Open research questions in RNA modifications and cancer

What the literature leaves open

• Abstract Lenvatinib has been approved by the FDA as a front-line treatment for advanced hepatocellular carcinoma (HCC), but its survival benefits are limited by acquired drug resistance.

  Tumor-derived annexin A1 drives lenvatinib resistance by regulating SOX2/STAT3/S100A6 feedback loop and M2 macrophage polarization in hepatocellular carcinoma · 2026 · DOI

• Significance StatementEpitranscriptomic studies on TNBC predominantly focus on highly metastatic models, leaving less aggressive subtypes poorly understood.

  METTL3 modulates cell viability and motility in HCC1143 and MDA-MB-231 triple-negative breast cancer cells · 2026 · DOI

• The maternal-to-zygotic transition (MZT) requires coordinated clearance and deadenylation of maternally deposited mRNAs, yet the underlying molecular mechanisms remain poorly understood.

  Single-molecule m6A profiling reveals position-dependent mRNA regulation and non-canonical roles for Ythdf2 in early embryogenesis · 2026 · DOI

• One avenue garnering attention is targeting epigenetic regulators that promote AR-activity; however, the importance of epitranscriptomic regulators, like those that modify mRNAs, is not well understood.

  A non-enzymatic role for METTL3 as an Androgen Receptor co-regulator that promotes prostate cancer proliferation. · 2026 · DOI

• Challenges in tRNA profiling The exploration of the molecular mechanisms underlying the nuclear export pathways of tRNA represents a critical area for future research in cancer biology. Recent studies have highlighted the significance of tRNA modifications and their roles in various cellular processes, including translation regulation and stress responses (152). Understanding how tRNAs are exported from the nucleus to the cytoplasm is essential, as this process is tightly linked to protein synthesis and cellular growth, both of which are dysregulated in cancer. For instance, the nuclear basket proteins Mlp1 and Mlp2 have been identified as key regulators of tRNA export, and their expression levels have been correlated with cancer prognosis (48). Future research should focus on elucidating the detailed molecular interactions and regulatory networks involved in tRNA nuclear export, particularly in the context of tumorigenesis. Investigating the impact of oncogenic signaling pathways on tRNA export mechanisms may reveal novel therapeutic targets that can be exploited to disrupt the aberrant protein synthesis that characterizes many cancers. Additionally, the potential role of tRNA-derived fragments (tRFs) in modulating these pathways should be further explored, as their dysregulation has been implicated in cancer progression (63). Several specialized sequencing approaches have been developed to profile tRNAs and tsRNAs, including hydro-tRNA-seq, DMtRNA-seq, ARM-seq, YAMAT-seq, and mim-tRNA-seq. These methods were designed to address technical barriers that limit standard RNA-seq-based tRNA quantification. The compact secondary structure of mature tRNAs can reduce reverse-transcription efficiency and introduce library-construction bias. In addition, abundant post-transcriptional modifications, such as m1A and m3C, can cause reverse-transcription stops or misincorporation leading to inaccurate quantification. Further challenges events, include incomplete removal of modification-induced biases, mapping ambiguity among highly similar tRNA genes and isodecoders, and difficulty distinguishing mature tRNAs from tRNA-derived fragments. Therefore, differential tRNA expression data should be interpreted with caution, particularly when comparing datasets generated using different sequencing or preprocessing strategies. 7.2 Precision therapeutic strategies Furthermore, the development of precision therapeutic strategies based on the regulation of tRNA nuclear export holds great promise for cancer treatment. As the understanding of tRNA modifications and their influence on translation efficiency expands, researchers can begin to design targeted therapies that specifically modulate these pathways. For example, targeting the enzymes responsible for tRNA modifications, such as METTL1, has been shown to influence cancer cell proliferation and survival.

  Transfer RNA expression, modification, and derived small RNAs in cancer biology and clinical potential · 2026 · DOI

• These findings provide new mechanistic insight into APM-induced HCC, nominate actionable diagnostic/therapeutic targets, and furnish evidence to help refine APM’s carcinogenic classification, although experimental validation remains to be completed.

