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RNA modification identification is an emerging field in epigenetics due to its indispensable regulatory role in the cell life cycle. With advancements in identification methods, an increasing number of RNA modifications has been discovered, thereby driving the development of more efficient and accurate techniques for localizing modified RNAs and elucidating their functions. High-throughput sequencing approaches for modified RNA detection can be categorized into antibody-based, enzymatic-based, and chemical-labeling-based methods. Given the intrinsic chemical reactions involved in all biochemical processes, we provide a comprehensive review of recent advancements in artificial chemical labeling and transformations of ten distinct RNA modifications and their applications in sequencing. Our aim is to contribute to a deeper understanding of the mechanisms underlying these modifications. We focus on the chemical reactions associated with RNA modifications and briefly compare the advantages and disadvantages of detection methods based on these reactions. Additionally, we introduce several approaches that identify multiple modifications through chemical labeling. As the field of RNA modification research continues to expand, we anticipate that the techniques and insights presented in this review will serve as a valuable resource for future studies aimed at further elucidating the functional roles of RNA modifications in biological processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S -adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.

RNA modifications play crucial roles in regulating gene expression and cellular homeostasis. Modulating RNA modifications, particularly by targeting the enzymes responsible for their catalysis, has emerged as a promising therapeutic strategy. However, limitations, such as the lack of identified modifying enzymes and compensatory mechanisms, hinder targeted interventions. Chemical approaches independent of enzymatic activity offer an alternative strategy for RNA modification modulation. Here, we present the identification of 2-chloro-3,5-dinitrobenzoic acid as a highly effective photochemical deprenylase of i 6 A RNA. This method demonstrates exceptional selectivity towards i 6 A, converting its substituent into a "N-doped" ozonide, which upon hydrolysis releases natural adenine. We believe that this chemical approach will pave the way for a better understanding of RNA modification biology and the development of novel therapeutic modalities., (© 2024 Wiley-VCH GmbH.)

Over 170 chemical modifications have been discovered in various types of ribonucleic acids (RNAs), including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). These RNA modifications play crucial roles in a wide range of biological processes such as gene expression regulation, RNA stability maintenance, and protein translation. RNA modifications represent a new dimension of gene expression regulation known as the "epitranscriptome". The discovery of RNA modifications and the relevant writers, erasers, and readers provides an important basis for studies on the dynamic regulation and physiological functions of RNA modifications. Owing to the development of detection technologies for RNA modifications, studies on RNA epitranscriptomes have progressed to the single-base resolution, multilayer, and full-coverage stage. Transcriptome-wide methods help discover new RNA modification sites and are of great importance for elucidating the molecular regulatory mechanisms of epitranscriptomics, exploring the disease associations of RNA modifications, and understanding their clinical applications. The existing RNA modification sequencing technologies can be categorized according to the pretreatment approach and sequencing principle as direct high-throughput sequencing, antibody-enrichment sequencing, enzyme-assisted sequencing, chemical labeling-assisted sequencing, metabolic labeling sequencing, and nanopore sequencing technologies. These methods, as well as studies on the functions of RNA modifications, have greatly expanded our understanding of epitranscriptomics. In this review, we summarize the recent progress in RNA modification detection technologies, focusing on the basic principles, advantages, and limitations of different methods. Direct high-throughput sequencing methods do not require complex RNA pretreatment and allow for the mapping of RNA modifications using conventional RNA sequencing methods. However, only a few RNA modifications can be analyzed by high-throughput sequencing. Antibody enrichment followed by high-throughput sequencing has emerged as a crucial approach for mapping RNA modifications, significantly advancing the understanding of RNA modifications and their regulatory functions in different species. However, the resolution of antibody-enrichment sequencing is limited to approximately 100-200 bp. Although chemical crosslinking techniques can achieve single-base resolution, these methods are often complex, and the specificity of the antibodies used in these methods has raised concerns. In particular, the issue of off-target binding by the antibodies requires urgent attention. Enzyme-assisted sequencing has improved the accuracy of the localization analysis of RNA modifications and enables stoichiometric detection with single-base resolution. However, the enzymes used in this technique show poor reactivity, specificity, and sequence preference. Chemical labeling sequencing has become a widely used approach for profiling RNA modifications, particularly by altering reverse transcription (RT) signatures such as RT stops, misincorporations, and deletions. Chemical-assisted sequencing provides a sequence-independent RNA modification detection strategy that enables the localization of multiple RNA modifications. Additionally, when combined with the biotin-streptavidin affinity method, low-abundance RNA modifications can be enriched and detected. Nevertheless, the specificity of many chemical reactions remains problematic, and the development of specific reaction probes for particular modifications should continue in the future to achieve the precise localization of RNA modifications. As an indirect localization method, metabolic labeling sequencing specifically localizes the sites at which modifying enzymes act, which is of great significance in the study of RNA modification functions. However, this method is limited by the intracellular labeling of RNA and cannot be applied to biological samples such as clinical tissues and blood samples. Nanopore sequencing is a direct RNA-sequencing method that does not require RT or the polymerase chain reaction (PCR). However, challenges in analyzing the data obtained from nanopore sequencing, such as the high rate of false positives, must be resolved. Discussing sequencing analysis methods for various types of RNA modifications is instructive for the future development of novel RNA modification mapping technologies, and will aid studies on the functions of RNA modifications across the entire transcriptome.

Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner-both in vitro and in vivo-are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site-specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid-phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N 4 -acetylcytidine, 5-hydroxymethylcytidine, 3-methylcytidine, 2'-OCF 3 , and 2'-N 3 ribose modifications. We also discuss the all-chemical synthesis of 5'-capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single-molecule FRET studies. Finally, we highlight promising developments in RNA-catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Inosine (I), resulting from the deamination of adenosine (A), is a prominent modification in the human transcriptome. The enzymes responsible for the conversion of adenosine to inosine in human mRNAs are the ADARs (adenosine deaminases acting on RNA). Inosine modification introduces a layer of complexity to mRNA processing and function, as it can impact various aspects of RNA biology, including mRNA stability, splicing, translation, and protein binding. The relevance of this process is emphasized in the growing number of human disorders associated with dysregulated A-to-I editing pathways. Here, we describe the impact of the A-to-I conversion on the structure and stability of duplex RNA and on the consequences of this modification at different locations in mRNAs. Furthermore, we highlight specific open questions regarding the interplay between inosine formation in duplex RNA and the innate immune response., (© 2024 Mendoza and Beal; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The potential presence of 5-methylcytosine as a sparse internal modification of mRNA was first raised in 1975, and a first map of the modification was also part of the epitranscriptomics "big bang" in 2012. Since then, the evidence for its presence in mRNA has firmed up, and initial insights have been gained into the molecular function and broader biological relevance of 5-methylcytosine when present in mRNA. Here, we summarize the status quo of the field, outline some of its current challenges, and suggest how to address them in future work., (© 2024 Guarnacci and Preiss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA 2'- O -methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field., (© 2024 Zhou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

N 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m 1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A. In this study, we introduce a reduction-based m 1 A sequencing (red-m 1 A-seq). We report that NaBH 4 reduction of m 1 A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m 1 A to m 6 A to provide good controls, allowing the detection of m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A sites, but also new m 1 A sites in mt-tRNA Asn-GTT and 5.8S rRNA., (© 2024 Pajdzik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA modification, a posttranscriptional regulatory mechanism, significantly influences RNA biogenesis and function. The accurate identification of modification sites is paramount for investigating their biological implications. Methods for encoding RNA sequence into numerical data play a crucial role in developing robust models for predicting modification sites. However, existing techniques suffer from limitations, including inadequate information representation, challenges in effectively integrating positional and sequential information, and the generation of irrelevant or redundant features when combining multiple approaches. These deficiencies hinder the effectiveness of machine learning models in addressing the performance challenges associated with predicting RNA modification sites. Here, we introduce a novel RNA sequence feature representation method, named BiPSTP, which utilizes bidirectional trinucleotide position-specific propensities. We employ the parameter ξ to denote the interval between the current nucleotide and its adjacent forward or backward dinucleotide, enabling the extraction of positional and sequential information from RNA sequences. Leveraging the BiPSTP method, we have developed the prediction model mRNAPred using support vector machine classifier to identify multiple types of RNA modification sites. We evaluate the performance of our BiPSTP method and mRNAPred model across 12 distinct RNA modification types. Our experimental results demonstrate the superiority of the mRNAPred model compared to state-of-art models in the domain of RNA modification sites identification. Importantly, our BiPSTP method enhances the robustness and generalization performance of prediction models. Notably, it can be applied to feature extraction from DNA sequences to predict other biological modification sites., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m 6 A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development., (© 2024 Wiley Periodicals LLC.)

RNA modification plays a crucial role in cellular regulation. However, traditional high-throughput sequencing methods for elucidating their functional mechanisms are time-consuming and labor-intensive, despite extensive research. Moreover, existing methods often limit their focus to specific species, neglecting the simultaneous exploration of RNA modifications across diverse species. Therefore, a versatile computational approach is necessary for interpretable analysis of RNA modifications across species. A multi-scale biological language-based deep learning model is proposed for interpretable, sequential-level prediction of diverse RNA modifications. Benchmark comparisons across species demonstrate the model's superiority in predicting various RNA methylation types over current state-of-the-art methods. The cross-species validation and attention weight visualization also highlight the model's capability to capture sequential and functional semantics from genomic backgrounds. Our analysis of RNA modifications helps us find the potential existence of "biological grammars" in each modification type, which could be effective for mapping methylation-related sequential patterns and understanding the underlying biological mechanisms of RNA modifications.

