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The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target. [ABSTRACT FROM AUTHOR]

RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA's operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis. [ABSTRACT FROM AUTHOR]

Despite the highly conserved nature of the genetic code, the frequency of usage of each codon can vary significantly. The evolution of codon usage is shaped by two main evolutionary forces: mutational bias and selection pressures. These pressures can be driven by environmental factors, but also by the need for efficient translation, which depends heavily on the concentration of transfer RNAs (tRNAs) within the cell. The data presented here supports the proposal that tRNA modifications play a key role in shaping the overall preference of codon usage in proteobacteria. Interestingly, some codons, such as CGA and AGG (encoding arginine), exhibit a surprisingly low level of variation in their frequency of usage, even across genomes with differing GC content. These findings suggest that the evolution of GC content in proteobacterial genomes might be primarily driven by changes in the usage of a specific subset of codons, whose usage is itself influenced by tRNA modifications. [ABSTRACT FROM AUTHOR]

RNA modifications affect key stages of the RNA life cycle, including splicing, export, decay, and translation. Epitranscriptomic regulations therefore significantly influence cellular physiology and pathophysiology. Here, we selected some of the most abundant modifications and reviewed their roles in the heart and in cardiovascular diseases: N6-methyladenosine (m6A), N6,2‘-O-dimethyladenosine (m6Am), N¹-methyladenosine (m¹A), pseudouridine (Ψ), 5-methylcytidine (m5C), and inosine (I). Dysregulation of epitranscriptomic machinery affecting these modifications vastly changes the cardiac phenotype and is linked with many cardiovascular diseases such as myocardial infarction, cardiomyopathies, or heart failure. Thus, a deeper understanding of these epitranscriptomic changes and their regulatory mechanisms can enhance our knowledge of the molecular underpinnings of prevalent cardiac diseases, potentially paving the way for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Various methods have been developed so far for detecting N6-methyladenosine (m6A). The total m6A level or the m6A status at individual positions on mRNA can be detected and quantified through some sequencing-independent biochemical methods, such as LC/MS, SCARLET, SELECT, and m6A-ELISA. However, the m6A-detection techniques relying on high-throughput sequencing have more effectively advanced the understanding about biological significance of m6A-containing mRNA and m6A pathway at a transcriptomic level over the past decade. Various SGS-based (Second Generation Sequencing-based) methods with different detection principles have been widely employed for this purpose. These principles include m6A-enrichment using antibodies, discrimination of m6A from unmodified A-base by nucleases, a fusion protein strategy relying on RNA-editing enzymes, and marking m6A with chemical/biochemical reactions. Recently, TGS-based (Third Generation Sequencing-based) methods have brought a new trend by direct m6A-detection. This review first gives a brief introduction of current knowledge about m6A biogenesis and function, and then comprehensively describes m6A-profiling strategies including their principles, procedures, and features. This will guide users to pick appropriate methods according to research goals, give insights for developing novel techniques in varying areas, and continue to expand our boundary of knowledge on m6A. [ABSTRACT FROM AUTHOR]

Abstract RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA’s operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis.

Recently, new data have been added to the interaction between non-coding RNAs (ncRNAs) and epigenetic machinery. Epigenetics includes enzymes involved in DNA methylation, histone modifications, and RNA modifications, and mechanisms underlying chromatin structure, repressive states, and active states operating in transcription. The main focus is on long ncRNAs (lncRNAs) acting as scaffolds to assemble protein complexes. This review does not cover RNA’s role in sponging microRNAs, or decoy functions. Several lncRNAs were shown to regulate chromatin activation and repression by interacting with Polycomb repressive complexes and mixed-lineage leukemia (MLL) activating complexes. Various groups reported on enhancer of zeste homolog 2 (EZH2) interactions with regulatory RNAs. Knowledge of the function of these complexes opens the perspective to develop new therapeutics for cancer treatment. Lastly, the interplay between lncRNAs and epitranscriptomic modifications in cancers paves the way for new targets in cancer therapy. The approach to inhibit lncRNAs interaction with protein complexes and perspective to regulate epitrascriptomics-regulated RNAs may bring new compounds as therapeuticals in various types of cancer.

Osteogenesis is modulated by multiple regulatory networks. Recent studies showed that RNA modifications and their reader, writer, and eraser (RWE) proteins are involved in regulating various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using LC-MS/MS in parallel-reaction monitoring (PRM) mode, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins throughout the entire time course of osteogenic differentiation in H9 human embryonic stem cells (ESCs). We found that approximately half of the 127 detected RWE proteins were down-regulated during osteogenic differentiation, and they included mainly proteins involved in RNA methylation and pseudouridylation. Protein-protein interaction (PPI) network analysis unveiled significant associations between the down-regulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis (GSEA) of publicly available RNA-seq data obtained from osteogenesis imperfecta patients suggested a potential role of METTL1 in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs, and our work unveiled potential regulatory roles of these proteins in osteogenesis. LC-MS/MS data were deposited on ProteomeXchange (PXD039249).

RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m7G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.

ABSTRACTThe identification of mechanisms capable of modifying genetic information by the addition of covalent RNA modifications distinguishes a level of complexity in gene expression which challenges key long-standing concepts of RNA biology. One of the current challenges of molecular biology is to properly understand the molecular functions of these RNA modifications, with more than 170 different ones having been identified so far. However, it has not been possible to map specific RNA modifications at a single-cell resolution until very recently. This review will highlight the technological advances in single-cell methodologies aimed at assessing and testing the biological function of certain RNA modifications, focusing on m6A. These advances have allowed for the development of novel strategies that enable the study of the ‘epitranscriptome’. Nevertheless, despite all these improvements, many challenges and difficulties still need fixing for these techniques to work efficiently.

ABSTRACTRNA modifications play crucial roles in various biological processes and diseases. Accurate prediction of RNA modification sites is essential for understanding their functions. In this study, we propose a hybrid approach that fuses a pre-trained sequence representation with various sequence features to predict multiple types of RNA modifications in one combined prediction framework. We developed MRM-BERT, a deep learning method that combined the pre-trained DNABERT deep sequence representation module and the convolutional neural network (CNN) exploiting four traditional sequence feature encodings to improve the prediction performance. MRM-BERT was evaluated on multiple datasets of 12 commonly occurring RNA modifications, including m6A, m5C, m1A and so on. The results demonstrate that our hybrid model outperforms other models in terms of area under receiver operating characteristic curve (AUC) for all 12 types of RNA modifications. MRM-BERT is available as an online tool (http://117.122.208.21:8501) or source code (https://github.com/abhhba999/MRM-BERT), which allows users to predict RNA modification sites and visualize the results. Overall, our study provides an effective and efficient approach to predict multiple RNA modifications, contributing to the understanding of RNA biology and the development of therapeutic strategies.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy. [ABSTRACT FROM AUTHOR]

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes. [ABSTRACT FROM AUTHOR]

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1' glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms' tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH2CH3) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens. [ABSTRACT FROM AUTHOR]

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a complex neurodegenerative disorder and the most common form of dementia. There are two main types of AD: familial and sporadic. Familial AD is linked to mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2). On the other hand, sporadic AD is the more common form of the disease and has genetic, epigenetic, and environmental components that influence disease onset and progression. Investigating the epigenetic mechanisms associated with AD is essential for increasing understanding of pathology and identifying biomarkers for diagnosis and treatment. Chemical covalent modifications on DNA and RNA can epigenetically regulate gene expression at transcriptional and posttranscriptional levels and play protective or pathological roles in AD and other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

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Threatening public health, pulmonary disease (PD) encompasses diverse lung injuries like chronic obstructive PD, pulmonary fibrosis, asthma, pulmonary infections due to pathogen invasion, and fatal lung cancer. The crucial involvement of RNA epigenetic modifications in PD pathogenesis is underscored by robust evidence. These modifications not only shape cell fates but also finely modulate the expression of genes linked to disease progression, suggesting their utility as biomarkers and targets for therapeutic strategies. The critical RNA modifications implicated in PDs are summarized in this review, including N6‐methylation of adenosine, N1‐methylation of adenosine, 5‐methylcytosine, pseudouridine (5‐ribosyl uracil), 7‐methylguanosine, and adenosine to inosine editing, along with relevant regulatory mechanisms. By shedding light on the pathology of PDs, these summaries could spur the identification of new biomarkers and therapeutic strategies, ultimately paving the way for early PD diagnosis and treatment innovation. [ABSTRACT FROM AUTHOR]

