Methylation of the N6 position of adenosine (m6A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity ...risk gene, encodes an m6A demethylase implicates m6A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m6A localization, which combines m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m6A, indicating that m6A is a common base modification of mRNA. The m6A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m6A sites are enriched near stop codons and in 3′ UTRs, and we uncover an association between m6A residues and microRNA-binding sites within 3′ UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.
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► m6A is a widespread RNA modification in many tissues with high levels in the brain ► MeRIP-Seq identifies m6A in 7,913 genes encoding both coding and noncoding RNAs ► m6A is enriched near stop codons and within 3′ UTRs in both mouse and human mRNAs ► The transcriptome-wide landscape of m6A provides important insights into m6A function
Adenosine methylation (m6A) is a widespread RNA modification found in >7,000 mammalian genes, encoding both mRNAs and noncoding RNAs, with an especially high prevalence in the developing brain. In both mouse and human mRNAs, m6A is enriched within 3′ UTRs that also contain miRNA-binding sites.
RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by ...low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
Reading, writing and erasing mRNA methylation Zaccara, Sara; Ries, Ryan J; Jaffrey, Samie R
Nature reviews. Molecular cell biology,
10/2019, Letnik:
20, Številka:
10
Journal Article
Recenzirano
RNA methylation to form N
-methyladenosine (m
A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression ...in diverse physiological processes. Transcriptome-wide m
A mapping has revealed the distribution and pattern of m
A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m
A, providing mechanistic links connecting m
A to cellular differentiation, cancer progression and other processes. The effects of m
A on mRNA are mediated by an expanding list of m
A readers and m
A writer-complex components, as well as potential erasers that currently have unclear relevance to m
A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts - in some cases, controversies - regarding our understanding of the mechanisms and functions of m
A readers, writers and erasers.
Steady-state RNA levels are controlled by the balance between RNA synthesis and RNA turnover. A selective RNA turnover mechanism that has received recent attention in neurons is nonsense-mediated RNA ...decay (NMD). NMD has been shown to influence neural development, neural stem cell differentiation decisions, axon guidance and synaptic plasticity. In humans, NMD factor gene mutations cause some forms of intellectual disability and are associated with neurodevelopmental disorders, including schizophrenia and autism spectrum disorder. Impairments in NMD are linked to neurodegenerative disorders, including amyotrophic lateral sclerosis. We discuss these findings, their clinical implications and challenges for the future.
Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger ...RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we show that Robo3.2, a receptor for the Slit family of guidance cues, is synthesized locally within axons of commissural neurons. Robo3.2 translation is induced by floor-plate-derived signals as axons cross the spinal cord midline. Robo3.2 is also a predicted target of the nonsense-mediated mRNA decay (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding.
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•Robo3.2 is locally synthesized in growth cones of commissural axons•NMD proteins are enriched in growth cones of diverse types of neurons•NMD regulates the amount of Robo3.2 synthesized in growth cones•Defects in NMD lead to abnormal axonal trajectories in spinal cord
In growth cones of spinal-cord commissural neurons, the levels of the axon guidance receptor Robo3.2 are regulated by nonsense-mediated mRNA decay (NMD), with defects in NMD leading to abnormal axonal trajectories.
Nucleotide modifications can markedly influence mRNA processing and metabolism. New studies, one in PLOS Biology, show that approximately 130 yeast mRNAs contain dihydrouridine, a derivative of ...uridine. Functional studies show that dihydrouridine, in some cases, can affect mRNA splicing.
The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide ...or both the first and second transcribed nucleotides, respectively
. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.
Human hnRNP A2/B1 is an RNA-binding protein that plays important roles in many biological processes, including maturation, transport, and metabolism of mRNA, and gene regulation of long noncoding ...RNAs. hnRNP A2/B1 was reported to control the microRNAs sorting to exosomes and promote primary microRNA processing as a potential m
A "reader." hnRNP A2/B1 contains two RNA recognition motifs that provide sequence-specific recognition of RNA substrates. Here, we determine crystal structures of tandem RRM domains of hnRNP A2/B1 in complex with various RNA substrates, elucidating specific recognitions of AGG and UAG motifs by RRM1 and RRM2 domains, respectively. Further structural and biochemical results demonstrate multivariant binding modes for sequence-diversified RNA substrates, supporting a RNA matchmaker mechanism in hnRNP A2/B1 function. Moreover, our studies in combination with bioinformatic analysis suggest that hnRNP A2/B1 may mediate effects of m
A through a "m
A switch" mechanism, instead of acting as a direct "reader" of m
A modification.
Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD+) exhibits axon-protective ...effects in cultured neurons; however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD+ levels in mice by administering nicotinamide riboside (NR), a recently described NAD+ precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD+-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD+ biosynthetic enzymes. These findings reveal that administration of NR activates a NAD+-SIRT3 pathway that reduces neurite degeneration caused by noise exposure.
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•The NAD+ precursor NR prevents noise-induced hearing loss•NR can be administered after acoustic trauma to prevent hearing loss•Prevention of noise-induced hearing loss by NAD+ and NR is dependent upon SIRT3•NAD+ and NR prevent noise-induced neurite retraction in the cochlea
Intense noise exposure causes hearing loss due to degeneration of cochlear neurites. Brown et al. show that the NAD+ precursor NR prevents noise-induced hearing loss and neurite degeneration in the cochlea, even after noise exposure. These effects are mediated by the NAD+-dependent mitochondrial sirtuin, SIRT3.
Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding ...small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA–fluorophore complexes would extend the utility of this technology, the identification of novel RNA–fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.
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