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The methylation of adenosine residues on the long non-coding RNA XIST is essential for X-chromosome transcriptional repression during female mammalian development.

Fat mass and obesity-associated protein (FTO) preferentially demethylates m⁶A_m, a modified adenosine that, when present at the 5′ end of certain mRNAs, positively influences mRNA stability by preventing DCP2-mediated decapping.

Five experts discuss topical aspects of RNA modifications, highlighting where the research field stands, recommendations for best practices and visions for the future of epitranscriptomics studies.

In this Review Luo, Kharas and Jaffrey outline how N⁶-methyladenosine (m⁶A) RNA modification affects RNA stability, translation, splicing and immune responses to influence cancer biology. They discuss emerging evidence on how m⁶A may influence cancer metabolic reprogramming and outline the challenges and opportunities of targeting m⁶A writers, erasers and readers for cancer therapy.

This work introduces Cyclone, an acyclovir-controlled cassette that enables regulation of both transgene and endogenous gene expression with minimal background activity.

A highly photostable RNA mimic of red fluorescent protein, Corn, was designed and used to image RNA polymerase III transcription. Quantitative imaging of Corn-tagged transcripts revealed transcription dynamics in live cells following mTOR inhibition.

The results of transcriptome-wideN⁶-methyladenosine (m⁶A) mapping techniques have resolved many of the long-standing concerns regarding the physiological relevance of m⁶A, which suggests that this modification regulates mRNA fate and function. The identification of adenosine methylases and demethylases provides insights into the cellular pathways that involve m⁶A and indicates a role of m⁶A in physiological processes.

The adenine riboswitch was evolved into a fluorogenic aptamer by randomizing the sequence and size of the ligand-binding pocket. The resulting fluorogenic aptamer, Squash, is highly folded and was adapted for ratiometric live imaging of SAM.

Here the authors show that the m⁶A modification on mRNAs mediates their length-dependent enrichment in stress granules, and that mRNAs with longer exons—which usually harbor more m⁶A sites—are preferentially enriched in the granules.

Dumelie et al. asked whether biomolecular phase-separated condensates can establish microenvironments with distinct metabolomes and found that amphipathic lipids are highly enriched in these microenvironments and influence the properties of the condensates.

A ribozyme-based circularization strategy improves the stability of exogenously expressed RNA aptamers.

N⁶-methyladenosine is enriched in poly(A) tails of VSG transcripts in Trypanosoma brucei, and when lacking result in mRNA degradation.

Cap2 methylation increases on transcripts as they age, reducing activation of innate immunity.

Imaging of single mRNA molecules undergoing translation provides insights into the regulation of protein synthesis.

N¹-methyladenosine (m¹A) was recently reported as a new mRNA modification but its prevalence has been controversial. Here the authors showed that m¹A, if present in mRNA, is at very low stoichiometry, with the notable exception of MT-ND5. Further, they show that the previously reported enrichment of m¹A near the start of transcripts are false-positive identifications due to cross-reactivity of the commonly used m1A antibody with mRNA caps.

An RNA aptamer specific for a protein of interest, when fused to an RNA sensor that activates a small-molecule fluorophore, can quantitate protein expression in live bacteria.

X-ray crystallography and cryo-electron microscopy analyses of Lettuce—a DNA mimic of GFP—bound to various fluorophores reveal previously unknown structures of DNA that rival analogous RNAs in complexity.

Proteome-wide identification of ubiquitination events reveals their functional classes and identifies substrates for ubiquitin ligases.

The fate of an mRNA is regulated by internal base modifications that generate the modified nucleotides N⁶-methyladenosine, 5-methylcytosine and inosine. Three new studies show that yeast and human mRNAs also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA.

The recently discovered aptamer Beetroot is a homodimeric RNA that binds and activates DFAME, a conditional, red-shifted fluorophore derived from GFP. Here the authors determine the Beetroot-DFAME co-crystal structure, which is distinctively different from that of similar RNA aptamer Corn.