Over 170 chemical modifications naturally occur on RNA and alter diverse aspects of RNA life. Recently developed tools based on the RNA-modifying machinery have enabled the exogenous introduction of RNA modifications into cells. RNA recorders are tools that utilize RNA-binding proteins (RBPs) to guide the RNA-modifying machinery to target sites with the aim of recording the RNA–RBP interactome or RNA modification maps. RNA modulators are tools that redirect the RNA-modifying machinery towards endogenous RNAs to edit RNA bases, thus correcting genetic disease-associated mutations. RNA sensors direct the RNA-modifying machinery together with a reporter to an RNA molecule of interest to enable cell type- and tissue-specific RNA sensing. Engineering both the RNA-binding and -modifying modules of the RNA-modifying machinery is necessary to reach sufficient on-target activity while minimizing off-target activity. Over 170 chemical modifications can be naturally installed on RNA, all of which are catalyzed by dedicated machineries. These modifications can alter RNA sequence structure, stability, and translation as well as serving as quality control marks that record aspects of RNA processing. The diverse roles played by RNAs within cells has motivated endeavors to exogenously introduce RNA modifications at target sites for diverse purposes ranging from recording RNA:protein interactions to therapeutic applications. Here, we discuss these applications and the approaches that have been employed to engineer RNA-modifying machineries, and highlight persisting challenges and perspectives. Over 170 chemical modifications can be naturally installed on RNA, all of which are catalyzed by dedicated machineries. These modifications can alter RNA sequence structure, stability, and translation as well as serving as quality control marks that record aspects of RNA processing. The diverse roles played by RNAs within cells has motivated endeavors to exogenously introduce RNA modifications at target sites for diverse purposes ranging from recording RNA:protein interactions to therapeutic applications. Here, we discuss these applications and the approaches that have been employed to engineer RNA modification machineries, and highlight persisting challenges and perspectives. [ABSTRACT FROM AUTHOR]

Cells must change their properties in order to adapt to a constantly changing environment. Most of the cellular sensing and regulatory mechanisms described so far are based on proteins that serve as sensors, signal transducers, and effectors of signalling pathways, resulting in altered cell physiology. In recent years, however, remarkable examples of the critical role of non-coding RNAs in some of these regulatory pathways have been described in various organisms. In this review, we focus on all classes of non-coding RNAs that play regulatory roles during stress response, starvation, and ageing in different yeast species as well as in structured yeast populations. Such regulation can occur, for example, by modulating the amount and functional state of tRNAs, rRNAs, or snRNAs that are directly involved in the processes of translation and splicing. In addition, long non-coding RNAs and microRNA-like molecules are bona fide regulators of the expression of their target genes. Non-coding RNAs thus represent an additional level of cellular regulation that is gradually being uncovered. [ABSTRACT FROM AUTHOR]

Cardiomyopathy (CM) represents a heterogeneous group of diseases primarily affecting cardiac structure and function, with genetic and epigenetic dysregulation playing a pivotal role in its pathogenesis. Emerging evidence from the burgeoning field of epitranscriptomics has brought to light the significant impact of various RNA modifications, notably N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N1-methyladenosine (m1A), 2′-O-methylation (Nm), and 6,2′-O-dimethyladenosine (m6Am), on cardiomyocyte function and the broader processes of cardiac and vascular remodelling. These modifications have been shown to influence key pathological mechanisms including mitochondrial dysfunction, oxidative stress, cardiomyocyte apoptosis, inflammation, immune response, and myocardial fibrosis. Importantly, aberrations in the RNA methylation machinery have been observed in human CM cases and animal models, highlighting the critical role of RNA methylating enzymes and their potential as therapeutic targets or biomarkers for CM. This review underscores the necessity for a deeper understanding of RNA methylation processes in the context of CM, to illuminate novel therapeutic avenues and diagnostic tools, thereby addressing a significant gap in the current management strategies for this complex disease.

Despite the highly conserved nature of the genetic code, the frequency of usage of each codon can vary significantly. The evolution of codon usage is shaped by two main evolutionary forces: mutational bias and selection pressures. These pressures can be driven by environmental factors, but also by the need for efficient translation, which depends heavily on the concentration of transfer RNAs (tRNAs) within the cell. The data presented here supports the proposal that tRNA modifications play a key role in shaping the overall preference of codon usage in proteobacteria. Interestingly, some codons, such as CGA and AGG (encoding arginine), exhibit a surprisingly low level of variation in their frequency of usage, even across genomes with differing GC content. These findings suggest that the evolution of GC content in proteobacterial genomes might be primarily driven by changes in the usage of a specific subset of codons, whose usage is itself influenced by tRNA modifications.

Digestive tract cancers are among the most common malignancies worldwide and have high incidence and mortality rates. Thus, the discovery of more effective diagnostic and therapeutic targets is urgently required. The development of technologies to accurately detect RNA modification has led to the identification of numerous RNA chemical modifications in humans (epitranscriptomics) that are involved in the occurrence and development of digestive tract cancers. RNA modifications can cooperatively regulate gene expression to facilitate normal physiological functions of the digestive system. However, the dysfunction of relevant RNA-modifying enzymes (“writers,” “erasers,” and “readers”) can lead to the development of digestive tract cancers. Consequently, targeting dysregulated enzyme activity could represent a potent therapeutic strategy for the treatment of digestive tract cancers. In this review, we summarize the most widely studied roles and mechanisms of RNA modifications (m6A, m1A, m5C, m7G, A-to-I editing, pseudouridine [Ψ]) in relation to digestive tract cancers, highlight the crosstalk between RNA modifications, and discuss their roles in the interactions between the digestive system and microbiota during carcinogenesis. The clinical significance of novel therapeutic methods based on RNA-modifying enzymes is also discussed. This review will help guide future research into digestive tract cancers that are resistant to current therapeutics.

