer (32) and breast cancer (94, 95). For example, elevated levels of ALYREF in HCC patients were found to be responsible for upregulated eIF4A3 expression (86) and abnormal cell cycle and mitosis (72). In lung adenocarcinoma patients, ALYREF, together with NSUN2, promotes m⁵C modification of YAP (Yes-Associated Protein) mRNA in the 3’-UTR, thus increasing the stability of YAP mRNA and causing enhanced exosome secretion, tumor malignancy and drug resistance (91). In contrast, ALYREF is considered a protective factor against colon adenocarcinoma (81), but the mechanism remains to be elucidated. In addition, ALYREF also participates in the regulation of adipogenesis (96), myogenesis (96, 97) and retrovirus replication (98) and may have biological activity in the context of abdominal aortic aneurysm (AAA) (99).

YBX1 is another m⁵C reader that has multiple functions in cancer development and embryo development. Oncogenic effects of YBX1 are found in GC (51), bladder cancer (100), glioblastoma (101), CRC (76, 102), cholangiocarcinoma (103), clear cell renal cell carcinoma (ccRCC) (104), prostate cancer (56), epithelial ovarian cancer (105) and cervical cancer (57), mainly owing to YBX1-RNA interactions that have stabilizing effects on target RNAs. For example, in GC patients, YBX1 binds with FOXC2 (Forkhead box protein C2) mRNA, which is m⁵C-modulated by NSUN2, to enhance its tumor-promoting ability (51). In bladder cancer patients, YBX1 stabilizes oncogenic HDGF (heparin binding growth factor) mRNA by targeting the m⁵C-modified site on its 3′-UTR (100). Interestingly, in addition to its oncogenic effects, YBX1 is also important in normal cell proliferation and embryo development. For instance, animal experiments revealed that YBX1 is essential for embryonic brain development in mice (101), and YBX1 deficiency causes early gastrulation defects in zebrafish embryos (106).

Currently discovered m⁵C demethyltransferases are the TET family and ALKBH1. The TET family is a group of Fe(II) and alpha-ketoglutarate-dependent m⁵C dioxygenases that convert 5-methylcytosine (m⁵C) to 5-hydroxymethylcytosine (hm⁵C) (122). These enzymes were originally discovered in the translocation breakpoint of t(10;11) in patients with infant acute myeloid leukemia (AML), hence the name (123). Overall, the scarcity of research focusing on m⁵C erasers limits the comprehensive understanding of these proteins. TET1-mediated mRNA m⁵C demethylation is essential for completion of DNA repair and survival of cells in the context of DNA damage (107). TET2 mainly facilitates the conversion of m⁵C into hm⁵C, thus leading to the elimination of m⁵C modification in RNA (108, 109), but the effects are not as strong as ALKBH1 (109). TET2 expression was measured in patients with various types of cancers, and it was upregulated in low-grade glioma patients (63) but downregulated in ccRCC (110, 111), ovarian cancer (112) and prostate adenocarcinoma (113) patients. Note that the potential tumor-suppressive effect of TET2 in prostate adenocarcinoma is likely to be linked with enhanced immune cell infiltration (113). TET3-mediated m⁵C elimination has not been clarified to date. However, some researchers have reported that upregulated TET3 expression in prostate cancer patients might be associated with poor prognosis (38).

ALKBH1 has been identified as a demethyltransferase for both RNA and DNA (124), but most studies focused on ALKBH1-demethylated DNA modifications, with fewer researchers concentrating on the RNA part. So far, studies have revealed that ALKBH1 takes part in the transformation of m⁵C RNA modifications to either hm⁵C or f⁵C (5-formylcytidine) RNA modifications (109, 114). More specifically, in cytoplasmic tRNA, ALKBH1 targets the wobble position (position 34) of tRNA^Leu-CAA and converts m⁵C RNA modifications to hm⁵C or f⁵C, promoting the decoding of Leu codons under stress (109, 114). At the same position (position 34) of mitochondrial tRNA^Met, only the alteration from m⁵C to f⁵C was found, which was proved indispensable for the translation of AUA, a non-universal codon in mammalian mitochondria, indicating that ALKBH1-mediated m⁵C RNA modification removal is significant for mammalian mitochondrial functions (114). Interestingly, an in vitro experiment showed that ALKBH1 first hydroxylates m⁵C to form hm⁵C, and then oxidizes hm⁵C to form f⁵C, meaning ALKBH1-mediated biogenesis of hm⁵C and f⁵C is actually two relevant and coherent processes (114).