  Aspartame drives the continuous progression from MASLD to HCC · 2026 · DOI

• While histidine is an essential amino acid in humans, cancer-related pathways that increase histidine demand have not been described. However, their involvement in amino acid substitutions had not been previously investigated [29-32].

  Histidine to glutamine substitutants, a regulated process that impacts cell survival and suggests histidine shortage in cancer · 2026 · DOI

• Abstract Background Parkinson’s disease (PD) is featured by progressive neurodegeneration linked to iron-dependent ferroptosis, yet the functions of m 6 A RNA-binding proteins and deubiquitinating enzymes in this process remain poorly understood.

  Inhibition of YTHDF2-mediated CYLD mRNA degradation promotes neuronal ferroptosis and pain in Parkinson's disease through NOX4 deubiquitination · 2026 · DOI

• Many studies have implicated that tRNA‐derived fragment, tiRNAs, in a variety of biological processes, but their roles in hypoxia‐induced PASMC ERS and proliferation have not been investigated.

  A Specific <scp>tRNA</scp> Half, 3' <scp>tiRNA</scp> ‐ <scp>GlyGCC</scp> , Regulates Hypoxic Pulmonary Artery Smooth Muscle Cell Proliferation via Myrf‐Mediated Endoplasmic Reticulum Stress · 2026 · DOI

• Although flow-responsive transcription factors such as KLF2 and KLF4 are well recognized, how mechanical forces converge on epitranscriptomic mechanisms to regulate RNA fate remains incompletely understood.

  Epitranscriptomic regulation of endothelial plasticity under hemodynamic forces: insights from the KLF2/4–METTL3–H19 pathway · 2026 · DOI

• Phospholipid scramblase 1 (PLSCR1) has been implicated in breast cancer progression, yet its precise role and underlying mechanisms in TNBC chemoresistance remain elusive.

  PLSCR1 drives chemoresistance in TNBC via METTL3/IGF2BP3-mediated mRNA stabilization and EGFR-MAPK pathway activation · 2026 · DOI

• Despite advances in understanding the dynamic changes in gene regulatory networks and chromatin landscapes during tumorigenesis, the functional interplay between RNA epitranscriptomic modifications and nuclear events in ICC remains poorly elucidated.

  NAT10/ac ⁴ C drives intrahepatic cholangiocarcinoma by suppressing transposable elements via chromatin remodeling · 2026 · DOI

• Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A) methylation and adenosine to inosine (A-to-I) editing, are critical regulators of hematopoietic stem cell (HSC) self-renewal and differentiation, yet their precise contributions to malignant transformation are not fully elucidated.

  Uncovering a role for METTL13 in malignant transformation of human hematopoietic stem cells and in the progression of pediatric leukemia · 2026 · DOI

• Although the Mms1-Rtt101 E3 ligase is known to trigger 25S NRD to clear defective 60S subunits, the specific molecular marks and targets it recognizes remain unknown.

  Absence of nascent peptides triggers nonfunctional ribosome decay · 2026 · DOI

• Introduction: As the core scaffold of the GAP Activity Toward Rags 2 complex (GATOR2) complex, SEC13 regulates mTORC1 signaling and endoplasmic reticulum homeostasis to modulate cellular metabolism, exhibits elevated expression across various solid tumors driving malignant progression, yet its comprehensive molecular mechanisms and immunotherapeutic potential remain to be systematically elucidated.

  Integrated pan cancer analysis with breast cancer validation identifies SEC13 homolog as prognostic biomarker and immunotherapy target · 2026 · DOI

