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Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

Due to the spectacular number of studies focusing on epigenetics in the last few decades, and particularly for the last few years, the availability of a chronology of epigenetics appears essential. Indeed, our review places epigenetic events and the identification of the main epigenetic writers, readers and erasers on a historic scale. This review helps to understand the increasing knowledge in molecular and cellular biology, the development of new biochemical techniques and advances in epigenetics and, more importantly, the roles played by epigenetics in many physiological and pathological situations.

Methylation of adenosines at the N6 position (m6A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m6A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m6A modification in post-transcriptional genome regulation.

The m 6 A RNA methylation is the most prevalent internal modification in mammalian mRNAs which plays critical biological roles by regulating vital cellular processes. Dysregulations of the m 6 A modification due to aberrant expression of its regulatory proteins are frequently observed in many pathological conditions, particularly in cancer. Normal cells undergo malignant transformation via activation or modulation of different oncogenic signaling pathways through complex mechanisms. Accumulating evidence showing regulation of oncogenic signaling pathways at the epitranscriptomic level has added an extra layer of the complexity. In particular, recent studies demonstrated that, in many types of cancers various oncogenic signaling pathways are modulated by the m 6 A modification in the target mRNAs as well as noncoding RNA transcripts. m 6 A modifications in these RNA molecules control their fate and metabolism by regulating their stability, translation or subcellular localizations. In this review we discussed recent exciting studies on oncogenic signaling pathways that are modulated by the m 6 A RNA modification and/or their regulators in cancer and provided perspectives for further studies. The regulation of oncogenic signaling pathways by the m 6 A modification and its regulators also render them as potential druggable targets for the treatment of cancer.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Background N6-methyladenosine (m 6 A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. Results We show that m 6 A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m 6 A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m 6 A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m 6 A sites compared to mice. Differential m 6 A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m 6 A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m 6 A methylation in a mouse model of Alzheimer’s disease, which is correlated with reduced protein levels. Conclusions Our results suggest that m 6 A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m 6 A controls protein levels of key genes involved in Alzheimer’s disease-associated pathways, suggesting that m 6 A plays an important role in aging and neurodegenerative disease.

The broad application of next-generation sequencing technologies in conjunction with improved bioinformatics has helped to illuminate the complexity of the transcriptome, both in terms of quantity and variety. In humans, 70–90% of the genome is transcribed, but only ~2% carries the blueprint for proteins. Hence, there is a huge class of non-translated transcripts, called long non-coding RNAs (lncRNAs), which have received much attention in the past decade. Several studies have shown that lncRNAs are involved in a plethora of cellular signaling pathways and actively regulate gene expression via a broad selection of molecular mechanisms. Only recently, sequencing-based, transcriptome-wide studies have characterized different types of post-transcriptional chemical modifications of RNAs. These modifications have been shown to affect the fate of RNA and further expand the variety of the transcriptome. However, our understanding of their biological function, especially in the context of lncRNAs, is still in its infancy. In this review, we will focus on three epitranscriptomic marks, namely pseudouridine (Ψ), N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We will introduce writers, readers, and erasers of these modifications, and we will present methods for their detection. Finally, we will provide insights into the distribution and function of these chemical modifications in selected, cancer-related lncRNAs.

Background Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied. Results We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits. Conclusions As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.

Summary Plants inevitably encounter environmental adversities, including abiotic and biotic stresses, which significantly impede plant growth and reduce crop yield. Thus, fine‐tuning the fate and function of stress‐responsive RNAs is indispensable for plant survival under such adverse conditions. Recently, post‐transcriptional RNA modifications have been studied as a potent route to regulate plant gene expression under stress. Among over 160 mRNA modifications identified to date, N6‐methyladenosine (m6A) in mRNAs is notable because of its multifaceted roles in plant development and stress response. Recent transcriptome‐wide mapping has revealed the distribution and patterns of m6A in diverse stress‐responsive mRNAs in plants, building a foundation for elucidating the molecular link between m6A and stress response. Moreover, the identification and characterization of m6A writers, readers and erasers in Arabidopsis and other model crops have offered insights into the biological roles of m6A in plant abiotic stress responses. Here, we review the recent progress of research on mRNA modifications, particularly m6A, and their dynamics, distribution, regulation and biological functions in plant stress responses. Further, we posit potential strategies for breeding stress‐tolerant crops by engineering mRNA modifications and propose the future direction of research on RNA modifications to gain a much deeper understanding of plant stress biology. Post‐transcriptional RNA modifications, particularly N6‐methyladenosine (m6A), in mRNAs play a crucial role in plant stress responses, which could a potential strategy for breeding stress‐tolerant crops by engineering mRNA modifications.

Cerebral ischaemia‒reperfusion injury (IRI), during which neurons undergo oxygen-glucose deprivation/reoxygenation (OGD/R), is a notable pathological process in many neurological diseases. N1-methyladenosine (m 1 A) is an RNA modification that can affect gene expression and RNA stability. The m 1 A landscape and potential functions of m 1 A modification in neurons remain poorly understood. We explored RNA (mRNA, lncRNA, and circRNA) m 1 A modification in normal and OGD/R-treated mouse neurons and the effect of m 1 A on diverse RNAs. We investigated the m 1 A landscape in primary neurons, identified m 1 A-modified RNAs, and found that OGD/R increased the number of m 1 A RNAs. m 1 A modification might also affect the regulatory mechanisms of noncoding RNAs, e.g., lncRNA–RNA binding proteins (RBPs) interactions and circRNA translation. We showed that m 1 A modification mediates the circRNA/lncRNA‒miRNA–mRNA competing endogenous RNA (ceRNA) mechanism and that 3' untranslated region (3’UTR) modification of mRNAs can hinder miRNA–mRNA binding. Three modification patterns were identified, and genes with different patterns had intrinsic mechanisms with potential m 1 A-regulatory specificity. Systematic analysis of the m 1 A landscape in normal and OGD/R neurons lays a critical foundation for understanding RNA modification and provides new perspectives and a theoretical basis for treating and developing drugs for OGD/R pathology-related diseases.