A NPAS4-NuA4 complex couples synaptic activity to DNA repair.

Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1-5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4-TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4-NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4-NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4-NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.

Developmental dynamics of RNA translation in the human brain.

The precise regulation of gene expression is fundamental to neurodevelopment, plasticity and cognitive function. Although several studies have profiled transcription in the developing human brain, there is a gap in understanding of accompanying translational regulation. In this study, we performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human-specific and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem-cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and, thus, may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.

Maternal immune activation in mice disrupts proteostasis in the fetal brain.

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. Here, 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, which reduced global mRNA translation and altered nascent proteome synthesis. Moreover, blockade of ISR activation prevented the behavioral abnormalities as well as increased cortical neural activity in MIA male offspring. Our data suggest that sex-specific activation of the ISR leads to maternal inflammation-associated neurodevelopmental disorders.

Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice.

Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα 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 Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα 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 Pcdhα promoter choice.

Gaining insight into transcriptome-wide RNA population dynamics through the chemistry of 4-thiouridine.

Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e., altered rate of transcription vs. degradation). The dynamics of different RNA populations can be studied by targeted incorporation of noncanonical nucleosides. 4-Thiouridine (s4 U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s4 U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s4 U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s4 U metabolic labeling, as well as a discussion of considerations and challenges when designing s4 U metabolic labeling experiments. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Turnover and Surveillance > Regulation of RNA Stability.

Solid phase chemistry to covalently and reversibly capture thiolated RNA.

Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.

TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding.

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, 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.

m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive 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 has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, 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.

Enriching s4 U-RNA Using Methane Thiosulfonate (MTS) Chemistry.

Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s4 U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s4 U-RNA is the efficiency of the chemistry used to enrich s4 U-RNA. Here, we present a protocol for s4 U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s4 U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s4 U to study RNA biology. © 2016 by John Wiley & Sons, Inc.

Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.

We describe a chemical method to label and purify 4-thiouridine (s(4)U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s(4)U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s(4)U 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.