• Ubiquitination and m6A RNA methylation have emerged as two highly dynamic and interconnected regulatory systems shaping almost every aspect of cancer biology (110). Accumulating evidence demonstrates the interactions of the two pathways: They converge to modulate the stability, localization and activity of key oncogenic signaling proteins and RNAs (111). Through multilayered crosstalk, the two pathways jointly influence hallmark cancer processes, including proliferation, stemness, metabolic reprogramming, immune evasion and therapeutic resistance (112). Based on the existing evidence, the present review advances the field in several important ways. The multi‑layered crosstalk between ubiquitination and m6A modification at the protein, RNA and chromatin levels is systematically summarized, and how these two regulatory systems converge on core oncogenic signaling pathways to cooperatively modulate tumor cell proliferation, metastasis, metabolic reprogramming and therapeutic resistance has also been discussed. In addition, the present review highlights the context‑dependent nature of ubiquitin‑m6A crosstalk and identifies key regulatory nodes with diagnostic and therapeutic potential. By providing a unified conceptual framework, the present review deepens mechanistic knowledge of cancer signaling networks and offers clear directions for the develop‑ ment of novel targeted strategies and combination therapies in precision oncology. Unfortunately, current understanding remains fragmented. To date, the majority of studies have focused on individual molecules or pathways, leaving the broader regulatory network largely uncharacterized (113,114). The context‑dependent roles of m6A and ubiquitination further complicate interpretations, as the same enzyme can act as either an oncogene or a tumor suppressor, depending on the tumor type or micro‑environ‑ ment. In addition, how these pathways integrate signals from stress, inflammation and metabolism to rewire oncogenic circuits remains largely defined. The bidirectional interplay between ubiquitination and m6A modification provides a strong rationale for combi‑ nation therapies, such as PROTACs combined with m6A inhibitors. PROTACs leverage E3 ligases to induce targeted degradation of oncogenic proteins, while m6A inhibitors (for example, METTL3 inhibitors and YTHDF2 inhibitors such as DC‑Y13‑27) disrupt m6A‑dependent RNA metabolism. Combining these agents yields synergistic effects by simulta‑ neously targeting the ubiquitin‑m6A network at both the protein and RNA levels: For example, PROTACs directed against oncogenic E3 ligases (such as STUB1) can restore the stability of METTL14, while m6A inhibitors further normalize m6A modification profiles, collectively reversing the dysregulation of oncogenic signaling pathways.

  Ubiquitination and N ⁶ ‑methyladenosine in cancer: Convergent regulation of oncogenic signaling pathways (Review) · 2026 · DOI

• 1. Introduction Among the diverse regulatory mechanisms in cell biology, post‑translational modifications are vital in the process of tumorigenesis (1). Ubiquitination, a pivotal post‑translational modification (PTM), involves the covalent attachment of the 76‑amino‑acid ubiquitin to substrate proteins and thus regu‑ lates their stability, activity and intracellular signaling (2). Such modification is catalyzed by a multi‑step enzymatic cascade consisting of ubiquitin‑activating enzymes (E1), ubiquitin‑conjugating enzymes (E2) and ubiquitin ligases (E3) (3). Ubiquitination contributes to cell‑cycle control and genome stability, as well as influencing cellular growth, proliferation and death. The dysregulation of ubiquitination in cancer frequently degrades tumor suppressors such as p53, thereby facilitating the proliferation and metastasis of tumor cells (4). Furthermore, counterbalancing ubiquitination, deubiquitinating enzymes (DUBs) remove ubiquitin moieties and disassemble ubiquitin chains, thereby playing a key role in maintaining ubiquitin system homeostasis and restraining 2 LI and LU: UBIQUITINATION AND M6A signal transduction, thus preventing pathway overactivation in cancer (5). N6‑methyladenosine (m6A) is among the most prevalent RNA modifications, gaining increasing attention in recent years (6). m6A modulates the stability and translational effi‑ ciency of mRNA, and is also indispensable in RNA splicing and decay (7). Accumulating evidence indicates that m 6A regulates gene expressions implicated in tumorigenesis, thereby directly influencing malignant phenotypes (8). For instance, m6A‑modified circNEK11 has been reported to promote hepatocellular carcinoma (HCC) progression via the miR‑1236‑3p/glutathione peroxidase 2 axis (9). m6A is also engaged in tumor micro‑environment remodeling by altering intercellular signaling networks (10). Recent studies have uncovered extensive crosstalk between ubiquitin‑dependent and m6A‑dependent regulation, such that these pathways do not act in isolation but converge on shared molecular nodes and signaling circuits (11,12). Mechanisms of interplay include ubiquitin‑mediated control of the abundance and activity of m6A writers/erasers/readers, m6A‑dependent regulation of mRNAs encoding components of ubiquitin path‑ ways, and coordinated actions at the chromatin‑transcription interface where co‑transcriptional m6A deposition and histone ubiquitination collectively influence RNAPII dynamics and nascent RNA processing (13). Through these interac‑ tions, ubiquitination and m6A cooperatively modulate core oncogenic signaling networks, for example, PI3K‑AKT (14), Wnt/β‑catenin (15) and NK‑κB (16), thereby governing proliferation, metastasis, immune interactions and resistance to therapy. Hence, a systematic dissection of their reciprocal regulation and pathogenic mechanisms helps deepen the current understandings of tumor biology and may uncover new therapeutic avenues. The present review synthesizes current knowledge on the molecular mechanisms by which ubiquitination and m6A intersect to regulate oncogenic signaling. The core enzymology and functional consequences of each modifica‑ tion were first summarized, and mechanistic examples of crosstalk at the levels of protein, RNA and chromatin were subsequently summarized. The ways through which these interactions impinge on major cancer signaling pathways were also addressed. Ultimately, the present review considers translational implications and outlines challenges and future directions, emphasizing the need for integrated multi‑omics and refined functional models to disentangle context speci‑ ficity and to exploit the ubiquitin‑m6A axis for cancer therapy.