Previous studies have demonstrated a clear relationship between high m⁵C levels and HCC development, migration and malignancy. Recent studies have mainly focused on lncRNAs. For example, during HCC, the expression levels of m⁵C-associated genes, including NSUN2 (35, 54), NSUN4 (71), NSUN5 (75) and ALYREF (86), increase. NSUN2-mediated m⁵C modulation of H19 lncRNA increases its stability, leading to enhanced recruitment of the G3BP1 (Ras-GTPase-activating protein-binding protein 1) oncoprotein, a potential enhancer of MYC accumulation (35). NSUN2 also modulates the Ras signaling pathway as well as the cell cycle, thus allowing for tumor escape from chemotherapy (54). A bioinformatics analysis discovered that NSUN5 overexpression was positively associated with enhanced ribosome functions and protein translation within HCC cells (75). ALYREF dysfunction is responsible for aberrant cell cycle regulation and mitosis of HCC cells (72) and promotes HCC possibly via stimulation of eIF4A3 expression (86). Thus, suppressors for ALYREF and eIF4A3, such as miR-4666a-5p and miR-6124, are promising therapeutic agents (86). Moreover, m⁵C modulation of circRNA is also important in HCC development (125). In addition, alterations in the tumor microenvironment (TME) and immune cell infiltration also contribute to m⁵C-mediated HCC development (135, 136). Currently, researchers are investigating new methods for prognosis prediction in HCC patients and constructed speculating models based on m⁵C-related modulators, such as the NSUN family, TET1, and YBX1 (72, 137, 138). These findings may have profound clinical implications.

The role of m⁵C RNA modification in GC is generally oncogenic, with high levels of m⁵C indicating poor prognosis and a low overall survival (OS) rate (126). Modifications of both mRNA (30, 34, 51) and lncRNA (126, 139) have been observed during GC progression, and risk models based on m⁵C levels were developed for prognosis prediction (139). NSUN2 is the main oncogenic m⁵C-methyltransferase in GC, targeting the mRNAs of PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1) (34), PCYT1A (phosphate cytidylyltransferase 1 choline-alpha) (34), FOXC2 (Forkhead box protein C2) (51) and p57^Kip2 (a type of cyclin-dependent kinase (CDK) inhibitor) (30). After m⁵C modulation and binding to m⁵C readers, such as YBX1, the transcriptional activity of PIK3R1, PCYT1A and FOXC2 mRNA was increased, while tumor-suppressive p57^Kip2 mRNA was destabilized as a result of m⁵C modulation in the 3’-UTR. Consequently, the elevated NSUN2 levels in GC patients lead to enhanced proliferation, migration, and invasion of cancerous cells. NSUN2 activators, such as small ubiquitin-like modifier (SUMO)-2/3, which directly interact with NSUN2 to stabilize and mediate its nuclear transport, promote the development of GC (34). The oncogenic interaction between NSUN2 and FOXC2 mRNA can be facilitated by lncRNA FOXC2-AS1 (FOXC2 antisense RNA 1) (51). The oncogenic role of m⁵C modulation may also be linked to immune suppression, as patients with lower levels of m⁵C modulation were found to have higher levels of immune activation and longer progression-free survival (PFS) and OS (126).

