Nucleotide-resolution Mapping of RNA N6-Methyladenosine (m6A) modifications and comprehensive analysis of global polyadenylation events in mRNA 3' end processing in malaria pathogen .

is an obligate human parasite of the phylum Apicomplexa and is the causative agent of the most lethal form of human malaria. Although N6-methyladenosine modification is thought to be one of the major post-transcriptional regulatory mechanisms for stage-specific gene expression in apicomplexan parasites, the precise base position of m6A in mRNAs or noncoding RNAs in these parasites remains unknown. Here, we report global nucleotide-resolution mapping of m6A residues in using DART-seq technology, which quantitatively displayed a stage-specific, dynamic distribution pattern with enrichment near mRNA 3' ends.

Single-cell mA profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation.

N-methyladenosine (mA) is an abundant mRNA modification in the brain that has important roles in neurodevelopment and brain function. However, because of technical limitations, global profiling of mA sites within the individual cell types that make up the brain has not been possible. Here, we develop a mouse model that enables transcriptome-wide mA detection in any tissue of interest at single-cell resolution.

Glia multitask to compensate for neighboring glial cell dysfunction.

As glia mature, they undergo glial tiling to abut one another without invading each other's boundaries. Upon the loss of the secreted neurotrophin Spätzle3 (Spz3), cortex glia transform morphologically and lose their intricate interactions with neurons and surrounding glial subtypes. Here, we reveal that all neighboring glial cell types (astrocytes, ensheathing glia, and subperineurial glia) react by extending processes into the previous cortex glial territory to compensate for lost cortex glial function and reduce the buildup of neuronal debris.

Studying mA in the brain: a perspective on current methods, challenges, and future directions.

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. -methyladenosine (mA) 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 mA is critical for neurodevelopment, learning and memory, and the response to injury, and mA dysregulation has been implicated in a variety of neurological disorders.

mRNA epitranscriptomics.

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.

Simultaneous In Situ Detection of mA-Modified and Unmodified RNAs Using DART-FISH.

N-methyladenosine (mA) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish mA-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both mA-modified and unmodified target transcripts.

Programmable protein expression using a genetically encoded mA sensor.

The N-methyladenosine (mA) modification is found in thousands of cellular mRNAs and is a critical regulator of gene expression and cellular physiology. mA dysregulation contributes to several human diseases, and the mA methyltransferase machinery has emerged as a promising therapeutic target. However, current methods for studying mA require RNA isolation and do not provide a real-time readout of mRNA methylation in living cells.

Exploring the brain epitranscriptome: perspectives from the NSAS summit.

Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma.

In situ visualization of m6A sites in cellular mRNAs.

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.

The Nab2 RNA binding protein inhibits mA methylation and male-specific splicing of transcript in female neuronal tissue.

The polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, dendritic arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor () that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (mA) modification by the Mettl3 complex.

The Proteins of mRNA Modification: Writers, Readers, and Erasers.

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, -methyladenosine (mA).

Epitranscriptomics in parasitic protists: Role of RNA chemical modifications in posttranscriptional gene regulation.

"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.

RBM45 is an mA-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

N-methyladenosine (mA) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. mA is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which mA contributes to brain development are incompletely understood.

Detection of mA in single cultured cells using scDART-seq.

Most techniques for mapping mA-methylated RNAs transcriptome-wide require large amounts of RNA and have been limited to bulk cells and tissues. Here, we provide a detailed protocol for the identification of mA sites in single HEK293T cells using single-cell DART-seq (scDART-seq). The protocol details how to generate cell lines with inducible expression of the APOBEC1-YTH transgene and the use of important controls for minimizing false positives.

Improved Methods for Deamination-Based mA Detection.

-methyladenosine (mA) is a critical regulator of gene expression and cellular function. Much of our knowledge of mA has been enabled by the identification of mA sites transcriptome-wide. However, global mA profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making mA profiling from rare cell types or scarce tissue samples infeasible.

m6A and YTHDF proteins contribute to the localization of select neuronal mRNAs.

The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a novel regulator of this process.

scDART-seq reveals distinct mA signatures and mRNA methylation heterogeneity in single cells.

N-methyladenosine (mA) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global mA profiling has revealed important aspects of mA distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify mA sites transcriptome-wide in single cells.

Detecting mA with In Vitro DART-Seq.

Recent studies have uncovered that cellular mRNAs contain a diverse epitranscriptome comprising chemically modified bases which play important roles in gene expression regulation. Among these is mA, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for mA profiling have used mA antibodies to immunoprecipitate methylated RNAs.

Mapping of mA and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-Low Induction of Therapy Resistance.

Recent evidence has highlighted the role of -methyladenosine (mA) in the regulation of mRNA expression, stability, and translation, supporting a potential role for posttranscriptional regulation mediated by mA in cancer. Here, we explore prostate cancer as an exemplar and demonstrate that low levels of -adenosine-methyltransferase () is associated with advanced metastatic disease. To investigate this relationship, we generated the first prostate mA maps, and further examined how METTL3 regulates expression at the level of transcription, translation, and protein.

RNA N-Methyladenosine and the Regulation of RNA Localization and Function in the Brain.

A major challenge in neurobiology in the 21st century is to understand how the brain adapts with experience. Activity-dependent gene expression is integral to the synaptic plasticity underlying learning and memory; however, this process cannot be explained by a simple linear trajectory of transcription to translation within a specific neuronal population. Many other regulatory mechanisms can influence RNA metabolism and the capacity of neurons to adapt.

DART-seq: an antibody-free method for global mA detection.

N-methyladenosine (mA) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of mA has been facilitated by the development of global mA 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.

The epitranscriptome and synaptic plasticity.

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.