  Ubiquitination and N ⁶ ‑methyladenosine in cancer: Convergent regulation of oncogenic signaling pathways (Review) · 2026 · DOI

• In conclusion, m6A and m5C RNA methylation are dynamic and reversible post-transcriptional modifications that play pivotal roles in cardiovascular homeostasis and disease. These modifications exhibit temporal and spatial specificity, influencing cardiac development, IRI, and pathological remodeling. Additionally, the regulatory factors associated with m6A and m5C display disease-specific expression patterns, positioning them as potential biomarkers for early diagnosis, disease monitoring, and prognosis assessment. However, despite notable progress, significant challenges Molecular Biology Reports (2026) 53:717 1 3 Page 13 of 18 717 Fig. 3 Translational pipeline of RNA methylation-based therapies in cardiovascular disease improvements in cardiovascular disease prevention and treatment. remain. Our current understanding of RNA methylation dynamics at the single-cell and subpopulation levels is limited, hindering precise disease classification and targeted interventions. Furthermore, the interactions between m6A, m5C, and other regulatory layers, including DNA methylation, histone modification, and non-coding RNA regulation, need further elucidation. The development of efficient, cardiac-specific delivery systems for RNA methylation modulation remains underdeveloped, posing a challenge for clinical translation. Looking forward, the integration of mechanistic studies with translational and clinical research will be crucial. Advances in high-resolution sequencing, single-cell multiomics, and precision gene editing are expected to accelerate discoveries in this field. By combining these innovations with therapeutic development, RNA methylation holds great promise for enhancing early diagnosis, improving risk stratification, and facilitating more personalized interventions.

  RNA methylation in cardiovascular remodeling: Molecular mechanisms, biomarkers, and therapeutic strategies · 2026 · DOI

• pressure overload models, While this review provides an overview of three major forms of methylation regulation, the impact of metabolic factors on methylation-related enzymes, and the underlying methylation mechanisms in DCM, several important limitations should be acknowledged. First, a major challenge lies in the heterogeneity of disease models. Many studies included in this review are derived from diverse experimental systems, including genetic DCM, doxorubicin-induced cardiomyopathy, and general heart failure. Although these models share certain pathological features, such as fibrosis and ventricular remodeling, their underlying mechanisms differ substantially, which may limit the applicability of the findings to other forms of DCM. Second, differences between human myocardial tissue, animal models, and in vitro systems are not always systematically addressed, and inconsistencies across studies are often difficult to reconcile. Third, although this review attempts integrate DNA methylation, histone methylation, and m6A RNA modification, studies investigating the crosstalk among these layers remain limited. In addition, the integration of metabolic remodeling with epigenetic regulation remains incomplete. direct evidence from human myocardial limited. Finally, the metabolomic studies translational potential of targeting methylation pathways remains constrained. Most available medicines are not DCMspecific, but are treatment of atherosclerosis, pressure overload, or general heart failure. While as these inflammation, fibrosis, and pathological remodeling, their underlying etiologies differ, making it uncertain whether these findings can be directly extrapolated to DCM. Despite encouraging preclinical results, methylation-targeting agents, inhibitors and histone methyltransferase including DNMT inhibitors, have not yet entered clinical trials for DCM, highlighting a significant gap between experimental research and clinical application. instead applied overlapping conditions in DCM features is still share such the to in Future studies should focus on DCM-specific, multi-omics approaches integrating epigenetic, transcriptomic, and metabolic data, as well as the development of cell-type–targeted and therapies.