Therapeutic mRNAs are generated using modified nucleotides, namely N 1 -methylpseudouridine (m 1 Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N 6 -methyladenosine (m 6 A), pseudouridine (Ψ), or m 1 Ψ. UV thermal denaturation assays showed that m 6 A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m 1 Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m 6 A, Ψ, or m 1 Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA modifications, including N -7-methylguanosine (m 7 G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m 7 G modifications is of paramount significance, given recent associations between altered m 7 G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m 7 G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m 7 G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m 7 G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH 4 , neutral pH and the addition of 7-methylguanosine monophosphate (m 7 GMP) during the reducing reaction. Notably, compared to NaBH 4 -based methods, Bo-Seq enhances the m 7 G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m 7 G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m 7 G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m 7 G in gene expression and its implications in cancer biology.

Acute liver failure (ALF) is a rare and complicated disease with a high mortality rate. Emergency liver transplantation is the only treatment method that can improve the ALF prognosis. However, its clinical application remains limited owing to the aggressive nature of liver transplantation, limited donors, and high postoperative mortality. The study investigated the effect of m6A on the immune microenvironment of hepatitis B virus-related ALF (HBV-ALF). In this study, the gene expression data of 47 normal people and 42 HBV-ALF patients were downloaded from the Gene Expression Omnibu (GEO) database. The known 23 m6A regulators which mediated RNA modification patterns were compared and analyzed in these two groups, and the gene diagnosis model of HBV-ALF patients was established based on the analysis results. In addition, we used unsupervised clustering to identify different m6A RNA methylation modification patterns in HBV-ALF based on m6A regulators, and evaluated the immune infiltration and biological differences in these subtypes. In addition, the relationship between m6A genes and immune cell activation in HBV-ACLF patients was explored by immune infiltration analysis. Nineteen m6A regulators mediated RNA methylation (m6A regulators for short) were differentially expressed in HBV-ALF and control groups. m6A regulators could well distinguish control samples from HBV-ALF samples, and m6A regulators might be used as a basis for diagnosing HBV-ALF patients. Immune cells such as activated CD8 T cells, activated B cells, and activated CD4 T cells might play important roles in HBV-ALF, and m6A regulators were closely associated with immune cell infiltration. ALKBH15, CBLL1, IGF2BP2, IGF2BP3, and ZC3H13 were significantly associated with immune cells. Considering 23 m6A regulators, HBV-ALF patients could be classified into two subtypes (cluster 1 and cluster 2) based on different immune cell infiltration. m6A regulators of the IGFBP and YHDF families have extremely different levels in these two subtypes. Differential immune cell infiltration among these subtypes was observed, a total of 913 differentially expressed genes among different m6A modification patterns was identified, and their biological functions were explored. m6A modification might play a crucial role in the diverse and complex immune microenvironment of HBV-ALF patients., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Combinations of biological macromolecules can provide researchers with precise control and unique methods for regulating, studying, and manipulating cellular processes. For instance, combining the unmatched encodability afforded by nucleic acids with the diverse functionality of proteins has transformed our approach to solving several problems in chemical biology. Despite these benefits, there remains a need for new methods to site-specifically generate conjugates between different classes of biomolecules. Here we present a fully enzymatic strategy for combining nucleic acids and proteins using SNAP-tag and RNA-TAG (transglycosylation at guanosine) technologies via a bifunctional preQ1-benzylguanine small molecule probe. We demonstrate the robust ability of this technology to assemble site-specific SNAP-tag - RNA conjugates with RNAs of varying length and use our conjugation strategy to recruit an endonuclease to an RNA of interest for targeted degradation. We foresee that combining SNAP-tag and RNA-TAG will facilitate researchers to predictably engineer novel macromolecular complexes., (© 2023 Wiley-VCH GmbH.)

Ovarian cancer (OC) is the most common female cancer worldwide. Patients with OC have high mortality because of its complex and poorly understood pathogenesis. RNA epigenetic modifications, such as m 6 A, m 1 A, and m 5 C, are closely associated with the occurrence and development of OC. RNA modifications can affect the stability of mRNA transcripts, nuclear export of RNAs, translation efficiency, and decoding accuracy. However, there are few overviews that summarize the link between m 6 A RNA modification and OC. Here, we discuss the molecular and cellular functions of different RNA modifications and how their regulation contributes to the pathogenesis of OC. By improving our understanding of the role of RNA modifications in the etiology of OC, we provide new perspectives for their use in OC diagnosis and treatment. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease., (© 2023 Wiley Periodicals LLC.)

N6-methyladenosine (m6A) is one of the most common and well-known internal RNA modifications that occur on mRNAs or ncRNAs. It affects various aspects of RNA metabolism, including splicing, stability, translocation, and translation. An abundance of evidence demonstrates that m6A plays a crucial role in various pathological and biological processes, especially in tumorigenesis and tumor progression. In this article, we introduce the potential functions of m6A regulators, including "writers" that install m6A marks, "erasers" that demethylate m6A, and "readers" that determine the fate of m6A-modified targets. We have conducted a review on the molecular functions of m6A, focusing on both coding and noncoding RNAs. Additionally, we have compiled an overview of the effects noncoding RNAs have on m6A regulators and explored the dual roles of m6A in the development and advancement of cancer. Our review also includes a detailed summary of the most advanced databases for m6A, state-of-the-art experimental and sequencing detection methods, and machine learning-based computational predictors for identifying m6A sites.