Various methods have been developed so far for detecting N6-methyladenosine (m6A). The total m6A level or the m6A status at individual positions on mRNA can be detected and quantified through some sequencing-independent biochemical methods, such as LC/MS, SCARLET, SELECT, and m6A-ELISA. However, the m6A-detection techniques relying on high-throughput sequencing have more effectively advanced the understanding about biological significance of m6A-containing mRNA and m6A pathway at a transcriptomic level over the past decade. Various SGS-based (Second Generation Sequencing-based) methods with different detection principles have been widely employed for this purpose. These principles include m6A-enrichment using antibodies, discrimination of m6A from unmodified A-base by nucleases, a fusion protein strategy relying on RNA-editing enzymes, and marking m6A with chemical/biochemical reactions. Recently, TGS-based (Third Generation Sequencing-based) methods have brought a new trend by direct m6A-detection. This review first gives a brief introduction of current knowledge about m6A biogenesis and function, and then comprehensively describes m6A-profiling strategies including their principles, procedures, and features. This will guide users to pick appropriate methods according to research goals, give insights for developing novel techniques in varying areas, and continue to expand our boundary of knowledge on m6A.

Posttranscriptional RNA modifications by adenosine-to-inosine (A-to-I) editing are abundant in the brain, yet elucidating functional sites remains challenging. To bridge this gap, we investigate spatiotemporal and genetically regulated A-to-I editing sites across prenatal and postnatal stages of human brain development. More than 10,000 spatiotemporally regulated A-to-I sites were identified that occur predominately in 3' UTRs and introns, as well as 37 sites that recode amino acids in protein coding regions with precise changes in editing levels across development. Hyper-edited transcripts are also enriched in the aging brain and stabilize RNA secondary structures. These features are conserved in murine and non-human primate models of neurodevelopment. Finally, thousands of cis-editing quantitative trait loci (edQTLs) were identified with unique regulatory effects during prenatal and postnatal development. Collectively, this work offers a resolved atlas linking spatiotemporal variation in editing levels to genetic regulatory effects throughout distinct stages of brain maturation.

Sequencing for RNA modifications with the nanopore direct RNA sequencing platform provides ionic current levels, helicase dwell times, and base call data that differentiate the modifications from the canonical form. Herein, model RNAs were synthesized with site‐specific uridine (U) base modifications that enable the study of increasing an alkyl group size, halogen identity, or a change in base acidity to impact the nanopore data. The analysis concluded that increases in alkyl size trend with greater current blockage but a similar change in base‐call error was not found. The addition of a halogen series to C5 of U revealed that the current levels recorded a trend with the water‐octanol partition coefficient of the base, as well as the base call error. Studies with U modifications that are deprotonated (i. e., anionic) under the sequencing conditions gave broad current levels that influenced the base call error. Some modifications led to helicase dwell time changes. These insights provide design parameters for modification‐specific chemical reagents that can shift nanopore signatures to minimize false positive reads, a known issue with this sequencing approach. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most abundant internal eukaryotic mRNA modification, and is involved in the regulation of various biological processes. Direct Nanopore sequencing of native RNA (dRNA-seq) emerged as a leading approach for its identification. Several software were published for m6A detection and there is a strong need for independent studies benchmarking their performance on data from different species, and against various reference datasets. Moreover, a computational workflow is needed to streamline the execution of tools whose installation and execution remains complicated. We developed NanOlympicsMod, a Nextflow pipeline exploiting containerized technology for comparing 14 tools for m6A detection on dRNA-seq data. NanOlympicsMod was tested on dRNA-seq data generated from in vitro (un)modified synthetic oligos. The m6A hits returned by each tool were compared to the m6A position known by design of the oligos. In addition, NanOlympicsMod was used on dRNA-seq datasets from wild-type and m6A-depleted yeast, mouse and human, and each tool's hits were compared to reference m6A sets generated by leading orthogonal methods. The performance of the tools markedly differed across datasets, and methods adopting different approaches showed different preferences in terms of precision and recall. Changing the stringency cut-offs allowed for tuning the precision-recall trade-off towards user preferences. Finally, we determined that precision and recall of tools are markedly influenced by sequencing depth, and that additional sequencing would likely reveal additional m6A sites. Thanks to the possibility of including novel tools, NanOlympicsMod will streamline the benchmarking of m6A detection tools on dRNA-seq data, improving future RNA modification characterization. [ABSTRACT FROM AUTHOR]