m⁵C-mediated cell proliferation is considered one of causes of bladder cell malignancy. Overexpressed ALYREF in bladder cancer cells interacts with the 3’-UTR of PKM2 (pyruvate kinase M2) mRNA, causing its stabilization and enhanced PKM2-associated glycolysis (32). ALYREF stimulators, such as hypoxia‐inducible factor‐1alpha (HIF‐1α), significantly increase the expression levels of ALYREF and PKM2 and are correlated with poor prognosis (32). Another m⁵C reader, YBX1,maintains the stability of its target mRNA, oncogenic HDGF mRNA methylated by NSUN2, by forming a 3’-UTR-binding complex with ELAVL1 (ELAV like RNA binding protein 1) (100). Moreover, high expression of immune cells, including regulatory T cells (Tregs), CD8+ T cells, plasma cells and activated dendritic cells, is related to a good prognosis, while high expression of resting CD4+ memory T cells, M0 macrophages, M1 macrophages, M2 macrophages and neutrophils show the opposite trend (140).

Several prognostic models based on m⁵C modulators (38) or m⁵C-related lncRNAs (141) have been developed for prostate cancer patients. Specifically, experimental results showed that increased NSUN2 (56), YBX1 (56) and TET3 (38) levels correlate with a poor prognosis. Posttranscriptional m⁵C modification of androgen receptor (AR) mRNA by NSUN2 is recognized by YBX1, increasing AR mRNA stability and translation (56). Interestingly, AR positively regulates NSUN2 at the transcriptional level (56), forming a reciprocal activation loop. High NSUN2 expression is also associated with low chemotherapeutic sensitivity and immune cell infiltration (37). In addition to NSUN2, immune cell infiltration characteristics are associated with many other m⁵C regulators, such as NSUN6 and TET1-3 (38, 113).

RNA m⁵C modifications affect tumor malignancy and drug resistance not only in solid tumors but also in nonparenchymal tumors, including leukemia. In 2018, David G. Courtney et al. determined that NSUN1 is partly responsible for the formation of the 5-AZA (5-azacitidine)-insensitive chromatin structure during leukemia, which causes drug resistance (127). Mechanistically, NSUN1 forms an active chromatin structure with BRD4 (bromodomain-containing protein 4) and RNA-polymerase-II, which responds poorly to 5-AZA but well to the BRD4 inhibitor JQ1 or miRNA targeting NSUN1. In contrast, another two m⁵C regulators, NSUN3 and DNMT2, bind directly with heterogeneous nuclear ribonucleoprotein K (hnRNPK), a conserved RNA-binding protein that interacts with the lineage-determining transcription factors GATA binding protein 1 (GATA1), recombinant spleen focus forming virus proviral integration 1/purine rich box-1 (SPI1/PU.1) and cyclin-dependent kinase 9 (CDK9)/positive transcription elongation factor b (P-TEFb) to recruit RNA-polymerase-II to RNA precursors, forming chromatin structures that are sensitive to 5-AZA (127).

HNSCC refers to a group of epithelium-derived cancers that occur in the mucosal surfaces of the head and neck, including the oral and nasal cavity, oropharynx, nasopharynx, larynx and hypopharynx. To date, statistics have revealed that almost all m⁵C regulators show elevated expression levels during HNSCC, with the exception of NSUN7 and TET2 (93, 142, 143), suggesting that they play different roles in HNSCC tumorigenesis. Mechanistically, NSUN3 promotes tumor progression by regulating immune infiltration (64), and ALYREF enhances mitochondrial activity and intracellular energy metabolism, which ensures continuous energy supplies for timorous tissues (92). In nasopharyngeal carcinoma (NPC) specifically, NSUN2 negatively regulates immune cell infiltration in the TME (58). In addition, NSUN2 promotes hypopharyngeal squamous cell carcinoma (HPSCC) by m⁵C-methylating oncogenic TEAD1 (TEA domain transcription factor 1) mRNA, which upregulates its expression level (55).

m⁵C RNA modification has dual effects on breast cancer development. Recent studies suggest NSUN2 (36), ALYREF (94, 95) and DNMT3B (95) as risk factors, while NSUN5 (128), NSUN6 (36), TET2 (128), and DNMT2 (128)are protective factors. Mechanistically, ALYREF, which was found amplified both at the mRNA and protein levels, binds with the oncogenic NEAT1 lncRNA promoter region, enhancing its transcription (94). Additionally, enrichment analysis revealed that vascular endothelial growth factor A (VEGFA) and enhancer of zeste homolog 2 (EZH2) were potential targets of DNMT3B (95). In addition, NSUN5, TET2, and DNMT2 modified three lncRNAs, namely, AP005131.2, AL121832.2, and LINC01152, to be protective factors against breast cancer (128).

