Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

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.

Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκß pathway.

BACKGROUND AND AIMS: Mitochondrial antiviral signaling protein (MAVS) is a critical regulator that activates the host's innate immunity against RNA viruses, and its signaling pathway has been linked to the secretion of proinflammatory cytokines. However, the actions of MAVS on inflammatory pathways during the development of metabolic dysfunction-associated steatotic liver disease (MASLD) have been little studied. APPROACH AND RESULTS: Liver proteomic analysis of mice with genetically manipulated hepatic p63, a transcription factor that induces liver steatosis, revealed MAVS as a target downstream of p63. MAVS was thus further evaluated in liver samples from patients and in animal models with MASLD. Genetic inhibition of MAVS was performed in hepatocyte cell lines, primary hepatocytes, spheroids, and mice. MAVS expression is induced in the liver of both animal models and people with MASLD as compared with those without liver disease. Using genetic knockdown of MAVS in adult mice ameliorates diet-induced MASLD. In vitro, silencing MAVS blunts oleic and palmitic acid-induced lipid content, while its overexpression increases the lipid load in hepatocytes. Inhibiting hepatic MAVS reduces circulating levels of the proinflammatory cytokine TNFα and the hepatic expression of both TNFα and NFκß. Moreover, the inhibition of ERK abolished the activation of TNFα induced by MAVS. The posttranslational modification O -GlcNAcylation of MAVS is required to activate inflammation and to promote the high lipid content in hepatocytes. CONCLUSIONS: MAVS is involved in the development of steatosis, and its inhibition in previously damaged hepatocytes can ameliorate MASLD.

Inhibition of hepatic p63 ameliorates steatohepatitis with fibrosis in mice.

OBJECTIVE: p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of liver TAp63 in the progression of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis is unknown. METHODS: We evaluated the hepatic p63 levels in different mouse models of steatohepatitis with fibrosis induced by diet. Next, we used virogenetic approaches to manipulate the expression of TAp63 in adult mice under diet-induced steatohepatitis with fibrosis and characterized the disease condition. Finally, we performed proteomics analysis in mice with overexpression and knockdown of hepatic TAp63. RESULTS: Levels of TAp63, but not of ΔN isoform, are increased in the liver of mice with diet-induced steatohepatitis with fibrosis. Both preventive and interventional strategies for the knockdown of hepatic TAp63 significantly ameliorated diet-induced steatohepatitis with fibrosis in mice fed a methionine- and choline-deficient diet (MCDD) and choline deficient and high fat diet (CDHFD). The overexpression of hepatic TAp63 in mice aggravated the liver condition in mice fed a CDHFD. Proteomic analysis in the liver of these mice revealed alteration in multiple proteins and pathways, such as oxidative phosphorylation, antioxidant activity, peroxisome function and LDL clearance. CONCLUSIONS: These results indicate that liver TAp63 plays a critical role in the progression of diet-induced steatohepatitis with fibrosis, and its inhibition ameliorates the disease.

Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

RATIONALE: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable. METHODS: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns. RESULTS: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families. CONCLUSIONS: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.

N6-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity, and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model, the Ts65Dn, through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types. Remarkably, we observed a significant shift in the transcriptomic profile of granule cells in the dentate gyrus (DG) associated with trisomy. We identified the downregulation of a specific small nucleolar RNA host gene, Snhg11, as the primary driver behind this observed shift in the trisomic DG. Notably, reduced levels of Snhg11 in this region were also observed in a distinct DS mouse model, the Dp(16)1Yey, as well as in human postmortem brain tissue, indicating its relevance in Down syndrome. To elucidate the function of this long non-coding RNA (lncRNA), we knocked down Snhg11 in the DG of wild-type mice. Intriguingly, this intervention alone was sufficient to impair synaptic plasticity and adult neurogenesis, resembling the cognitive phenotypes associated with trisomy in the hippocampus. Our study uncovers the functional role of Snhg11 in the DG and underscores the significance of this lncRNA in intellectual disability. Furthermore, our findings highlight the importance of DG in the memory deficits observed in Down syndrome.

p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway.

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-ß1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance.

