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.
Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications ...transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification,
N
6
-methyladenosine (m
6
A). We first describe the discovery of the key enzymes that deposit and remove m
6
A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m
6
A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.
mRNA epitranscriptomics Meyer, Kate D; Pan, Tao
RNA (Cambridge),
05/2024, Letnik:
30, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Epitranscriptomics refers to chemical changes in RNAs and includes numerous chemical types with varying stoichiometry and functions. RNA modifications are highly diverse in chemistry and respond in ...cell-type- and cell-state-dependent manners that enable and facilitate the execution of a wide array of biological functions. This includes roles in the regulation of transcription, translation, chromatin maintenance, immune response, and many other processes. This special issue presents the past, present, and future of epitranscriptomics research with a focus on mRNA. It includes perspectives from experts in the field, with the goal of encouraging discussions and debates that will further advance this area of research and enable it to realize its full potential in basic research and applications to human health and disease.
Abstract
N
6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is spatially regulated ...remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m6A sites in target RNAs which enables simultaneous detection of both m6A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m6A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m6A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m6A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m6A-modified transcript dynamics and investigating methylated RNA localization in individual cells.
Graphical Abstract
Graphical Abstract
Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent ...manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.
N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m6A has been facilitated by the development of ...global m6A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.
"Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There ...are several RNA base modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites-Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.
RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of ...RBP targets are limited, since they examine only a single RBP at a time and do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We applied TRIBE-STAMP to the cytoplasmic m
A reader proteins YTHDF1, YTHDF2, and YTHDF3 and discovered that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.
•Loss of mRNA methylation impairs synaptic development.•Activity-dependent methylation contributes to learning and memory.•mRNA modification machinery influences synaptic signaling and ...potentiation.•mRNA methylation contributes to local gene expression control at synapses.
RNA modifications, collectively referred to as ‘the epitranscriptome,’ have recently emerged as a pervasive feature of cellular mRNAs which have diverse impacts on gene expression. In the last several years, technological advances improving our ability to identify mRNA modifications, coupled with the discovery of proteins that add and remove these marks, have substantially expanded our knowledge of how the epitranscriptome shapes gene expression. Efforts to uncover functional roles for mRNA modifications have begun to reveal important roles for some marks within the nervous system, and animal models have emerged which demonstrate severe neurodevelopmental and neurocognitive abnormalities resulting from the loss of mRNA modification machinery. Here, we review the recent advances in the field of neuroepitranscriptomics, with a particular emphasis on how modifications to mRNAs within the brain contribute to synaptic activity.
A major mechanism of post-transcriptional RNA regulation in cells is the addition of chemical modifications to RNA nucleosides, which contributes to nearly every aspect of the RNA life cycle. N 6 ...-methyladenosine (m 6 A) is a highly prevalent modification in cellular mRNAs and non-coding RNAs, and it plays important roles in the control of gene expression and cellular function. Within the brain, proper regulation of m 6 A is critical for neurodevelopment, learning and memory, and the response to injury, and m 6 A dysregulation has been implicated in a variety of neurological disorders. Thus, understanding m 6 A and how it is regulated in the brain is important for uncovering its roles in brain function and potentially identifying novel therapeutic pathways for human disease. Much of our knowledge of m 6 A has been driven by technical advances in the ability to map and quantify m 6 A sites. Here, we review current technologies for characterizing m 6 A and highlight emerging methods. We discuss the advantages and limitations of current tools as well as major challenges going forward, and we provide our perspective on how continued developments in this area can propel our understanding of m 6 A in the brain and its role in brain disease.
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