As in many other types of cancers, NSUN2 regulates pancreatic tumorigenesis and epithelial differentiation through mRNA methylation (129). In contrast, NSUN6 (31) and DNMT3A (131) have inhibitory effects on pancreatic cancer and suppress the proliferation of cancerous cells, but the mechanisms remain to be elucidated. m⁵C modification profoundly influences the tumor immune microenvironment (130, 144), interfering with the infiltration of CD8+ T cells and upregulating PD-L1 expression (130). Risk models based on m⁵C-related lncRNAs have also been constructed to provide prognostic information (145).

m⁵C modification has dual effects on ccRCC development. YBX1 negatively modulates ccRCC by binding and stabilizing PEBR1 mRNA, a tumor suppressor gene (104). Other m⁵C-related genes, such as DNMT3B, NSUN1, NSUN2 and NSUN5, are highly expressed in ccRCC patients and correlate with worse prognosis (110, 111, 132), while NSUN6 and TET2 mainly function as protective factors (111). Notably, the role of NSUN4 in ccRCC remains controversial, as studies have obtained opposing results (110, 111).

In CRC patients, increased levels of m⁵C-related regulators, such as NSUN5, NSUN6, ALYREF and YBX1, were found (76, 81). The m⁵C levels of peripheral blood immune cells showed higher CRC diagnostic value than that of common blood tumor biomarkers (76), which is correlated with the discovery that m⁵C modification inhibits tumor infiltration of immune cells (133). In cholangiocarcinoma patients, the m⁵C-modified functional lncRNA NKILA (NF-kappa B interacting lncRNA), which is recognized and stabilized by YBX1, is associated with advanced TNM stage and poor prognosis (103). In glioma patients, m⁵C-associated genes, including NSUN1-5, NSUN7, DNMT1, DNMT3B and YBX-1, are upregulated, with the exception of NSUN6 (63, 101, 134). In patients with glioblastoma, the most aggressive diffuse glioma, upregulated ALYREF plays an oncogenic role by activating the Wnt/β-catenin signaling pathway and stabilizing MYC mRNA (87, 88). Interestingly, MYC also exerts positive impacts on ALYREF, forming a positive feedback loop (87). In neuroblastoma patients, the m⁵C reader ALYREF forms a nuclear coactivator complex with MYCN to stimulate USP3 transcription, which promotes the tumorigenesis of neuroblastoma (89). In lung adenocarcinoma patients, NSUN2 and ALYREF were found to be oncogenic through interacting with YAP mRNA. The m⁵C modification in the 328-331 3’-UTR of YAP mRNA increases its stability, enhances exosome secretion, and stimulates the transcription of seven downstream exosome-promoting genes. Together, m⁵C-mediated YAP stimulation leads to increased tumor malignancy and drug resistance (91). In addition, risk models based on m⁵C regulators or m⁵C-related lncRNAs were also developed for prognosis prediction (11, 90, 146). In patients with ESCC, NSUN2 promotes ESCC progression and chemoresistance by promoting TIGAR (TP53 induced glycolysis regulatory phosphatase) (53) and GRB2 (growth factor receptor bound protein 2) (52). The positive influence on GRB2 is achieved by NSUN2-mediated LIN28B-dependent m⁵C modification of GRB2 mRNA, which indirectly activates the PI3K/AKT and ERK/MAPK signaling pathways (52). In cervical cancer patients, NSUN2 and YBX1, which catalyze and recognize methylation sites, respectively, induce KRT13 mRNA methylation and translational activation (57). In ovarian cancer patients, YBX1 modulates the expression of a variety of downstream targets, including CD44, thus enhancing chemoresistance (105). In contrast, RNA m⁵C modification mediated by the methyltransferase NSUN6 suppresses testis, thyroid and ovary cancers (79). Finally, NSUN2-mediated RNA m⁵C modification modulates uveal melanoma cell proliferation and migration, although the exact mechanisms remain unknown (59).

