Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH.

N6-methyladenosine (m6A) 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 m6A-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 m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.

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. 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.

Pathogenic DDX3X Mutations Impair RNA Metabolism and Neurogenesis during Fetal Cortical Development.

De novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.

Dosage-dependent requirements of Magoh for cortical interneuron generation and survival.

Embryonic interneuron development underlies cortical function and its disruption contributes to neurological disease. Yet the mechanisms by which viable interneurons are produced from progenitors remain poorly understood. Here, we demonstrate dosage-dependent requirements of the exon junction complex component Magoh for interneuron genesis in mouse. Conditional Magoh ablation from interneuron progenitors, but not post-mitotic neurons, depletes cortical interneuron number through adulthood, with increased severity in homozygotes. Using live imaging, we discover that Magoh deficiency delays progenitor mitotic progression in a dosage-sensitive fashion, with 40% of homozygous progenitors failing to divide. This shows that Magoh is required in progenitors for both generation and survival of newborn progeny. Transcriptome analysis implicates p53 signaling; moreover, p53 ablation in Magoh haploinsufficient progenitors rescues apoptosis, completely recovering interneuron number. In striking contrast, in Magoh homozygotes, p53 loss fails to rescue interneuron number and mitotic delay, further implicating mitotic defects in interneuron loss. Our results demonstrate that interneuron development is intimately dependent upon progenitor mitosis duration and uncover a crucial post-transcriptional regulator of interneuron fate relevant for neurodevelopmental pathologies.This article has an associated 'The people behind the papers' interview.