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RNA modifications have recently emerged as critical posttranscriptional regulators of gene expression programmes. Significant advances have been made in understanding the functional role of RNA modifications in regulating coding and non-coding RNA processing and function, which in turn thoroughly shape distinct gene expression programmes. They affect diverse biological processes, and the correct deposition of many of these modifications is required for normal development. Alterations of their deposition are implicated in several diseases, including cancer. In this Review, we focus on the occurrence of N 6 -methyladenosine (m 6 A), 5-methylcytosine (m 5 C) and pseudouridine (Ψ) in coding and non-coding RNAs and describe their physiopathological role in cancer. We will highlight the latest insights into the mechanisms of how these posttranscriptional modifications influence tumour development, maintenance, and progression. Finally, we will summarize the latest advances on the development of small molecule inhibitors that target specific writers or erasers to rewind the epitranscriptome of a cancer cell and their therapeutic potential.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

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.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the AC A GA box is modified by N 6 methylation, but the role of this m 6 A modification is poorly understood. Here, we show that m 6 A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m 6 A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m 6 A influences 3’ splice site usage. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA AC A GA box. We conclude that U6 snRNA m 6 A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing. All the information necessary to build the proteins that perform the biological processes required for life is encoded in the DNA of an organism. Making these proteins requires the DNA sequence of a gene to be transcribed into a ‘messenger RNA’ (mRNA), which is then processed into a final, mature form. This blueprint is then translated to assemble the corresponding protein. When an mRNA is processed, segments of the sequence that do not code for protein are removed and the remaining coding sequences are joined together in the right order. An intricate molecular machine known as the spliceosome controls this mechanism by recognising the ‘splice sites’ where coding and non-coding sequences meet. Depending on external conditions, the spliceosome can ‘pick-and-mix’ the coding sequences to create different processed mRNAs (and therefore proteins) from a single gene. This alternative splicing mechanism is often used to regulate when certain biological processes take place based on environmental cues; for example, the splicing of genes which control the timing of plant flowering is sensitive to ambient temperatures. To investigate this mechanism, Parker et al. focused on Arabidopsis thaliana , a plant that blooms later when temperatures are low. This precise timing partly relies on a gene whose mRNA is efficiently spliced in the cold, resulting in an active form of its protein that blocks blooming. Parker et al. grew and screened many A. thaliana plants to find individuals that could flower early in the cold, in which splicing of this gene was disrupted. A mutant fitting these criteria was identified and subjected to further investigation, which revealed that it could not produce FIONA1. In non-mutant plants, this enzyme chemically modifies one of the components of the spliceosome, a small nuclear RNA known as U6. Parker et al found that there are two types of splice site – one more likely to interact with U6 and another that preferentially interacts with another small nuclear RNA, U5. When FIONA1 is inactive (such as in the mutant identified by Parker et al.), splice sites that tend to strongly interact with U5 are selected. However, when the enzyme is active, splice sites that tend to bind with the chemically modified U6 are used instead. Further work by Parker et al. showed that these two types of splice sites (‘preferring’ either U5 or U6) are found in equal proportions in the genomes of many species, including humans. This suggests that Parker et al. have uncovered an essential feature of how genomes are organised and splicing is controlled.

RNA methylation at position N6 in adenosine (m6A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m6A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m6A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m6A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m6A/RNA co-immunoprecipitation. Impact of m6A depletion on RNA stability was assessed by Actinomycin D assay. The association of m6A-levels with PCa prognosis was examined in clinical samples. Higher m6A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m6A-levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m6A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911–43.183, p = 0.006). VIRMA is a critical factor sustaining m6A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m6A-levels decreasing stability and abundance of oncogenic lncRNAs.

5-methylcytosine (m⁵C) is an abundant RNA modification that's presence is reported in a wide variety of RNA species, including cytoplasmic and mitochondrial ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as messenger RNAs (mRNAs), enhancer RNAs (eRNAs) and a number of non-coding RNAs. In eukaryotes, C5 methylation of RNA cytosines is catalyzed by enzymes of the NOL1/NOP2/SUN domain (NSUN) family, as well as the DNA methyltransferase homologue DNMT2. In recent years, substrate RNAs and modification target nucleotides for each of these methyltransferases have been identified, and structural and biochemical analyses have provided the first insights into how each of these enzymes achieves target specificity. Functional characterizations of these proteins and the modifications they install have revealed important roles in diverse aspects of both mitochondrial and nuclear gene expression. Importantly, this knowledge has enabled a better understanding of the molecular basis of a number of diseases caused by mutations in the genes encoding m⁵C methyltransferases or changes in the expression level of these enzymes.

Summary N6‐methyladenosine (m6A) RNA methylation, one of the most pivotal internal modifications of RNA, is a conserved post‐transcriptional mechanism to enrich and regulate genetic information in eukaryotes. The scope and function of this modification in plants has been an intense focus of study, especially in model plant systems. The characterization of plant m6A writers, erasers and readers, as well as the elucidation of their functions, is currently one of the most fascinating hotspots in plant biology research. The functional analysis of m6A in plants will be booming in the foreseeable future, which could contribute to crop genetic improvement through epitranscriptome manipulation. In this review, we systematically analysed and summarized recent advances in the understanding of the structure and composition of plant m6A regulatory machinery, and the biological functions of m6A in plant growth, development and stress response. Finally, our analysis showed that the evolutionary relationships between m6A modification components were highly conserved across the plant kingdom.