Subgroups of T lymphocytes (mainly CD4+ and CD8+ T cells) are distinguished in T cells by surface markers using flow cytometry (154). Upon leaving the thymus, naïve CD4+ T cells further differentiate into T cell subsets according to different stimulating signals, such as T helper (Th) cells (e.g., Th1, Th2, Th9, Th17, Th22), T follicular helper (Tfh) cells and regulatory T cells (Tregs) (155).

Recent studies have mainly focused on the m⁵C RNA modification of T cells in the context of cancer. For instance, the abundance of CD4+ T cells is higher in patients with soft tissue sarcoma (STS) (149), ccRCC (110) and HCC (148) but the opposite is found in glioma patients (156). Tregs appear to be slightly different from common CD4+ T cells, as they correlate with poor prognosis in STS (149) and HCC (138) patients and positive outcomes in HNSCC (147) and bladder cancer (140) patients. The role of CD8+ T cells in cancer is also controversial since they are considered protective factors in bladder cancer (140) and lung adenocarcinoma (157) patients but risk factors in ccRCC (110) patients.

For CD4+ T cells, associated m⁵C-related regulators include NSUN1 (46), NSUN2 (37, 60, 151, 152), DNMT3A (80), TET1 (150) and TET2 (150). Downregulated NSUN2 expression, along with decreased mRNA m⁵C levels of CD4+ T cells, was observed in systemic lupus erythematosus (SLE) patients, while the number of m⁵C-containing RNAs increased. In addition, m⁵C sites were mainly distributed in mRNA translation initiation sites, and hypermethylated m⁵C and/or upregulated genes in SLE were enriched immune-related and inflammatory pathways, including immune system signaling pathway, cytokine signaling pathway, and interferon signaling pathway (151). In HIV-1-infected CD4+ T cells, NSUN2, as the primary HIV-1 m⁵C methyltransferase, facilitates HIV-1 transcript methylation as well as viral replication (152). Note that NSUN2 inactivation did not reduce HIV-1 mRNA expression levels but did downregulate protein expression, suggesting the role of m⁵C in HIV-1 translation. Additionally, m⁵C loss dysregulates the alternative splicing of viral RNAs (152). In contrast, NSUN1 deficiency caused latently infected HIV-1 proviruses to reactivate, revealing the viral suppressive effects of NSUN1 (46). Moreover, NSUN2 enhances IL-17A secretion by T cells by methylating IL-17A mRNA at C466, stimulating its translation (60). Finally, the m⁵C erasers TET1 and TET2 regulate pre-mRNA splicing in a CCCTC‐binding factor (CTCF)-dependent manner.

Moreover, m⁵C-related regulators affecting CD8+ T cells include NSUN3, NSUN6, TET1 and TET3 (38, 66, 80), but no further studies were found.

B cells are derived from hematopoietic stem cells (HSCs) in the bone marrow. Naïve B cells, once properly activated, mature into plasma cells, the antibody-secreting form of B cells, following an intrinsic developmental process (158). Recent studies on B cells mainly concentrated on alterations in m⁵C RNA modifications during tumor pathology. In HNSCC patients, a higher number of naïve B cells is negatively correlated with the risk score for poor prognosis (147). Activated plasma cells exert similar effects in bladder cancer (140) and lung adenocarcinoma (157) patients. Also, a prognostic model for pancreatic cancer based on m⁶A/m⁵C/m¹A-associated lncRNAs showed that the low-risk group has a significantly higher concentration of naïve B cells and plasma cells within the TME (159), suggesting the protective role of B cells and plasma cells. Genes associated with m⁵C RNA modifications in B cells include NSUN2, NSUN6, DNMT3A, TET1 and TET3 (37, 38, 80). Specifically, although NSUN6 is dispensable for germinal center (GC) B-cell formation, it plays vital roles in the formation of antibody-secreting plasma cells (83).

