m6A-mediated translation regulation Meyer, Kate D.
Biochimica et biophysica acta. Gene regulatory mechanisms,
03/2019, Letnik:
1862, Številka:
3
Journal Article
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•m6A regulates both cap-dependent and cap-independent translation in cells.•Various m6A reader proteins control protein production through distinct mechanisms.•m6A regulates the translation of select ...mRNAs in response to cellular stress.
Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, ...encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.
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. In particular, the RNA modification N6-methyladenosine (m6A) has recently been shown to regulate RNA processing through alternative splicing, RNA stability, and translation. Here, we discuss the emerging idea that m6A could also coordinate the transport, localization, and local translation of key mRNAs in learning and memory and expand on the notion of dynamic functional RNA states in the brain.
Epitranscriptomics or ‘RNA modifications’ contribute to RNA localization processes in the brain.N6-methyladenosine (m6A) is an RNA modification potentially critical for the localization of RNA.m6A not only has roles in the nuclear processing and export of RNA molecules but is also likely to contribute to the phase separation of RNA–protein complexes into RNA reservoirs e.g., RNA granules, stress granules, processing (P)-bodies and local translation at specific neuronal compartments.Many new techniques are capable of profiling m6A-modified transcripts at single-base resolution. However, these techniques need to be adapted to determine transcripts localized to various locations of the neuron including activated synapses, axons, and dendritic compartments.Investigations into the roles of m6A in RNA biology are rapidly expanding but require new techniques to fully understand m6A’s role in RNA localization in the brain.
N6-methyladenosine (m6A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m6A profiling has revealed important aspects of m6A distribution and ...function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m6A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m6A sites across individual cells and identify differentially methylated mRNAs across the cell cycle. Additionally, we show that cellular subpopulations can be distinguished based on their RNA methylation signatures, independent from gene expression. These studies reveal fundamental features of m6A that have been missed by m6A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m6A deposition.
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•scDART-seq achieves global m6A profiling in thousands of single cells•Most mRNAs are methylated in a small fraction of cells•RNAs have many more m6A sites than previously reported, but most occur rarely•m6A stoichiometry is highly variable across individual cells of a population
Tegowski et al. use single-cell m6A profiling to map the methylomes of thousands of individual cells. They find that m6A is highly heterogeneous at the single-cell level, with most m6A sites occurring in a small proportion of cells. They also uncover differential methylation within subpopulations of cells.
The first book to argue for the concept of tragic
dilemmas in Christian ethics
Moral dilemmas arise when individuals are unable to fulfill all
of their ethical obligations. Tragic dilemmas are moral ...dilemmas
that involve great tragedy. The existence of moral and tragic
dilemmas is debated in philosophy and often dismissed in theology
based on the notion that there are effective strategies that
completely solve hard ethical situations. Yet cases from real-life
events in war and bioethics offer compelling evidence for the
existence of tragic dilemmas.
In Tragic Dilemmas in Christian Ethics , Jackson-Meyer
expertly explores the thought of Augustine and Aquinas to show the
limits of their treatment of hard cases, as well as where their
thought can be built on and expanded in relation to tragic
dilemmas. She recognizes and develops a new theological
understanding of tragic dilemmas rooted in moral philosophy,
contemporary case studies, and psychological literature on moral
injury. Jackson-Meyer argues that in tragic dilemmas moral agents
choose between conflicting nonnegotiable moral obligations rooted
in Christian commitments to protect human life and the vulnerable.
Personal culpability is mitigated due to constrained situations and
society is also culpable when tragic dilemmas are a result of
structural sin. In response, Jackson-Meyer implores Christian
communities to offer individual and communal healing after tragic
dilemmas and to acknowledge their own participation in
injustice.
Tragic Dilemmas in Christian Ethics offers practical
strategies that Christian communities can use to provide healing to
those who have acted in tragic dilemmas and to transform the unjust
structures that often cause these tragedies.
Most techniques for mapping m6A-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 m6A 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. We also describe the bioinformatic analysis to identify m6A sites.
For complete details on the use and execution of this protocol, please refer to Tegowski et al. (2022).
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•Generation of transgenic cell lines expressing inducible APOBEC1-YTH•Transgene induction and sorting of individual cells•Preparation of sequencing libraries from single cells•Processing data to identify m6A sites from C-to-U mutations in the sequencing data
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Most techniques for mapping m6A-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 m6A 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. We also describe the bioinformatic analysis to identify m6A sites.
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. The inaugural NSAS Challenge Workshop on Brain Epitranscriptomics hosted in Crans-Montana, Switzerland in 2023 assembled a group of experts from the field, to discuss the current state of the field and provide novel translational perspectives. A summary of the discussions at the workshop is presented here to simulate broader engagement from the general neuroscience field.
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