Head and neck squamous cell carcinoma (HNSCC) constitute approximately 4% of all cancers worldwide. In this study, we analyzed the expression profile of the long noncoding RNA (lncRNA) of 502 HNSCC patients from The Cancer Genome Atlas database. Among the differentially expressed lncRNAs between HNSCC and normal samples, LNCAROD is overexpressed in HNSCC and associated with advanced T stage and shortened overall survival. The N6‐methyladenosine (m6A) modification mediated by METTL3 and METTL14 enhanced the stability of LNCAROD in HNSCC cells. Depletion of LNCAROD attenuated cell proliferation, mobility in vitro, and tumorigenicity in vivo, whereas overexpression of LNCAROD exerted opposite effects. LNCAROD is mainly distributed in nucleus and binds with YBX1 and HSPA1A proteins. Silencing either YBX1 or HSPA1A did not affect the level of LNCAROD. However, loss of LNCAROD led to shortened half‐life of YBX1 protein. Mechanistically, LNCAROD protected YBX1 from proteasomal degradation by facilitating YBX1‐HSPA1A protein–protein interaction. Depletion of HSPA1A in LNCAROD‐overexpressing cells resulted in accelerated proteasomal degradation of YBX1 protein. Moreover, re‐expression of Flag‐YBX1 in LNCAROD‐silenced cells rescued malignant behavior of HNSCC cells. Our study indicates that LNCAROD is an oncogenic lncRNA and dysregulation of m6A modification might account for aberrant expression of LNCAROD in HNSCC. LNCAROD acts as a scaffold for the interaction between YBX1 and HSPA1A, preventing proteasomal degradation of YBX1 in HNSCC cells. Dysregulation of long noncoding RNAs (lncRNAs) is implicated in cancer development and progression. In this study, Ban et al. demonstrated that an oncogenic lncRNA, LNCAROD, is stabilized by m6A methylation and overexpressed in head and neck squamous cell carcinoma (HNSCC). LNCAROD forms a ternary complex with HSPA1A and YBX1, preventing proteasomal degradation of YBX1. This study provides a new paradigm of the mechanisms of lncRNAs in HNSCC development.

The alphabet of building blocks for RNA molecules is much larger than the standard four nucleotides. The diversity is achieved by the post-transcriptional biochemical modification of these nucleotides into distinct chemical entities that are structurally and functionally different from their unmodified counterparts. Some of these modifications are constituent and critical for RNA functions, while others serve as dynamic markings to regulate the fate of specific RNA molecules. Together, these modifications form the epitranscriptome, an essential layer of cellular biochemistry. As of the time of writing this review, more than 300 distinct RNA modifications from all three life domains have been identified. However, only a few of the most well-established modifications are included in most reviews on this topic. To provide a complete overview of the current state of research on the epitranscriptome, we analyzed the extent of the available information for all known RNA modifications. We selected 25 modifications to describe in detail. Summarizing our findings, we describe the current status of research on most RNA modifications and identify further developments in this field.

Common variable immunodeficiency (CVID) is frequently complicated by duodenal inflammation, but the underlying molecular mechanisms remain poorly understood. While epigenetic alterations have been described in CVID, the epitranscriptome is largely unexplored. We therefore investigated whether RNA N6-methyladenosine (m6A) modifications in duodenal tissue are altered in CVID and whether such changes are associated with the local microbiota or m6A-related enzymes. m6A modification levels were analysed in snap-frozen duodenal biopsies from CVID patients with intraepithelial lymphocytosis and inflammation (CVID_IEL; n = 5), CVID patients with normal duodenal histology (CVID_N; n = 5) and controls with normal biopsies (n = 5) using m6A-RNA immunoprecipitation followed by microarray profiling and gene set enrichment analysis. Duodenal bacterial microbiota from the same anatomical region were characterised by 16S ribosomal RNA gene sequencing, and selected m6A-regulating enzymes were quantified in biopsies by targeted proteomics. In total, 4,134 differentially methylated transcripts were identified, and unsupervised principal component analyses revealed partially overlapping, but clearly divergent m6A signatures for CVID_IEL, CVID_N and controls, with a gradient along the first principal component. Pathway analysis showed relative hypermethylation of mitochondria- and ribosome-related gene sets in both CVID subgroups versus controls, and hypomethylation of pathways linked to ubiquitination, proteasomal degradation, glycosylation and post-transcriptional gene silencing in CVID_IEL versus CVID_N. Sparse canonical correlation models demonstrated significant associations between specific duodenal bacterial genera and m6A-modified transcripts in CVID, but not in controls, whereas expression levels of the examined m6A-regulating enzymes did not differ between groups. These findings suggest that duodenal inflammation in CVID may be associated with a distinct m6A epitranscriptomic signature that is linked to specific features of the mucosal microbiota, providing preliminary, hypothesis-generating evidence for a potential interaction between microbiota, epitranscriptomic regulation and local immune dysregulation in CVID.