  Methylation-centric epigenetic regulation in dilated cardiomyopathy: mechanisms, metabolic interplay, and translational potential · 2026 · DOI

• including tumor growth, metastasis, m6A dynamically modulates circRNA biogenesis, stability, and translation, while circRNAs reciprocally regulate the m6A machinery and act as scaffolds to fine-tune gene expression (Qin et al., 2021). Functionally, this interplay governs key cancer hallmarks, ferroptosis, stemness, therapy resistance, metabolic reprogramming, and immune microenvironment remodeling (Du et al., 2022). Clinically, m6A-modified circRNAs hold great promise as diagnostic biomarkers due to their stability, tissue specificity, and reflection of disease states (Xu et al., 2024). Furthermore, targeting the m6A–circRNA axis offers innovative therapeutic avenues, either by modulating m6A regulators or by manipulating circRNAs directly through RNA-based interventions (Androsavich, 2024). Overall, elucidating the m6A–circRNA network not only enhances our understanding of lung cancer pathogenesis but also provides a foundation for precision diagnostics and tailored therapeutic strategies. Firstly, the regulatory modes by which m6A affects circRNAs are highly diverse. m6A can influence circRNA biogenesis, stability, subcellular localization, and translational potential, while different m6A writers, erasers, and readers may exert distinct or even opposing effects depending on the cell type or physiological and pathological context. For instance, m6A writers may promote back- splicing and circRNA generation in some cases, whereas in others they may suppress circRNA expression. Similarly, m6A readers can either stabilize circRNAs or facilitate their degradation, exhibiting bidirectional regulatory features. Such multilayered regulation suggests that the m6A–circRNA network is not a simple linear pathway, but rather a highly dynamic regulatory system. Secondly, the functional outcomes of m6A-mediated circRNA regulation vary markedly across different cancers and even among subtypes of the same cancer, which is closely linked to tumor heterogeneity. The same m6A–circRNA axis may act as an oncogenic driver in one tumor type while functioning as a tumor suppressor in another. This divergence may be attributed to differences in genetic mutation backgrounds, microenvironmental contexts, expression profiles of m6A regulators, and downstream target networks. Therefore, future studies should integrate tumor subtypes, staging, and treatment status for refined analysis to elucidate the specific roles of m6A–circRNA interactions under distinct pathological conditions. Thirdly, m6A modification can promote the translation of circRNA-encoded peptides or microproteins, providing a new dimension for circRNA function. Increasing evidence indicates that some circRNAs possess translational potential, and their encoded peptides play key roles in tumor cell proliferation, apoptosis, migration, and drug resistance. As a translational initiation mark, m6A may facilitate peptide production by recruiting translation initiation complexes or altering circRNA structures to enhance ribosome recognition. This field is still in its infancy, and more systematic studies are needed to validate the biological significance and clinical relevance of m6A-dependent circRNA translation. Finally, beyond m6A, it remains an important unresolved question whether other RNA modifications, such as m5C and ac4C, similarly regulate circRNA expression and function. Existing studies have shown that m5C and ac4C modifications significantly influence mRNA stability, translation efficiency, and nuclear export; however, their roles in circRNA biology are relatively underexplored. Future multi-omics analyses may uncover a more comprehensive “circRNA epitranscriptome” and elucidate how different modifications interact to shape tumor biology. Overall, the m6A–circRNA interaction network offers a novel perspective for cancer research, but its complexity suggests that higher-throughput and more refined research strategies—combining single-cell sequencing, structural biology, and clinical validation—are required to translate discoveries into precise diagnostic and therapeutic approaches.