OBJECTIVE: Free fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance. METHODS: Central FFAR1 agonism and virogenic knockdown were performed in mice. Energy balance studies, infrared thermographic analysis of brown adipose tissue (BAT) and molecular analysis of the hypothalamus, BAT, white adipose tissue (WAT) and liver were carried out. RESULTS: Pharmacological stimulation of FFAR1, using central administration of its agonist TUG-905 in diet-induced obese mice, decreases body weight and is associated with increased energy expenditure, BAT thermogenesis and browning of subcutaneous WAT (sWAT), as well as reduced AMP-activated protein kinase (AMPK) levels, reduced inflammation, and decreased endoplasmic reticulum (ER) stress in the hypothalamus. As FFAR1 is expressed in distinct hypothalamic neuronal subpopulations, we used an AAV vector expressing a shRNA to specifically knockdown Ffar1 in proopiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) of obese mice. Our data showed that knockdown of Ffar1 in POMC neurons promoted hyperphagia and body weight gain. In parallel, these mice developed hepatic insulin resistance and steatosis. CONCLUSIONS: FFAR1 emerges as a new hypothalamic nutrient sensor regulating whole body energy balance. Moreover, pharmacological activation of FFAR1 could provide a therapeutic advance in the management of obesity and its associated metabolic disorders.

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types. Remarkably, we observed a significant shift in the transcriptomic profile of granule cells in the dentate gyrus (DG) associated with trisomy. We identified the downregulation of a specific small nucleolar RNA host gene, Snhg11, as the primary driver behind this observed shift in the trisomic DG. Notably, reduced levels of Snhg11 in this region were also observed in a distinct DS mouse model, the Dp(16)1Yey, as well as in human postmortem tissue, indicating its relevance in Down syndrome. To elucidate the function of this long non-coding RNA (lncRNA), we knocked down Snhg11 in the DG of wild-type mice. Intriguingly, this intervention alone was sufficient to impair synaptic plasticity and adult neurogenesis, resembling the cognitive phenotypes associated with trisomy in the hippocampus. Our study uncovers the functional role of Snhg11 in the DG and underscores the significance of this lncRNA in intellectual disability. Furthermore, our findings highlight the importance of the DG in the memory deficits observed in Down syndrome.

Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

Hepatocyte-specific O-GlcNAc transferase downregulation ameliorates nonalcoholic steatohepatitis by improving mitochondrial function.

OBJECTIVE: O-GlcNAcylation is a post-translational modification that directly couples the processes of nutrient sensing, metabolism, and signal transduction, affecting protein function and localization, since the O-linked N-acetylglucosamine moiety comes directly from the metabolism of glucose, lipids, and amino acids. The addition and removal of O-GlcNAc of target proteins are mediated by two highly conserved enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA), respectively. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, and cardiovascular diseases. The contribution of deregulated O-GlcNAcylation to the progression and pathogenesis of NAFLD remains intriguing, and a better understanding of its roles in this pathophysiological context is required to uncover novel avenues for therapeutic intervention. By using a translational approach, our aim is to describe the role of OGT and O-GlcNAcylation in the pathogenesis of NAFLD. METHODS: We used primary mouse hepatocytes, human hepatic cell lines and in vivo mouse models of steatohepatitis to manipulate O-GlcNAc transferase (OGT). We also studied OGT and O-GlcNAcylation in liver samples from different cohorts of people with NAFLD. RESULTS: O-GlcNAcylation was upregulated in the liver of people and animal models with steatohepatitis. Downregulation of OGT in NAFLD-hepatocytes improved diet-induced liver injury in both in vivo and in vitro models. Proteomics studies revealed that mitochondrial proteins were hyper-O-GlcNAcylated in the liver of mice with steatohepatitis. Inhibition of OGT is able to restore mitochondrial oxidation and decrease hepatic lipid content in in vitro and in vivo models of NAFLD. CONCLUSIONS: These results demonstrate that deregulated hyper-O-GlcNAcylation favors NAFLD progression by reducing mitochondrial oxidation and promoting hepatic lipid accumulation.

Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart.

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.

Ribosomes are macromolecular machines responsible for protein synthesis in all living beings. Recent studies have shown that ribosomes can be heterogeneous in their structure, possibly leading to a specialized function. Here, we focus on RPL3L, a ribosomal protein expressed exclusively in striated muscles. We find that the deletion of the Rpl3l gene in a mouse model triggers a compensation mechanism, in which the missing RPL3L protein is replaced by its paralogue, RPL3. Furthermore, we find that RPL3-containing ribosomes establish closer interactions with mitochondria, cellular organelles responsible for energy production, leading to higher energy production when compared with RPL3L-containing ribosomes. Finally, we show that the RPL3­RPL3L compensation mechanism is also triggered in heart disease conditions, such as hypertrophy and myocardial infarction.

Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores.

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies. We introduce the key concepts of MoP2 and provide a step-by-step fully reproducible and complete example of how to use the workflow for the analysis of S. cerevisiae total RNA samples sequenced using MinION flowcells. The workflow starts with the pre-processing of raw FAST5 files, which includes basecalling, read quality control, demultiplexing, filtering, mapping, estimation of per-gene/transcript abundances, and transcriptome assembly, with support of the GPU computing for the basecalling and read demultiplexing steps. The secondary analyses of the workflow focus on the estimation of RNA poly(A) tail lengths and the identification of RNA modifications. The MoP2 code is available at https://github.com/biocorecrg/MOP2 and is distributed under the MIT license.

Hepatic p63 regulates glucose metabolism by repressing SIRT1.

OBJECTIVE: p63 is a transcription factor within the p53 protein family that has key roles in development, differentiation and prevention of senescence, but its metabolic actions remain largely unknown. Herein, we investigated the physiological role of p63 in glucose metabolism. DESIGN: We used cell lines and mouse models to genetically manipulate p63 in hepatocytes. We also measured p63 in the liver of patients with obesity with or without type 2 diabetes (T2D). RESULTS: We show that hepatic p63 expression is reduced on fasting. Mice lacking the specific isoform TAp63 in the liver (p63LKO) display higher postprandial and pyruvate-induced glucose excursions. These mice have elevated SIRT1 levels, while SIRT1 knockdown in p63LKO mice normalises glycaemia. Overexpression of TAp63 in wild-type mice reduces postprandial, pyruvate-induced blood glucose and SIRT1 levels. Studies carried out in hepatocyte cell lines show that TAp63 regulates SIRT1 promoter by repressing its transcriptional activation. TAp63 also mediates the inhibitory effect of insulin on hepatic glucose production, as silencing TAp63 impairs insulin sensitivity. Finally, protein levels of TAp63 are reduced in obese persons with T2D and are negatively correlated with fasting glucose and homeostasis model assessment index. CONCLUSIONS: These results demonstrate that p63 physiologically regulates glucose homeostasis.

Myeloid p38 activation maintains macrophage-liver crosstalk and BAT thermogenesis through IL-12-FGF21 axis.

Obesity features excessive fat accumulation in several body tissues and induces a state of chronic low-grade inflammation that contributes to the development of diabetes, steatosis, and insulin resistance. Recent research has shown that this chronic inflammation is crucially dependent on p38 pathway activity in macrophages, suggesting p38 inhibition as a possible treatment for obesity comorbidities. Nevertheless, we report here that lack of p38 activation in myeloid cells worsens high-fat diet-induced obesity, diabetes, and steatosis. Deficient p38 activation increases macrophage IL-12 production, leading to inhibition of hepatic FGF21 and reduction of thermogenesis in the brown fat. The implication of FGF21 in the phenotype was confirmed by its specific deletion in hepatocytes. We also found that IL-12 correlates with liver damage in human biopsies, indicating the translational potential of our results. Our findings suggest that myeloid p38 has a dual role in inflammation and that drugs targeting IL-12 might improve the homeostatic regulation of energy balance in response to metabolic stress.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Exploring the epitranscriptome by native RNA sequencing.

Chemical RNA modifications, collectively referred to as the "epitranscriptome," are essential players in fine-tuning gene expression. Our ability to analyze RNA modifications has improved rapidly in recent years, largely due to the advent of high-throughput sequencing methodologies, which typically consist of coupling modification-specific reagents, such as antibodies or enzymes, to next-generation sequencing. Recently, it also became possible to map RNA modifications directly by sequencing native RNAs using nanopore technologies, which has been applied for the detection of a number of RNA modifications, such as N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). However, the signal modulations caused by most RNA modifications are yet to be determined. A global effort is needed to determine the signatures of the full range of RNA modifications to avoid the technical biases that have so far limited our understanding of the epitranscriptome.