While the existence of post-transcriptional modifications of RNA nucleotides has been known for decades, in most RNA species the exact positions of these modifications and their physiological function have been elusive until recently. Technological advances, such as high-throughput next-generation sequencing (NGS) methods and nanopore-based mapping technologies, have made it possible to map the position of these modifications with single nucleotide accuracy, and genetic screens have uncovered the "writer", "reader" and "eraser" proteins that help to install, interpret and remove such modifications, respectively. These discoveries led to intensive research programmes with the aim of uncovering the roles of these modifications during diverse biological processes. In this review, we assess novel discoveries related to the role of post-transcriptional modifications during animal development, highlighting how these discoveries can affect multiple aspects of development from fertilization to differentiation in many species., (© 2022. The Author(s).)

Post- and co-transcriptional RNA modifications are found to play various roles in regulating essential biological processes at all stages of RNA life. Precise identification of RNA modification sites is thus crucial for understanding the related molecular functions and specific regulatory circuitry. To date, a number of computational approaches have been developed for in silico identification of RNA modification sites; however, most of them require learning from base-resolution epitranscriptome datasets, which are generally scarce and available only for a limited number of experimental conditions, and predict only a single modification, even though there are multiple inter-related RNA modification types available. In this study, we proposed AdaptRM, a multi-task computational method for synergetic learning of multi-tissue, type and species RNA modifications from both high- and low-resolution epitranscriptome datasets. By taking advantage of adaptive pooling and multi-task learning, the newly proposed AdaptRM approach outperformed the state-of-the-art computational models (WeakRM and TS-m6A-DL) and two other deep-learning architectures based on Transformer and ConvMixer in three different case studies for both high-resolution and low-resolution prediction tasks, demonstrating its effectiveness and generalization ability. In addition, by interpreting the learned models, we unveiled for the first time the potential association between different tissues in terms of epitranscriptome sequence patterns. AdaptRM is available as a user-friendly web server from http://www.rnamd.org/AdaptRM together with all the codes and data used in this project., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

N 6 -Methyladenosine (m 6 A) is the most abundant internal chemical modification in eukaryotic mRNA and plays important roles in gene expression regulation, including transcriptional and post-transcriptional regulation. m 6 A is a reversible modification that is installed, removed, and recognized by methyltransferases (writers), demethylases (erasers), and m 6 A-binding proteins (readers), respectively. Recently, the breadth of research on m 6 A in plants has expanded, and the vital roles of m 6 A in plant development, biotic and abiotic stress responses, and crop trait improvement have been investigated. In this review, we discuss recent developments in research on m 6 A and highlight the detection methods, distribution, regulatory proteins, and molecular and biological functions of m 6 A in plants. We also offer some perspectives on future investigations, providing direction for subsequent research on m 6 A in plants., (Copyright © 2023. Published by Elsevier Inc.)

Characterization of RNA modifications has identified their distribution features and molecular functions. Dynamic changes in RNA modification on various forms of RNA are essential for the development and function of the immune system. In this review, we discuss the value of innovative RNA modification profiling technologies to uncover the function of these diverse, dynamic RNA modifications in various immune cells within healthy and diseased contexts. Further, we explore our current understanding of the mechanisms whereby aberrant RNA modifications modulate the immune milieu of the tumor microenvironment and point out outstanding research questions.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics., Competing Interests: Declaration of interests A patent has been filed relating to the data presented., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT 4 . Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2'-O-methyl, 2'-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.

Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.

Each day it is becoming increasingly difficult not to notice the completely new, fast growing, extremely intricate and challenging world of epitranscriptomics as the understanding of RNA methylation is expanding at a hasty rate. Writers (methyltransferases), erasers (demethylases) and readers (RNA-binding proteins) are responsible for adding, removing and recognising methyl groups on RNA, respectively. Several methyltransferases identified in plants are now being investigated and recent studies have shown a connection between RNA-methyltransferases (RNA-MTases) and stress and development processes. However, compared to their animal and bacteria counterparts, the understanding of RNA methyltransferases is still incipient, particularly those located in organelles. Comparative and systematic analyses allowed the tracing of the evolution of these enzymes suggesting the existence of several methyltransferases yet to be characterised. This review outlines the functions of plant nuclear and organellar RNA-MTases in plant development and stress responses and the comparative and evolutionary discoveries made on RNA-MTases across kingdoms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