  Emerging functions of m6A-modified circRNAs and their targeting strategies in lung cancer · 2026 · DOI

• The β-ethynylserine chemical synthesis achieved 34% overall yield through 5 steps with specific attention to acetyl ester deprotection; large-scale synthesis protocols for multi-gram quantities and scalability of the Jones oxidation step for industrial production of β-ethynylserine have not been addressed.

  tRNA-deacylase-directed discovery of biosynthetic pathways · 2026 · DOI

• The proteomics visualization of alkyne-labelled S. cattleya using TAMRA-azide derivatization was conducted with spent media-derived βes; direct quantification of β-ethynylserine incorporation stoichiometry across the proteome and identification of which specific proteins preferentially incorporate βes versus canonical threonine is absent.

  tRNA-deacylase-directed discovery of biosynthetic pathways · 2026 · DOI

• The bioinformatic SSN analysis of the AlaX family tRNA-deacylases used a sequence alignment score threshold of 43% determined empirically; validation of this threshold against functionally characterized tRNA-deacylases from organisms outside the dataset and demonstration of predictive accuracy for novel cluster assignments is needed.

  tRNA-deacylase-directed discovery of biosynthetic pathways · 2026 · DOI

• The non-haem Fe(II)/αKG hydroxylase screening of AlfC and FuzA was conducted at room temperature for 2-18 h timepoints; the effect of temperature variation (37°C vs room temperature), pH optimization beyond 7.5, and extended reaction kinetics on hydroxylase substrate conversion rates requires investigation.

  tRNA-deacylase-directed discovery of biosynthetic pathways · 2026 · DOI

• The in vitro IVTT experiments with β-ethynylserine incorporation used only a single model peptide sequence (METRSKNFL); systematic evaluation of how βes incorporation efficiency varies across diverse peptide sequences, secondary structures, and β-ethynylserine positioning within proteins is lacking.

  tRNA-deacylase-directed discovery of biosynthetic pathways · 2026 · DOI

Most-cited papers in RNA modifications and cancer

• Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis · Cell · 2024 · 505 citations
• Histone lactylation-regulated METTL3 promotes ferroptosis via m6A-modification on ACSL4 in sepsis-associated lung injury · Redox Biology · 2024 · 251 citations
• SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms · Nature Methods · 2024 · 190 citations
• Cell surface RNAs control neutrophil recruitment · Cell · 2024 · 163 citations
• Rfam 15: RNA families database in 2025 · Nucleic Acids Research · 2024 · 160 citations
• Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer · Drug Resistance Updates · 2024 · 145 citations
• Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA · Nature Biotechnology · 2024 · 134 citations
• DNA damage induces p53-independent apoptosis through ribosome stalling · Science · 2024 · 133 citations
• Lactylation-driven FTO targets CDK2 to aggravate microvascular anomalies in diabetic retinopathy · EMBO Molecular Medicine · 2024 · 126 citations
• The role of RNA methylation in tumor immunity and its potential in immunotherapy · Molecular Cancer · 2024 · 126 citations

Most recent work

• A functional map of the human intrinsically disordered proteome · Proceedings of the National Academy of Sciences · 2026
• Nucleolar dynamics are determined by the ordered assembly of the ribosome · bioRxiv · 2026
• Assessment of nanopore RNA modification calling in human cell lines and synthetic systems · Genome Biology · 2026
• The YTHDF proteins shape the brain gene signatures of Alzheimer's disease · bioRxiv · 2026
• SWARM resolves nanopore signal interference between RNA modification types and reveals splicing-shaped pseudouridylation · bioRxiv · 2026
• CDS-localized m6A drives co-translational RNA decay to relieve biotic and abiotic endoplasmic reticulum stresses · Nature Plants · 2026
• m6A RNA Methylation in Plants: From Molecular Insights to Applications · Annual Review of Plant Biology · 2026
• Clarifying the role of METTL1-associated m7G modification in acute kidney injury · Kidney International · 2026
• tRNA-derived fragments elevated in Alzheimer’s disease facilitate Tau aggregation · bioRxiv (Cold Spring Harbor Laboratory) · 2026
• How ascorbate tunes the activity and substrate selectivity of Fe(II)-dependent dioxygenase superfamily enzymes · bioRxiv · 2026

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