Various types of RNA modification in parasites affect parasite virulence, viability, differentiation and reproduction. Epitranscriptomics represents a novel regulatory layer and a new hallmark in parasites. Prediction of parasite homologs of the regulators of RNA modifications reported in humans facilitates prediction of the types of RNA modification and related regulators in parasites and elucidates the evolution of the parasitic epitranscriptional machinery. Multiple forms of crosstalk affect epitranscriptome variations and may be an important mechanism for the regulation of RNA modifications in parasites. RNA modifications of host cells are also an important component of the host–parasite interface, which is involved in the upregulation of host antiparasite immunity and influences the transmission and progression of parasitic diseases. RNA modifications (epitranscriptome) – such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ) – modulate RNA processing, stability, interaction, and translation, thereby playing critical roles in the development, replication, virulence, metabolism, and life cycle adaptations of parasitic protozoa. Here, we summarize potential homologs of the major human RNA modification regulatory factors in parasites, outline current knowledge on how RNA modifications affect parasitic protozoa, highlight the regulation of RNA modifications and their crosstalk, and discuss current progress in exploring RNA modifications as potential drug targets. This review contributes to our understanding of epitranscriptomic regulation of parasitic protozoa biology and pathogenesis and provides new perspectives for the treatment of parasitic diseases. [ABSTRACT FROM AUTHOR]

Despite a long history of research, neurodegenerative diseases and malignant brain tumor gliomas are both considered incurable, facing challenges in the development of treatments. Recent evidence suggests that RNA modifications, previously considered as static components of intracellular RNAs, are in fact dynamically regulated across various RNA species in cells and play a critical role in major biological processes in the nervous system. Innovations in next-generation sequencing have enabled the accurate detection of modifications on bases and sugars within various RNA molecules. These RNA modifications influence the stability and transportation of RNA, and crucially affect its translation. This review delves into existing knowledge on RNA modifications to offer a comprehensive inventory of these modifications across different RNA species. The detailed regulatory functions and roles of RNA modifications within the nervous system are discussed with a focus on neurodegenerative diseases and gliomas. This article presents a comprehensive overview of the fundamental mechanisms and emerging roles of RNA modifications in these diseases, which can facilitate the creation of innovative diagnostics and therapeutics for these conditions. [ABSTRACT FROM AUTHOR]

Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is one of the most common modifications in both eukaryotic and prokaryotic mRNAs. It has been experimentally confirmed that m6A methylation is involved in the regulation of stability and translation of various mRNAs. Until recently, the majority of m6A-related studies have been focused on the cytoplasmic functions of this modification. Here, we review new data on the role of m6A in several key biological processes taking place in the cell nucleus, such as transcription, chromatin organization, splicing, nuclear-cytoplasmic transport, and R-loop metabolism. Based on analysis of these data, we suggest that m6A methylation of nuclear RNAs is another level of gene expression regulation which, together with DNA methylation and histone modifications, controls chromatin structure and functioning in various biological contexts. [ABSTRACT FROM AUTHOR]

Advanced detection of RNA modifications, especially N 6-methyladenosine, paves the way for transcriptome-wide, single-cell, and stoichiometric modification profiling at single-nucleotide resolution. RNA modification databases, tumor atlases, drug-like compound libraries, and machine learning have accelerated therapeutic development focused on epitranscriptomics. Novel RNA modification engineering tools based on the CRISPR/Cas technology or endogenous RNA modification machinery can be applied to uncover causal relationships between individual modification sites and phenotypic outcomes. The epitranscriptomics field has undergone tremendous growth since the discovery that the RNA N 6-methyladenosine (m6A) modification is reversible and is distributed throughout the transcriptome. Efforts to map RNA modifications transcriptome-wide and reshape the epitranscriptome in disease settings have facilitated mechanistic understanding and drug discovery in the field. In this review we discuss recent advancements in RNA modification detection methods and consider how these developments can be applied to gain novel insights into the epitranscriptome. We also highlight drug discovery efforts aimed at developing epitranscriptomic therapeutics for cancer and other diseases. Finally, we consider engineering of the epitranscriptome as an emerging direction to investigate RNA modifications and their causal effects on RNA processing at high specificity. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (aHSCT) is utilized as a potential curative treatment for various hematologic malignancies. However, graft-versus-host disease (GVHD) post-aHSCT is a severe complication that significantly impacts patients' quality of life and overall survival, becoming a major cause of non-relapse mortality. In recent years, the association between epigenetics and GVHD has garnered increasing attention. Epigenetics focuses on studying mechanisms that affect gene expression without altering DNA sequences, primarily including DNA methylation, histone modifications, non-coding RNAs (ncRNAs) regulation, and RNA modifications. This review summarizes the role of epigenetic regulation in the pathogenesis of GVHD, with a focus on DNA methylation, histone modifications, ncRNA, RNA modifications and their involvement and applications in the occurrence and development of GVHD. It also highlights advancements in relevant diagnostic markers and drugs, aiming to provide new insights for the clinical diagnosis and treatment of GVHD.