Monocytes and macrophages stem from hematological precursors in the bone marrow and are important in the innate immune system due to their phagocytic and antigen-presenting activity (160). Monocytes accumulate in peripheral blood, while macrophages are tissue-resident mature monocytes (161). Classically activated macrophages, or M1 macrophages, are proinflammatory, while alternatively activated macrophages, or M2 macrophages, are anti-inflammatory (162). Similar to T cells and B cells, most studies on m⁵C RNA modifications in macrophages are in the context of cancer development, especially macrophage infiltration in the TME. The risk scores based on m⁵C-related genes of patients with four types of cancer (HCC, HNSCC, glioma and pancreatic cancer) were positively correlated with infiltration of resting macrophages (M0), M1 or M2 macrophages (138, 147, 148, 156, 159). Analysis of the TME in prostate cancer patients showed differentially expressed NSUN6, TET1 and TET3 in M1 and M2 macrophages (38). NSUN3 promotes infiltration of M2 macrophages but suppresses M1 macrophage infiltration in HNSCC patients (64). Moreover, in AAA patients, ALYREF-interacting lncRNAs are involved in immune system processes and macrophage infiltration (99).

Granulocytes refer to a group of leukocytes with specific cytoplasmic granules distinguished by Romanowsky staining into three main subsets, namely, neutrophils, eosinophils and basophils (163). Currently, the scarcity of research focusing on m⁵C RNA modification of eosinophils and basophils makes it difficult to conclude m⁵C-related alterations in these two types of granulocytes. Only in lung adenocarcinoma patients is eosinophil infiltration correlated with a favorable prognostic pattern (157). Neutrophil abundance is generally associated with poor prognosis (138, 147), and a study on lung squamous cell carcinoma patients discovered that NSUN4 exerts a regulatory effect on neutrophil m⁵C RNA modification (66). Studies have also reported that TET2 and TET3 influence neutrophil granulation, phagocytosis and cytokine signaling by demethylating and destabilizing socs3b (suppressors of cytokine signaling 3b) mRNA, a member of the suppression of cytokine signaling gene family (153). TET2/3-defective embryos showed aberrant granule formation, defective phagocytosis and dysregulation of cytokine signaling in neutrophils due to accumulation of socs3b mRNA, which binds the Jak receptor to prevent Stat phosphorylation and downstream signaling via the Jak/Stat pathway (153).

Natural killer (NK) cells constitute a first line of innate immunity against tumors due to their capabilities of killing aberrant cells (164). Regarding m⁵C RNA modifications in NK cells, both resting and activated NK cells are correlated with positive outcomes in HNSCC (147), glioma (156) and lung adenocarcinoma (157) patients, with NSUN2 being the most closely associated m⁵C gene (37).

Dendritic cells (DCs) are generally regarded as the most potent antigen-presenting cells, thus modulating both immunity and tolerance (165). In lung adenocarcinoma patients, more DC infiltration was discovered in the low-risk group, suggesting the protective role of m⁵C in DCs (157). In contrast, a prognostic model for pancreatic cancer based on m⁶A/m⁵C/m¹A-associated lncRNAs showed that the high-risk group has a significantly higher concentration of activated DCs within the TME (159), showing the tumor-promoting effects of activated DCs. The conflicting results might result from the differences in cancer types, DC subtypes, activation extent and patient characteristics. Consequently, rigorous future studies are needed in this regard. In CRC patients, the m⁵C writer DNMT3A was found to be involved in regulating DCs (80).

Mast cells are tissue-resident cells that function in inflammatory responses and tissue homeostasis (166). These cells are usually recognized clinically for their roles in IgE-mediated degranulation and allergic inflammation (167). A risk model based on m⁶A/m¹A/m⁵C-regulated genes in HCC patients indicated the links between poor prognosis and high infiltration of resting mast cells (148).

In summary, the roles of m⁵C RNA modification of immune cells in the context of tumorigenesis largely remain to be further clarified. A better understanding of the mechanisms by which alterations in cell expression, infiltration and activation are regulated by methylation can be extremely helpful for the development of novel methods for tumor diagnosis as well as treatment.