Local protein translation regulates growth cone dynamics in response to guidance cues. Here the authors show that locally translated proteins are the main targets of the ubiquitin-proteasome system, and that local signalling promotes the ubiquitination and degradation of RhoA, leading to growth cone collapse.

Conventional CRISPR-based approaches to monitor genomic loci can be hampered by high background and nonspecific nucleolar signal. Here, the authors propose a fluorogenic CRISPR (fCRISPR) tool that allows for high-contrast and sensitive imaging of genomic DNA.

Truong et al. report crystal structures of Squash, a fluorophore-activating aptamer RNA evolved from the adenine riboswitch. Squash preserves the overall scaffold of the adenine riboswitch, yet has a highly divergent ligand-binding site.

Nease and colleagues demonstrate that the methyltransferase FTSJ1 mediates selenocysteine-specific tRNA methylation of selenoproteins, which protects melanoma cells against oxidative stress and promotes metastasis.

Blander and colleagues show that concurrent detection of LPS and bacterial RNA triggers the interaction of procaspase-11 with NLRP3, upstream of the activation of either receptor and before NLRP3–ASC oligomerization.

The crystal structure of the fluorogenic RNA aptamer Corn reveals an unexpected homodimer. One molecule of the cognate fluorophore DFHO is encapsulated at the locally asymmetric RNA interface, which is notable for lacking intermolecular base pairs.

Structural elucidation of the RNA aptamer 'Spinach' reveals that a new G-quadruplex structure forms the fluorophore-binding site that confers the ability of the RNA to function as a GFP mimic.

Salisbury et al. show that hepatic lipogenic mRNA is regulated by m⁶A modifications in a sex-dependent and dietary-dependent manner, contributing to sex-specific differences in hepatic lipid metabolism.

Two different methylation states of the adenosine adjacent to the snRNA cap are found in the biogenesis process of snRNAs, Am and m6Am, whose levels are regulated by FTO and are related to alternative pre-mRNA splicing.

Nonsense-mediated RNA decay — a highly selective and conserved RNA turnover pathway — is shown to have roles in both brain development and function. In this Review, Jaffrey and Wilkinson discuss how this pathway affects the nervous system.

N⁶-methyladenosine (m⁶A) is a conserved RNA modification that has recently emerged as an important regulator of messenger RNA processing and activity. Here, the authors provide evidence that m6A pathway facilitates female-specific splicing of Sxl, regulating sex determination in Drosophila.

The Spinach2 RNA aptamer provides substantially improved imaging over its predecessor Spinach, enabling live-cell imaging of many tagged RNA species including toxic trinucleotide repeat–containing RNAs.

RNA-binding protein hnRNP A2/B1 is suggested to promote miRNA processing as a m⁶A 'reader'. Here, the authors determine crystal structures of RRM domains of hnRNP A2/B1 in complex with various RNA substrates and determine that hnRNP A2/B1 may function as an auxiliary factor in 'm⁶A switch' instead.

The cytosolic N⁶-methyladenosine (m⁶A)-binding proteins YTHDF1, YTHDF2 and YTHDF3 undergo liquid–liquid phase separation in vitro and in cells; this is enhanced by polymethylated mRNAs to form complexes that partition into different cellular phase-separated compartments.

Unique mutational signatures induced by cross-linking of m6A-specific antibodies to RNA identify m6A and m6Am residues at single-nucleotide resolution, transcriptome-wide.

Previous studies have shown genome-wide associations between polymorphisms in the gene FTO (fat mass and obesity associated) and type 2 diabetes and obesity, and genetic manipulation of Fto in mice causes feeding dysregulation and body weight changes. Here Hess et al. show that FTO affects the activity and function of midbrain dopaminergic neurons and subsequent reward-related behaviors. The study also shows that FTO acts as a demethylating enzyme for specific mRNAs in vivo, including mRNAs in the dopaminergic signaling pathway.

The presence of N¹ methyl groups on adenine bases was thought to be widespread in messenger RNAs. It now seems that these modifications are much less prevalent, and occur on mRNAs that structurally mimic transfer RNA. See Letter p.251