RNA modifications regulate multiple aspects of cellular function including RNA splicing, translation, export, decay, stability, and phase separation. One of the comprehensive ways to detect such modifications is by the recent advancement of direct RNA sequencing from Oxford Nanopore Technologies (ONT). However, this method obtains a large amount of data with high complexity in the form of raw current signal that poses a new informatics challenge to accurately detect those modifications. Here, we provide nanoDoc2, a software to detect multiple types of RNA modification from nanopore direct RNA sequencing data. The nanoDoc2 includes a novel signal segmentation algorithm based on the trace value-a base probability feature that is added by the Guppy basecalling program from ONT during processing of the raw signal. The core of nanoDoc2 includes a machine learning algorithm in which a 6-mer segmented raw current signal is analyzed by deep one-class classification using a WaveNet-based neural network. As an output, an RNA modification is detected by a statistical score in each candidate position. Herein, we describe the detailed instructions on how to use nanoDoc2 for signal segmentation, train/test the neural network, and finally predict RNA modifications present in nanopore direct RNA sequencing data., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cancer immunotherapy, which modulates immune responses against tumors using immune-checkpoint inhibitors or adoptive cell transfer, has emerged as a novel and promising therapy for tumors. However, only a minority of patients demonstrate durable responses, while the majority of patients are resistant to immunotherapy. The immune system can paradoxically constrain and promote tumor development and progression. This process is referred to as cancer immunoediting. The mechanisms of resistance to immunotherapy seem to be that cancer cells undergo immunoediting to evade recognition and elimination by the immune system. RNA modifications, specifically N 6 -methyladenosine (m 6 A) methylation, have emerged as a key regulator of various post-transcriptional gene regulatory processes, such as RNA export, splicing, stability, and degradation, which play unappreciated roles in various physiological and pathological processes, including immune system development and cancer pathogenesis. Therefore, a deeper understanding of the mechanisms by which RNA modifications impact the cancer immunoediting process can provide insight into the mechanisms of resistance to immunotherapies and the strategies that can be used to overcome such resistance. In this chapter, we briefly introduce the background of cancer immunoediting and immunotherapy. We also review and discuss the roles and mechanisms of RNA m 6 A modifications in fine-tuning the innate and adaptive immune responses, as well as in regulating tumor escape from immunosurveillance. Finally, we summarize the current strategies targeting m 6 A regulators for cancer immunotherapy., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In addition to A, C, G and U, RNA contains over 100 additional chemically distinct residues. An abundant modified base frequently found in tRNAs, dihydrouridine (D) has recently been mapped to over 100 positions in mRNAs in yeast and human cells. Multiple highly conserved dihydrouridine synthases associate with and modify mRNA, suggesting there are many D sites yet to be found. Because D alters RNA structure, installation of D in mRNA is likely to effect multiple steps in mRNA metabolism including processing, trafficking, translation, and degradation. Here, we introduce D-seq, a method to chart the D landscape at single nucleotide resolution. The included protocols start with RNA isolation and carry through D-seq library preparation and data analysis. While the protocols below are tailored to map Ds in mRNA, the D-seq method is generalizable to any RNA type of interest, including non-coding RNAs, which have also recently been identified as dihydrouridine synthase targets., (Copyright © 2023. Published by Elsevier Inc.)

As one of the main characteristics of neoplasia, metabolic reprogramming provides nutrition and energy to enhance cell proliferation and maintain environment homeostasis. Glycolysis is one of the most important components of cancer metabolism and the Warburg effect contributes to the competitive advantages of cancer cells in the threatened microenvironment. Studies show strong links between N 6 -methyladenosine (m 6 A) modification and metabolic recombination of cancer cells. As the most abundant modification in eukaryotic RNA, m 6 A methylation plays important roles in regulating RNA processing, including splicing, stability, transportation, translation and degradation. The aberration of m 6 A modification can be observed in a variety of diseases such as diabetes, neurological diseases and cancers. This review describes the mechanisms of m 6 A on cancer glycolysis and their applications in cancer therapy and prognosis evaluation, aiming to emphasize the importance of targeting m 6 A in modulating cancer metabolism., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Posttranscriptional RNA modifications occur in almost all types of RNA in all life forms. As an abundant RNA modification in mammals, pseudouridine (Ψ) regulates diverse biological functions of different RNA species such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. However, the functional investigation of mRNA pseudouridine (Ψ) has been hampered by the lack of a quantitative method that can efficiently map Ψ transcriptome-wide. We developed bisulfite-induced deletion sequencing (BID-seq), with the optimized bisulfite-based chemical reaction to convert pseudouridine selectively and completely into Ψ-BS adduct without cytosine deamination. The Ψ-BS adduct can be further read out as deletion signatures during reverse transcription. The deletion ratios induced by Ψ sites were used for estimating the modification stoichiometry at each modified site. BID-seq starts with 10-20 ng polyA + RNA and detects thousands of mRNA Ψ sites with stoichiometry information in cell lines and tissues. We uncovered consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to individual Ψ deposition. BID-seq also confirmed the presence of Ψ within stop codons of mammalian mRNA. BID-seq set the stage for future investigations of Ψ functions in diverse biological processes., (Copyright © 2023. Published by Elsevier Inc.)