Abstract Threatening public health, pulmonary disease (PD) encompasses diverse lung injuries like chronic obstructive PD, pulmonary fibrosis, asthma, pulmonary infections due to pathogen invasion, and fatal lung cancer. The crucial involvement of RNA epigenetic modifications in PD pathogenesis is underscored by robust evidence. These modifications not only shape cell fates but also finely modulate the expression of genes linked to disease progression, suggesting their utility as biomarkers and targets for therapeutic strategies. The critical RNA modifications implicated in PDs are summarized in this review, including N6‐methylation of adenosine, N1‐methylation of adenosine, 5‐methylcytosine, pseudouridine (5‐ribosyl uracil), 7‐methylguanosine, and adenosine to inosine editing, along with relevant regulatory mechanisms. By shedding light on the pathology of PDs, these summaries could spur the identification of new biomarkers and therapeutic strategies, ultimately paving the way for early PD diagnosis and treatment innovation.

Abstract Glioblastoma (GBM) comprises 45.6% of all primary malignant brain cancers and is one of the most common and aggressive intracranial tumors in adults. Intratumoral heterogeneity with a wide range of proteomic, genetic, and epigenetic dysregulation contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. To date, numerous clinical trials have been developed to target the proteome and epigenome of high-grade gliomas with promising results. However, studying RNA modifications, or RNA epitranscriptomics, is a new frontier within neuro-oncology. RNA epitranscriptomics was discovered in the 1970s, but in the last decade, the extent of modification of mRNA and various non-coding RNAs has emerged and been implicated in transposable element activation and many other oncogenic processes within the tumor microenvironment. This review provides background information and discusses the therapeutic potential of agents modulating epitranscriptomics in high-grade gliomas. A particular emphasis will be placed on how combination therapies that include immune agents targeting hERV-mediated viral mimicry could improve the treatment of GBM.

Abstract Background RNA modifications, especially N6-methyladenosine, N1-methyladenosine and 5–methylcytosine, play an important role in the progression of cardiovascular disease. However, its regulatory function in dilated cardiomyopathy (DCM) remains to be undefined. Methods In the study, key RNA modification regulators (RMRs) were screened by three machine learning models. Subsequently, a risk prediction model for DCM was developed and validated based on these important genes, and the diagnostic efficiency of these genes was assessed. Meanwhile, the relevance of these genes to clinical traits was explored. In both animal models and human subjects, the gene with the strongest connection was confirmed. The expression patterns of important genes were investigated using single-cell analysis. Results A total of 4 key RMRs were identified. The risk prediction models were constructed basing on these genes which showed a good accuracy and sensitivity in both the training and test set. Correlation analysis showed that insulin-like growth factor binding protein 2 (IGFBP2) had the highest correlation with left ventricular ejection fraction (LVEF) (R = -0.49, P = 0.00039). Further validation expression level of IGFBP2 indicated that this gene was significantly upregulated in DCM animal models and patients, and correlation analysis validation showed a significant negative correlation between IGFBP2 and LVEF (R = -0.87; P = 6*10–5). Single-cell analysis revealed that this gene was mainly expressed in endothelial cells. Conclusion In conclusion, IGFBP2 is an important biomarker of left ventricular dysfunction in DCM. Future clinical applications could possibly use it as a possible therapeutic target.

The world of small noncoding RNAs (sncRNAs) is ever-expanding, from small interfering RNA, microRNA and Piwi-interacting RNA to the recently emerging non-canonical sncRNAs derived from longer structured RNAs (for example, transfer, ribosomal, Y, small nucleolar, small nuclear and vault RNAs), showing distinct biogenesis and functional principles. Here we discuss recent tools for sncRNA identification, caveats in sncRNA expression analysis and emerging methods for direct sequencing of sncRNAs and systematic mapping of RNA modifications that are integral to their function.

