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Specific chemical modifications of biological molecules are an efficient way of regulating molecular function, and a plethora of downstream signalling pathways are influenced by the modification of DNA and proteins. Many of the enzymes responsible for regulating protein and DNA modifications are targets of current cancer therapies. RNA epitranscriptomics, the study of RNA modifications, is the new frontier of this arena. Despite being known since the 1970s, eukaryotic RNA modifications were mostly identified on transfer RNA and ribosomal RNA until the last decade, when they have been identified and characterized on mRNA and various non-coding RNAs. Increasing evidence suggests that RNA modification pathways are also misregulated in human cancers and may be ideal targets of cancer therapy. In this Review we highlight the RNA epitranscriptomic pathways implicated in cancer, describing their biological functions and their connections to the disease.After synthesis, all RNA molecules are subject to covalent modifications. This Review presents the evidence that RNA modification pathways are misregulated in cancer and suggests that they may be ideal targets for cancer therapy.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10–20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different ‘writer’ proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes. Pseudouridine sites in mRNA are detected at base resolution and functionally investigated.

RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid–liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble–function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.The functions of many regulatory RNAs depend on how their 3D structure changes in response to cellular conditions. Recent studies have revealed that RNA exists as a dynamic ensemble of conformations, which form with different probabilities in different cellular conditions and thus modulate RNA function.

RNA tailing, or the addition of non-templated nucleotides to the 3′ end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.The non-canonical addition of non-templated nucleotides to RNA 3′ ends (tailing) by terminal nucleotidyltransferases includes uridylation, mixed-nucleotide tailing and post-transcriptional polyadenylation. Recent studies of human terminal nucleotidyltransferases have revealed their distinct specificities for substrates, including mRNAs, microRNAs and other non-coding RNAs, and how they control RNA stability and activity.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression by altering the translation efficiency and/or stability of targeted mRNAs. In vertebrates, more than 50 % of all protein-coding RNAs are assumed to be subject to miRNA-mediated control, but current high-throughput methods that reliably measure miRNA–mRNA interactions either require prior knowledge of target mRNAs or elaborate preparation procedures. Consequently, experimentally validated interactions are relatively rare. Furthermore, in silico prediction based on sequence complementarity of miRNAs and their corresponding target sites suffers from extremely high false positive rates. Apparently, sequence complementarity alone is often insufficient to reflect the complex post-transcriptional regulation of mRNAs by miRNAs, which is especially true for animals. Therefore, combined analysis of small non-coding and protein-coding RNAs is indispensable to better understand and predict the complex dynamics of miRNA-regulated gene expression. Single-nucleotide polymorphisms (SNPs) and alternative polyadenylation (APA) can affect miRNA binding of a given transcript from different individuals and tissues, and especially APA is currently emerging as a major factor that contributes to variations in miRNA–mRNA interplay in animals. In this review, we focus on the influence of APA and SNPs on miRNA-mediated gene regulation and discuss the computational approaches that take these mechanisms into account.

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m⁵C, Ψ, and m¹A at single-base resolution transcriptome-wide. With RBS-Seq, m⁵C and m¹A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m⁵C, m¹A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m⁵C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m⁵C sites in noncoding and coding RNAs and the absence of substantial m¹A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Key Points MicroRNAs (miRNAs) are small non-coding RNAs that guide post-transcriptional gene regulation to shape the rate at which genetic information is converted into proteins. Due to this, miRNAs contribute to the establishment of gene expression patterns that are required for normal development and physiology in plants and animals. RNase III enzymes, together with specific double-stranded RNA-binding partner proteins, produce miRNAs from genomically encoded precursor miRNAs (pre-miRNAs). In rare cases, nucleases from other cellular pathways can replace RNase III enzymes in the production of miRNAs. After the assembly of miRNA duplexes, these small RNAs are loaded into proteins from the Argonaute (AGO) protein family. AGO proteins organize small RNAs into subdomains, including the seed sequence, which mediates target RNA binding. The mechanisms by which miRNAs function include endonucleolytic cleavage, translational repression and mRNA turnover. Recent evidence suggests that small RNA stability can be influenced by miRNA sequence motifs, chemical modifications and interactions with target mRNAs. miRNAs are annotated as single sequences, but recent high-throughput efforts to catalogue small RNAs from various organisms, tissues and cell types reveal that most miRNAs comprise multiple isoforms. Several mechanisms have been shown to diversify miRNA sequence and function. The advent of next-generation sequencing technology has revealed the miRNAs of key model organisms, but the extent to which each miRNA contributes to the regulation of targets in the transcriptome of a given cell type remains unclear. The biochemical and biophysical properties of miRNA silencing complexes provide a quantitative framework for their reciprocal function and their targets, according to their abundance and relative stoichiometry inside the cell. The mechanisms that regulate miRNA stability and the generation of distinct miRNA isoforms are beginning to be elucidated. Better understanding of how such miRNAs mediate gene expression control will require quantitative analyses that dissect different models of miRNA function. MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are only now beginning to understand how they are generated, assembled into functional complexes and destroyed. Various mechanisms have now been identified that regulate miRNA stability and that diversify miRNA sequences to create distinct isoforms. The production of different isoforms of individual miRNAs in specific cells and tissues may have broader implications for miRNA-mediated gene expression control. Rigorously testing the many discrepant models for how miRNAs function using quantitative biochemical measurements made in vivo and in vitro remains a major challenge for the future.

Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2′-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2′-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation. Dynamic changes in 2′-O-methylation of rRNA in human cells lead to ribosome heterogeneity and result in altered translation of select mRNAs, correlating with changes in cellular phenotypes.

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3′-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3′ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d. In this extension to their NET-seq protocol, the authors combine isolation of 4sU-labeled chromatin-associated nascent RNA with long-read direct RNA sequencing on nanopores to profile the kinetics and patterns of co-transcriptional RNA processing.

Key Points MicroRNAs (miRNAs) are an extensive class of small non-coding RNAs that act as post-transcriptional regulators of gene expression in most cell types, including neurons. In animals, miRNAs mostly regulate gene expression at the level of mRNA translation through a mechanism that is still controversial. A subset of neuronal miRNAs are localized in the synaptodendritic compartment, where they participate in the local control of protein synthesis during synapse development and plasticity. Currently, more than 20 different miRNAs have been identified in dendrites of vertebrate neurons, but this is probably just the tip of the iceberg. The ability of miRNAs to regulate neuronal mRNA translation is itself subject to activity-dependent control. Neural activity controls miRNA transcription, subcellular localization and processing and the function of miRNA-associated multiprotein complexes. During the early stages of synaptogenesis, miRNA-regulated mechanisms are involved in the growth, remodelling and targeting of dendrites in both vertebrate and invertebrate model systems. The initial formation of synaptic contacts seems to be independent of miRNA activity. At least two miRNAs, miR-134 and miR-138, regulate the morphology of dendritic spines, the major sites of excitatory synaptic transmission in the vertebrate brain. miRNA-regulated pathways that are relevant for spine plasticity seem to converge on the actin cytoskeleton. Examples of a function of miRNAs in synaptic plasticity relevant to learning and memory have now been provided by several studies from invertebrates ( Caenorhabditis elegans , Drosophila melanogaster and Aplysia californica ). The miRNA-dependent regulation of important plasticity proteins, such as the neurotransmitter receptors CAMKII and CREB, might underlie this function. Several forms of higher-order processing, such as regulation of the circadian clock and neuroadaptations to drugs of abuse, have been shown to be influenced by miRNA activity. However, the relevance of miRNAs for synaptic plasticity in the mammalian system in vivo is yet to be determined. Several neuronal miRNA targets have been implicated in neurological diseases that are connected to synaptic dysfunction, for example autism-spectrum disorders and mental retardation. Impaired miRNA-dependent fine-tuning of synaptic proteins could result in defective neuronal homeostasis, a hallmark of these diseases. MicroRNAs are emerging as key modulators of post-transcriptional gene regulation in the synaptodendritic compartment. Here, Schratt reviews recent studies showing that neural activity controls microRNA transcription, subcellular localization, processing and function, and discusses the relevance of microRNAs for synapse development and plasticity. MicroRNAs (miRNAs) are emerging as key modulators of post-transcriptional gene regulation in a plethora of tissues, including the nervous system. Recent evidence points to a widespread role for neural miRNAs at various stages of synaptic development, including dendritogenesis, synapse formation and synapse maturation. Furthermore, studies from invertebrates indicate that miRNAs might contribute to the control of synapse function and plasticity in the adult. Key features of synapse-relevant miRNAs include their ability to regulate mRNA translation locally in the synaptodendritic compartment and the modulation of their expression and function by neuronal activity. The potentially huge impact of miRNA-based mechanisms on higher-order processing, memory and neuropsychiatric disorders in vertebrates is just starting to be recognized.