5-Formylcytidine (f 5 C) is one of the epigenetic nucleotides in tRNA. Despite the evident importance of f 5 C in gene expression regulation, little is known about its exact amount and position. To capture this information, we developed a modification-specific functionalization with a semi-stabilized ylide. The chemical labelling exhibited a high selectivity towards f 5 C and allowed distinction from similar 5-formyluridine. We realized a detection strategy based on the fluorescence signal of the cyclization product 4,5-pyridin-2-amine-cytidine paC, which exhibited a high quantum yield. The results clearly identified f 5 C with a limit of detection at 0.58 nM. This method altered the hydrogen bonding activities of f 5 C and modulated its reverse transcription signature in its sequencing profile. We showed that f 5 C can be detected from tRNA segments with a single-base resolution. Taken together, this approach is a sensitive, antibody-free, and applicable detection and sequencing method for f 5 C-containing RNA., (© 2022 Wiley-VCH GmbH.)

N 6 A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. m 6 A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. m 6 A methyltransferase complexes and demethylases are involved in the dynamic and reversible regulation of m 6 A modification, while m 6 A modification and regulate RNA metabolism, including splicing, translocation, stability, and translation. m 6 A modification and regulate RNA metabolism, including splicing, translocation, stability, and translation. m 6 A is found to play a vital role in all the fundamental biological processes, including the regulation of the immune system, from normal development to diseases. The normal development and functioning of the immune system rely on the precise regulation of the relevant gene expression, and m 6 A modification is crucial to the regulation. Herein, we reviewed the concept, the functions, and the mechanism of m 6 A modification, concentrating on its role in immune system function modulation. Furthermore, we highlighted present obstacles and future research directions in the field, intending to provide a theoretical foundation and reference for future study on how m 6 A modification is involved in immune regulation., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Post-transcriptional modifications play an important role in several processes, including translation, splicing, and RNA degradation in eukaryotic cells. To investigate the function of specific modifications it is of high interest to develop tools for sequence-specific RNA-targeting. This work focuses on two abundant modifications of eukaryotic mRNA, namely methylation of the guanine-N7 position of the 5'-cap and internal N 6 -methyladenosine (m 6 A). We describe the sequence-specific targeting of model RNA transcripts via RNA-binding proteins, such as nuclease-deficient RNA-targeting Cas9 (RCas9) and the Pumilio homology domain (PumHD) fused to two different effector enzymes, the dioxygenase FTO and the guanine-N7 methyltransferase Ecm1. With this tool, we were able to install and remove the methylation at the respective positions with high specificity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

AlkB homolog 5 (ALKBH5) is an RNA m 6 A demethylase involved in the regulation of genes transcription, translation and metabolism and has been considered as a promising therapeutic target for various human diseases, especially cancers. However, there is still a lack of potent and selective ALKBH5 inhibitors. Herein, we report a new class of ALKBH5 inhibitors containing the 1-aryl-1H-pyrazole scaffold, which were obtained through fluorescence polarization-based screening, structural optimization and structure-activity relationship analysis. Among these compounds, 20m was the most potent one, which showed an IC 50 value of 0.021 μM in fluorescence polarization assay. Compound 20m exhibited high selectivity towards ALKBH5 versus FTO as well as other AlkB subfamily members, indicating good selectivity for ALKBH5. Cellular thermal shift assay (CETSA) analysis showed that 20m could efficiently stabilize ALKBH5 in HepG2 cells. Dot blot assay demonstrated that 20m could increase m 6 A level in intact cells. Collectively, 20m is a potent, selective and cell active ALKBH5 inhibitor and could be used as a versatile chemical probe to explore the biological function of ALKBH5., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