Glioblastoma (GBM) comprises 45.6% of all primary malignant brain cancers and is one of the most common and aggressive intracranial tumors in adults. Intratumoral heterogeneity with a wide range of proteomic, genetic, and epigenetic dysregulation contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. To date, numerous clinical trials have been developed to target the proteome and epigenome of high-grade gliomas with promising results. However, studying RNA modifications, or RNA epitranscriptomics, is a new frontier within neuro-oncology. RNA epitranscriptomics was discovered in the 1970s, but in the last decade, the extent of modification of mRNA and various non-coding RNAs has emerged and been implicated in transposable element activation and many other oncogenic processes within the tumor microenvironment. This review provides background information and discusses the therapeutic potential of agents modulating epitranscriptomics in high-grade gliomas. A particular emphasis will be placed on how combination therapies that include immune agents targeting hERV-mediated viral mimicry could improve the treatment of GBM. Key points: In vivo studies link epitranscriptomic dysregulation with glioma pathogenesis. TCGA shows elevated regulator expression is associated with increased mortality in GBM patients. RNA modifications negatively correlate with GBM immunogenicity and thus reduce the efficacy of immunotherapies in GBM patients. RNA modification regulators represent promising therapeutic agents for reducing mortality and immunotherapy adjuvants for enhancing tumor immunogenicity. [ABSTRACT FROM AUTHOR]

Pseudouridylation, the conversion of uridine (U) to pseudouridine (Ѱ), is one of the most prevalent and evolutionary conserved RNA modifications, which is catalyzed by pseudouridine synthase (PUS) enzymes. Ѱs play a crucial epitranscriptomic role by regulating attributes of cellular RNAs across diverse organisms. However, the precise biological functions of PUSs in plants remain largely elusive. In this study, we identified and characterized 21 members in the rice PUS family which were categorized into six distinct subfamilies, with RluA and TruA emerging as the most extensive. A comprehensive analysis of domain structures, motifs, and homology modeling revealed that OsPUSs possess all canonical features of true PUS proteins, essential for substrate recognition and catalysis. The exploration of OsPUS promoters revealed presence of cis-acting regulatory elements associated with hormone and abiotic stress responses. Expression analysis of OsPUS genes showed differential expression at developmental stages and under stress conditions. Notably, OsTruB3 displayed high expression in salt, heat, and drought stresses. Several OsRluA members showed induction in heat stress, while a significant decline in expression was observed for various OsTruA members in drought and salinity. Furthermore, miRNAs predicted to target OsPUSs were themselves responsive to variable stresses, adding an additional layer of regulatory complexity of OsPUSs. Study of protein–protein interaction networks provided substantial support for the potential regulatory role of OsPUSs in numerous cellular and stress response pathways. Conclusively, our study provides functional insights into the OsPUS family, contributing to a better understanding of their crucial roles in shaping the development and stress adaptation in rice. [ABSTRACT FROM AUTHOR]

Pseudouridine is one of the well-known chemical modifications in various RNA species. Current advances to detect pseudouridine show that the pseudouridine landscape is dynamic and affects multiple cellular processes. Although our understanding of this post-transcriptional modification mainly depends on yeast and human models, the recent findings provide strong evidence for the critical role of pseudouridine in plants. Here, we review the current knowledge of pseudouridine in plant RNAs, including its synthesis, degradation, regulatory mechanisms, and functions. Moreover, we propose future areas of research on pseudouridine modification in plants. [ABSTRACT FROM AUTHOR]

Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma. The inaugural NSAS Challenge Workshop on Brain Epitranscriptomics hosted in Crans-Montana, Switzerland in 2023 assembled a group of experts from the field, to discuss the current state of the field and provide novel translational perspectives. A summary of the discussions at the workshop is presented here to simulate broader engagement from the general neuroscience field. [ABSTRACT FROM AUTHOR]

Background: RNA modifications, especially N6-methyladenosine, N1-methyladenosine and 5–methylcytosine, play an important role in the progression of cardiovascular disease. However, its regulatory function in dilated cardiomyopathy (DCM) remains to be undefined. Methods: In the study, key RNA modification regulators (RMRs) were screened by three machine learning models. Subsequently, a risk prediction model for DCM was developed and validated based on these important genes, and the diagnostic efficiency of these genes was assessed. Meanwhile, the relevance of these genes to clinical traits was explored. In both animal models and human subjects, the gene with the strongest connection was confirmed. The expression patterns of important genes were investigated using single-cell analysis. Results: A total of 4 key RMRs were identified. The risk prediction models were constructed basing on these genes which showed a good accuracy and sensitivity in both the training and test set. Correlation analysis showed that insulin-like growth factor binding protein 2 (IGFBP2) had the highest correlation with left ventricular ejection fraction (LVEF) (R = -0.49, P = 0.00039). Further validation expression level of IGFBP2 indicated that this gene was significantly upregulated in DCM animal models and patients, and correlation analysis validation showed a significant negative correlation between IGFBP2 and LVEF (R = -0.87; P = 6*10–5). Single-cell analysis revealed that this gene was mainly expressed in endothelial cells. Conclusion: In conclusion, IGFBP2 is an important biomarker of left ventricular dysfunction in DCM. Future clinical applications could possibly use it as a possible therapeutic target. [ABSTRACT FROM AUTHOR]

