N⁶-Methyladenosine (m⁶A) is the most prevalent RNA modification on mRNAs and lncRNAs. It plays a pivotal role during various biological processes and disease pathogenesis. We present here a comprehensive knowledgebase, m⁶A-Atlas, for unraveling the m⁶A epitranscriptome. Compared to existing databases, m⁶A-Atlas features a high-confidence collection of 442 162 reliable m⁶A sites identified from seven base-resolution technologies and the quantitative (rather than binary) epitranscriptome profiles estimated from 1363 high-throughput sequencing samples. It also offers novel features, such as; the conservation of m⁶A sites among seven vertebrate species (including human, mouse and chimp), the m⁶A epitranscriptomes of 10 virus species (including HIV, KSHV and DENV), the putative biological functions of individual m⁶A sites predicted from epitranscriptome data, and the potential pathogenesis of m⁶A sites inferred from disease-associated genetic mutations that can directly destroy m⁶A directing sequence motifs. A user-friendly graphical user interface was constructed to support the query, visualization and sharing of the m⁶A epitranscriptomes annotated with sites specifying their interaction with post-transcriptional machinery (RBP-binding, microRNA interaction and splicing sites) and interactively display the landscape of multiple RNA modifications. These resources provide fresh opportunities for unraveling the m⁶A epitranscriptomes. m⁶A-Atlas is freely accessible at: www.xjtlu.edu.cn/biologicalsciences/atlas.

N⁶-methyladenosine (m⁶A) is the most prevalent post-transcriptional modification in eukaryotes, and plays a pivotal role in various biological processes, such as splicing, RNA degradation and RNA–protein interaction. We report here a prediction framework WHISTLE for transcriptome-wide m⁶A RNA-methylation site prediction. When tested on six independent datasets, our approach, which integrated 35 additional genomic features besides the conventional sequence features, achieved a major improvement in the accuracy of m⁶A site prediction (average AUC: 0.948 and 0.880 under the full transcript or mature messenger RNA models, respectively) compared to the state-of-the-art computational approaches MethyRNA (AUC: 0.790 and 0.732) and SRAMP (AUC: 0.761 and 0.706). It also out-performed the existing epitranscriptome databases MeT-DB (AUC: 0.798 and 0.744) and RMBase (AUC: 0.786 and 0.736), which were built upon hundreds of epitranscriptome high-throughput sequencing samples. To probe the putative biological processes impacted by changes in an individual m⁶A site, a network-based approach was implemented according to the ‘guilt-by-association’ principle by integrating RNA methylation profiles, gene expression profiles and protein–protein interaction data. Finally, the WHISTLE web server was built to facilitate the query of our high-accuracy map of the human m⁶A epitranscriptome, and the server is freely available at: www.xjtlu.edu.cn/biologicalsciences/whistle and http://whistle-epitranscriptome.com.

5-Methylcytosine (m⁵C) is one of the most prevalent covalent modifications on RNA. It is known to regulate a broad variety of RNA functions, including nuclear export, RNA stability and translation. Here, we present m⁵C-Atlas, a database for comprehensive collection and annotation of RNA 5-methylcytosine. The database contains 166 540 m⁵C sites in 13 species identified from 5 base-resolution epitranscriptome profiling technologies. Moreover, condition-specific methylation levels are quantified from 351 RNA bisulfite sequencing samples gathered from 22 different studies via an integrative pipeline. The database also presents several novel features, such as the evolutionary conservation of a m⁵C locus, its association with SNPs, and any relevance to RNA secondary structure. All m⁵C-atlas data are accessible through a user-friendly interface, in which the m⁵C epitranscriptomes can be freely explored, shared, and annotated with putative post-transcriptional mechanisms (e.g. RBP intermolecular interaction with RNA, microRNA interaction and splicing sites). Together, these resources offer unprecedented opportunities for exploring m⁵C epitranscriptomes. The m⁵C-Atlas database is freely accessible at https://www.xjtlu.edu.cn/biologicalsciences/m5c-atlas.

N⁶-Methyladenosine (m⁶A) is one of the most abundant internal chemical modifications on eukaryote mRNA and is involved in numerous essential molecular functions and biological processes. To facilitate the study of this important post-transcriptional modification, we present here m⁶A-Atlas v2.0, an updated version of m⁶A-Atlas. It was expanded to include a total of 797 091 reliable m⁶A sites from 13 high-resolution technologies and two single-cell m⁶A profiles. Additionally, three methods (exomePeaks2, MACS2 and TRESS) were used to identify >16 million m⁶A enrichment peaks from 2712 MeRIP-seq experiments covering 651 conditions in 42 species. Quality control results of MeRIP-seq samples were also provided to help users to select reliable peaks. We also estimated the condition-specific quantitative m⁶A profiles (i.e. differential methylation) under 172 experimental conditions for 19 species. Further, to provide insights into potential functional circuitry, the m⁶A epitranscriptomics were annotated with various genomic features, interactions with RNA-binding proteins and microRNA, potentially linked splicing events and single nucleotide polymorphisms. The collected m⁶A sites and their functional annotations can be freely queried and downloaded via a user-friendly graphical interface at: http://rnamd.org/m6a.

With advanced technologies to map RNA modifications, our understanding of them has been revolutionized, and they are seen to be far more widespread and important than previously thought. Current next-generation sequencing (NGS)-based modification profiling methods are blind to RNA modifications and thus require selective chemical treatment or antibody immunoprecipitation methods for particular modification types. They also face the problem of short read length, isoform ambiguities, biases and artifacts. Direct RNA sequencing (DRS) technologies, commercialized by Oxford Nanopore Technologies (ONT), enable the direct interrogation of any given modification present in individual transcripts and promise to address the limitations of previous NGS-based methods. Here, we present the first ONT-based database of quantitative RNA modification profiles, DirectRMDB, which includes 16 types of modification and a total of 904,712 modification sites in 25 species identified from 39 independent studies. In addition to standard functions adopted by existing databases, such as gene annotations and post-transcriptional association analysis, we provide a fresh view of RNA modifications, which enables exploration of the epitranscriptome in an isoform-specific manner. The DirectRMDB database is freely available at: http://www.rnamd.org/directRMDB/.

Deciphering the biological impacts of millions of single nucleotide variants remains a major challenge. Recent studies suggest that RNA modifications play versatile roles in essential biological mechanisms, and are closely related to the progression of various diseases including multiple cancers. To comprehensively unveil the association between disease-associated variants and their epitranscriptome disturbance, we built RMDisease, a database of genetic variants that can affect RNA modifications. By integrating the prediction results of 18 different RNA modification prediction tools and also 303,426 experimentally-validated RNA modification sites, RMDisease identified a total of 202,307 human SNPs that may affect (add or remove) sites of eight types of RNA modifications (m⁶A, m⁵C, m¹A, m⁵U, Ψ, m⁶Am, m⁷G and Nm). These include 4,289 disease-associated variants that may imply disease pathogenesis functioning at the epitranscriptome layer. These SNPs were further annotated with essential information such as post-transcriptional regulations (sites for miRNA binding, interaction with RNA-binding proteins and alternative splicing) revealing putative regulatory circuits. A convenient graphical user interface was constructed to support the query, exploration and download of the relevant information. RMDisease should make a useful resource for studying the epitranscriptome impact of genetic variants via multiple RNA modifications with emphasis on their potential disease relevance. RMDisease is freely accessible at: www.xjtlu.edu.cn/biologicalsciences/rmd.