To date, over 150 different RNA modifications have been identified, playing crucial roles in biological processes and disease pathogenesis. Thanks to the advancement of high-throughput sequencing technologies employed for transcriptome-wide mapping, a bunch of RNA modification databases have emerged as an exciting area, which promotes further investigation of the mechanisms and functions of these modified ribonucleotides. This article introduces the high-throughput sequencing technique developed for transcriptome-wide mapping of RNA modifications, as well as the procedures and main techniques of building these databases from the developers' perspective. It also reviews existing RNA modification databases in terms of their main functions, species, the number of sites they collected, the annotations, and the tools they provided. From the view of users, we further analyze and compare these databases in terms of their functions. For instance, these databases can be applied to record chemical structures and biosynthetic pathways, or unravel the epi-transcriptome comprehensively, or only investigate specific features of RNA modifications. Additionally, the limitations of the existing approaches are discussed, and some future suggestions are offered., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Traditional epitranscriptome profiling approach relies on specific antibodies or chemical treatments to capture modified RNA molecules and then applies high throughput sequencing to identify their transcriptomic locations. However, due to the lack of suitable or high-quality antibodies, only a small proportion of the 170 documented RNA modifications were profiled with those approaches. Direct sequencing of native RNA molecules using Oxford Nanopore Technologies (ONT) enabled straight inspection of RNA modifications and offered a promising alternative solution. N6-methyladenosine (m 6 A) is known to cause characteristic changes and increased base call errors of ONT signals compared with non-modified adenosines, based on which, the m 6 A sites can be identified directly from ONT signals. Meanwhile, a number of studies have shown that it is possible to predict m 6 A sites from RNA primary sequences. Using the m 6 A sites revealed by Illumina technology as benchmark, we showed that, the accuracy of ONT-based m 6 A site prediction can be further increased by integrating additional information from the primary sequences of RNA (AUROC of 0.918), compared with using ONT signals only (AUROC 0.878 using Base call error features, and 0.804 using Tombo features), providing a new perspective for more reliable mining of the relatively noisy ONT signals., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m 6 A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m 6 A. Here we introduce m 6 A-REF-seq, a powerful and straightforward method to identify m 6 A at single-nucleotide resolution. m 6 A-REF-seq relies on the recognition of RNA endonuclease MazF towards m 6 A at the ACA motif, providing an orthogonal method independent of the m 6 A antibody being adopted by most of current methods. We describe a detailed protocol to perform m 6 A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m 6 A sites identified by m 6 A-REF-seq, which can also be applied to detect any candidate m 6 A sites., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Modified nucleotides within cellular RNAs significantly influence their biogenesis, stability, and function. As reviewed here, 3-methylcytidine (m 3 C) has recently come to the fore through the identification of the methyltransferases responsible for installing m 3 C 32 in human tRNAs. Mechanistic details of how m 3 C 32 methyltransferases recognize their substrate tRNAs have been uncovered and the biogenetic and functional relevance of interconnections between m 3 C 32 and modified adenosines at position 37 highlighted. Functional insights into the role of m 3 C 32 modifications indicate that they influence tRNA structure and, consistently, lack of m 3 C 32 modifications impairs translation. Development of quantitative, transcriptome-wide m 3 C mapping approaches and the discovery of an m 3 C demethylase reveal m 3 C to be dynamic, raising the possibility that it contributes to fine-tuning gene expression in different conditions., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (>160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS.

Competing Interests: Declaration of interests No interests are declared.

Pseudouridine is one of the most abundant post-transcriptional modifications in RNA. We have previously shown that the FF99-derived parameters for pseudouridine and some of its naturally occurring derivatives in the AMBER distribution either alone or in combination with the revised γ torsion parameters (parmbsc0) failed to reproduce their conformational characteristics observed experimentally (Deb et al. in J Chem Inf Model 54:1129-1142, 2014; Deb et al. in J Comput Chem 37:1576-1588, 2016; Dutta et al. in J Chem Inf Model 60:4995-5002, 2020). However, the application of the recommended bsc0 correction did lead to an improvement in the description not only of the distribution in the γ torsional space but also of the sugar pucker distributions. In an earlier study, we examined the transferability of the revised glycosidic torsion parameters (χ IDRP ) for Ψ to its derivatives. We noticed that although these parameters in combination with the AMBER FF99-derived parameters and the revised γ torsional parameters resulted in conformational properties of these residues that were in better agreement with experimental observations, the sugar pucker distributions were still not reproduced accurately. Here we report a new set of partial atomic charges for pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine and 2'-O-methylpseudouridine and a new set of glycosidic torsional parameters (χ ND ) based on chosen glycosidic torsional profiles that most closely corresponded to the NMR data for conformational propensities and studied their effect on the conformational distributions using REMD simulations at the individual nucleoside level. We have also studied the effect of the choice of water model on the conformational characteristics of these modified nucleosides. Our observations suggest that the current revised set of parameters and partial atomic charges describe the sugar pucker distributions for these residues more accurately and that the choice of a suitable water model is important for the accurate description of their conformational properties. We have further validated the revised sets of parameters by studying the effect of substitution of uridine with pseudouridine within single stranded RNA oligonucleotides on their conformational and hydration characteristics., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

