N6 -methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by ‘writers’, ‘erasers’ and ‘readers’ of this mark 1,2. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates 3,4. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that deletion of m6A ‘writer’ protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopenic mouse adoptive transfer model, naive Mettl3 deficient T cells failed to undergo homeostatic expansion and remarkably remained in the naïve state up through 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding STAT- signaling inhibitory proteins, Socs1, Socs3 and Cish, were marked by m6A, exhibited slower mRNA decay and increased mRNAs and protein expression levels in Mettl3 deficient naïve T cells. This increased SOCS family activity consequently inhibited IL-7 mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A plays important roles for inducible degradation of Socs mRNAs in response to IL-7 signaling in order to reprogram Naïve T cells for proliferation and differentiation. Our study elucidates for the first time the in vivo biological role of m6A modification in T cell mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, a chemical method that uses oxidative-nucleophilic-aromatic-substitution to convert 4-thiouridine into cytidine analogues, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single molecule approach that is adaptable to many applications, and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

SUMMARY We describe a chemical method to label and purify 4-thiouridine (s4U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA, such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.

Graphical Abstract Highlights d A conserved antisense promoter is located within each of the Pcdha alternate exons d Antisense lncRNA transcription leads to DNA demethylation of promoters and CBSs d CTCF/cohesin drive the assembly of Pcdha enhancer/ promoter complex via loop extrusion d Coupling lncRNA transcription to DNA demethylation ensures stochastic promoter choice SUMMARYStochastic activation of clustered Protocadherin (Pcdh) a, b, and g genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdha alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdha promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdha promoter choice.

Maternal infection and inflammation during pregnancy are associated with neurodevelopmental disorders in offspring, but little is understood about the molecular mechanisms underlying this epidemiologic phenomenon. We leveraged single-cell RNA sequencing to profile transcriptional changes in the mouse fetal brain in response to maternal immune activation (MIA) and identified perturbations in cellular pathways associated with mRNA translation, ribosome biogenesis, and stress signaling. We found that MIA activates the integrated stress response (ISR) in male, but not female, MIA offspring in an Interleukin-17a dependent manner, thereby reducing global mRNA translation and altering nascent proteome synthesis. Moreover, blockade of ISR activation prevented the behavioral abnormalities as well as an increase in cortical neural activity in MIA male offspring. Our data suggest that sex-specific activation of the ISR leads to maternal inflammation-associated neurodevelopmental disorders.