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications. [ABSTRACT FROM AUTHOR]

The dynamic balance between tRNA supply and codon usage demand is a fundamental principle in the cellular translation economy. However, the regulation and functional consequences of this balance remain unclear. Here, we use PARIS2 interactome capture, structure modeling, conservation analysis, RNA–protein interaction analysis, and modification mapping to reveal the targets of hundreds of snoRNAs, many of which were previously considered orphans. We identify a snoRNA–tRNA interaction network that is required for global tRNA modifications, including 2′-O-methylation and others. Loss of Fibrillarin, the snoRNA-guided 2′-O-methyltransferase, induces global upregulation of tRNA fragments, a large group of regulatory RNAs. In particular, the snoRNAs D97/D133 guide the 2′-O-methylation of multiple tRNAs, especially for the amino acid methionine (Met), a protein-intrinsic antioxidant. Loss of D97/D133 snoRNAs in human HEK293 cells reduced target tRNA levels and induced codon adaptation of the transcriptome and translatome. Both single and double knockouts of D97 and D133 in HEK293 cells suppress Met-enriched proliferation-related gene expression programs, including, translation, splicing, and mitochondrial energy metabolism, and promote Met-depleted programs related to development, differentiation, and morphogenesis. In a mouse embryonic stem cell model of development, knockdown and knockout of D97/D133 promote differentiation to mesoderm and endoderm fates, such as cardiomyocytes, without compromising pluripotency, consistent with the enhanced development-related gene expression programs in human cells. This work solves a decades-old mystery about orphan snoRNAs and reveals a function of snoRNAs in controlling the codon-biased dichotomous cellular states of proliferation and development. [ABSTRACT FROM AUTHOR]

White adipose tissue (WAT) lipolysis releases free fatty acids as a key energy substance to support metabolism in fasting, cold exposure, and exercise. Atgl, in concert with Cgi‐58, catalyzes the first lipolytic reaction. The sympathetic nervous system (SNS) stimulates lipolysis via neurotransmitter norepinephrine that activates adipocyte β adrenergic receptors (Adrb1‐3). In obesity, adipose Adrb signaling and lipolysis are impaired, contributing to pathogenic WAT expansion; however, the underling mechanism remains poorly understood. Recent studies highlight importance of N6‐methyladenosine (m6A)‐based RNA modification in health and disease. METTL14 heterodimerizes with METTL3 to form an RNA methyltransferase complex that installs m6A in transcripts. Here, this work shows that adipose Mettl3 and Mettl14 are influenced by fasting, refeeding, and insulin, and are upregulated in high fat diet (HFD) induced obesity. Adipose Adrb2, Adrb3, Atgl, and Cgi‐58 transcript m6A contents are elevated in obesity. Mettl14 ablation decreases these transcripts' m6A contents and increases their translations and protein levels in adipocytes, thereby increasing Adrb signaling and lipolysis. Mice with adipocyte‐specific deletion of Mettl14 are resistant to HFD‐induced obesity, insulin resistance, glucose intolerance, and nonalcoholic fatty liver disease (NAFLD). These results unravel a METTL14/m6A/translation pathway governing Adrb signaling and lipolysis. METTL14/m6A‐based epitranscriptomic reprogramming impairs adipose Adrb signaling and lipolysis, promoting obesity, NAFLD, and metabolic disease. [ABSTRACT FROM AUTHOR]

For years, we have taken a reductionist approach to understanding gene regulation through the study of one gene in one cell at a time. While this approach has been fruitful it is laborious and fails to provide a global picture of what is occurring in complex situations involving tightly coordinated immune responses. The emergence of whole-genome techniques provides a system-level view of a response and can provide a plethora of information on events occurring in a cell from gene expression changes to splicing changes and chemical modifications. As with any technology, this often results in more questions than answers, but this wealth of knowledge is providing us with an unprecedented view of what occurs inside our cells during an immune response. In this review, we will discuss the current RNA-sequencing technologies and what they are helping us learn about the innate immune system.

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.