RNA plays critical roles in a wide range of physiological processes. For example, it is well known that RNA plays an important role in regulating gene expression, cell proliferation, and differentiation, and many other chemical and biological processes. However, the research community still suffers from limited approaches that can be applied to readily visualize a specific RNA-of-interest (ROI). Several methods can be used to track RNAs; these rely mainly on biological properties, namely, hybridization, aptamer, reporter protein, and protein binding. With respect to covalent approaches, very few cases have been reported. Happily, several new methods for efficient labeling studies of ROIs have been demonstrated successfully in recent years. Additionally, methods employed for the detection of ROIs by RNA modifying enzymes have also proved feasible. Several approaches, namely, phosphoramidite chemistry, in vitro transcription reactions, co-transcription reactions, chemical post-modification, RNA modifying enzymes, ligation, and other methods targeted at RNA labeling have been revealed in the past decades. To illustrate the most recent achievements, this review aims to summarize the most recent research in the field of synthesis of RNAs-of-interest bearing a variety of unnatural nucleosides, the subsequent RNA labeling research via biocompatible ligation, and beyond., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Epigenetic modifications play important roles in genome evolution and innovation. However, most analyses have focused on the evolutionary role of DNA modifications, and little is understood about the influence of posttranscriptional RNA modifications on genome evolution. To explore the evolutionary significance of RNA modifications, we generated transcriptome-wide profiles of N6-methyladenosine (m6A), the most prevalent internal modification of mRNA, for 13 representative plant species spanning over half a billion years of evolution. These data reveal the evolutionary conservation and divergence of m6A methylomes in plants, uncover the preference of m6A modifications on ancient orthologous genes, and demonstrate less m6A divergence between orthologous gene pairs with earlier evolutionary origins. Further investigation revealed that the evolutionary divergence of m6A modifications is related to sequence variation between homologs from whole-genome duplication and gene family expansion from local-genome duplication. Unexpectedly, a significant negative correlation was found between the retention ratio of m6A modifications and the number of family members. Moreover, the divergence of m6A modifications is accompanied by variation in the expression level and translation efficiency of duplicated genes from whole- and local-genome duplication. Our work reveals new insights into evolutionary patterns of m6A methylomes in plant species and their implications, and provides a resource of plant m6A profiles for further studies of m6A regulation and function in an evolutionary context., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Due to the importance of the RNA chemical modifications, methods for the selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. We have developed functional artificial nucleic acids that modify a specific site of RNA in a site- and base-selective manner. In addition, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a polyethylene glycol (PEG) linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced to the 6-thioguanine base in the oligodeoxynucleotide (ODN) in high yields. The transfer groups 11 and 12 more efficiently underwent the cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer groups 12 and 13 were determined to be promising compounds for the modification of long RNAs.

Microscale thermophoresis (MST) is a technology that allows for quantitative analysis of interactions between biomolecules with low sample consumption. MST uses localized temperature fields to measure the diffusion rates of the free and bound states of a fluorescently labeled protein, and to determine the dissociation constant K D by fitting of the binding isotherm with a 1:1 binding model. Here, we describe the use of MST for quantitative analysis of the interaction of the N-terminal his-tagged 6-methyladenine (m 6 A) reader protein YTHDF2 with m 6 A modified and unmodified RNA, in single-strand configuration or with RNA:DNA hybrid substrates. The described protocol is also suitable for studies of interactions with proteins binding to double-stranded RNA or DNA substrates., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Hypobaric hypoxia as an extreme environment in a plateau may have deleterious effects on human health. Studies have indicated that rush entry into a plateau may reduce male fertility and manifest in decreased sperm counts and weakened sperm motility. RNA modifications are sensitive to environmental changes and have recently emerged as novel post-transcriptional regulators in male spermatogenesis and intergenerational epigenetic inheritance. In the present study, we generated a mouse hypoxia model simulating the environment of 5500 m in altitude for 35 days, which led to compromised spermatogenesis, decreased sperm counts, and an increased sperm deformation rate. Using this hypoxia model, we further applied our recently developed high-throughput RNA modification quantification platform based on liquid chromatography with tandem mass spectrometry, which exhibited the capacity to simultaneously examine 25 types of RNA modifications. Our results revealed an altered sperm RNA modifications signature in the testis (6 types) and mature sperm (11 types) under the hypoxia model, with 4 types showing overlap (Am, Gm, m7G, and m22G). Our data first drew the signature of RNA modification profiles and comprehensively analyzed the alteration of RNA modification levels in mouse testis and sperm under a mouse hypoxia model. These data may be highly related to human conditions under a similar hypoxia environment., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Versatile RNA modifications play important roles in post-transcriptional regulations of gene expression, among which glycosylation modifications on small RNAs emerge as a novel clade whose characteristics need further interrogations. Here, we demonstrated that the sequence pattern around RNA glycosylation sites was not random and could be exploited for glycosylation site prediction. A machine learning predictor, GlyinsRNA, which integrated multiple RNA sequence representation encodings, was established. GlyinsRNA achieved AUROC (area under the receiver operating characteristic curve) of 0.7933 and 0.7979 in five-fold cross-validation and independent tests, respectively. GlyinsRNA was implemented as an online webserver, where both the predicted glycosylation sites and the overrepresented RNA-binding protein (RBP)-related motifs were annotated to facilitate the users. GlyinsRNA webserver is freely available at http://www.rnanut.net/glyinsrna.

In addition to DNA methylation, reversible epigenetic modification occurring in RNA has been discovered recently. The most abundant type of RNA methylation is N6-methyladenosine (m6A) modification, which is dynamically regulated by methylases ("writers"), demethylases ("erasers") and m6A-binding proteins ("readers"). As an essential posttranscriptional regulator, m6A can control mRNA splicing, processing, stability, export and translation. Recent studies have revealed that m6A modification has the strongest tissue specificity for brain tissue and plays crucial roles in central nervous system (CNS) injures by affecting its downstream target genes or non-coding RNAs. This review focuses on the expression and function of m6A regulatory proteins in CNS trauma in vitro and in vivo. We also highlight the latest insights into the molecular mechanisms of pathological damage in the CNS. Understanding m6A dynamics, functions, and machinery will yield an opportunity for designing and developing novel therapeutic agents for CNS injuries., (Copyright © 2021 Elsevier Inc. All rights reserved.)