p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR).

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

Genetic Control of Expression and Splicing in Developing Human Brain Informs Disease Mechanisms.

Tissue-specific regulatory regions harbor substantial genetic risk for disease. Because brain development is a critical epoch for neuropsychiatric disease susceptibility, we characterized the genetic control of the transcriptome in 201 mid-gestational human brains, identifying 7,962 expression quantitative trait loci (eQTL) and 4,635 spliceQTL (sQTL), including several thousand prenatal-specific regulatory regions. We show that significant genetic liability for neuropsychiatric disease lies within prenatal eQTL and sQTL. Integration of eQTL and sQTL with genome-wide association studies (GWAS) via transcriptome-wide association identified dozens of novel candidate risk genes, highlighting shared and stage-specific mechanisms in schizophrenia (SCZ). Gene network analysis revealed that SCZ and autism spectrum disorder (ASD) affect distinct developmental gene co-expression modules. Yet, in each disorder, common and rare genetic variation converges within modules, which in ASD implicates superficial cortical neurons. More broadly, these data, available as a web browser and our analyses, demonstrate the genetic mechanisms by which developmental events have a widespread influence on adult anatomical and behavioral phenotypes.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.

Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.

Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Change history: In this Letter, the labels for splicing events A3SS and A5SS were swapped in column D of Supplementary Table 3a and b. This has been corrected online.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Autism spectrum disorder (ASD) involves substantial genetic contributions. These contributions are profoundly heterogeneous but may converge on common pathways that are not yet well understood. Here, through post-mortem genome-wide transcriptome analysis of the largest cohort of samples analysed so far, to our knowledge, we interrogate the noncoding transcriptome, alternative splicing, and upstream molecular regulators to broaden our understanding of molecular convergence in ASD. Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes. Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences. We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2-13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD. Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression. Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.

Genome-wide DNA methylation profiling identifies convergent molecular signatures associated with idiopathic and syndromic autism in post-mortem human brain tissue.

Autism spectrum disorder (ASD) encompasses a collection of complex neuropsychiatric disorders characterized by deficits in social functioning, communication and repetitive behaviour. Building on recent studies supporting a role for developmentally moderated regulatory genomic variation in the molecular aetiology of ASD, we quantified genome-wide patterns of DNA methylation in 223 post-mortem tissues samples isolated from three brain regions [prefrontal cortex, temporal cortex and cerebellum (CB)] dissected from 43 ASD patients and 38 non-psychiatric control donors. We identified widespread differences in DNA methylation associated with idiopathic ASD (iASD), with consistent signals in both cortical regions that were distinct to those observed in the CB. Individuals carrying a duplication on chromosome 15q (dup15q), representing a genetically defined subtype of ASD, were characterized by striking differences in DNA methylationacross a discrete domain spanning an imprinted gene cluster within the duplicated region. In addition to the dramatic cis-effects on DNA methylation observed in dup15q carriers, we identified convergent methylomic signatures associated with both iASD and dup15q, reflecting the findings from previous studies of gene expression and H3K27ac. Cortical co-methylation network analysis identified a number of co-methylated modules significantly associated with ASD that are enriched for genomic regions annotated to genes involved in the immune system, synaptic signalling and neuronal regulation. Our study represents the first systematic analysis of DNA methylation associated with ASD across multiple brain regions, providing novel evidence for convergent molecular signatures associated with both idiopathic and syndromic autism.

A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels. RESULTS: In agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes. CONCLUSIONS: Our findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.

Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses.

Adenosine deaminases acting on double-stranded RNA (ADARs) catalyze the deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA structures, a process known as A-to-I RNA editing. dsRNA is an important trigger of innate immune responses, including interferon (IFN) production and action. We examined the role of A-to-I RNA editing by two ADARs, ADAR1 and ADAR2, in the sensing of self-RNA in the absence of pathogen infection, leading to activation of IFN-induced, RNA-mediated responses in mouse embryo fibroblasts. IFN treatment of Adar1(-/-) cells lacking both the p110 constitutive and p150 IFN-inducible ADAR1 proteins induced formation of stress granules, whereas neither wild-type (WT) nor Adar2(-/-) cells displayed a comparable stress granule response following IFN treatment. Phosphorylation of protein synthesis initiation factor eIF2α at serine 51 was increased in IFN-treated Adar1(-/-) cells but not in either WT or Adar2(-/-) cells following IFN treatment. Analysis by deep sequencing of mouse exonic loci containing A-to-I-editing sites revealed that the majority of editing in mouse embryo fibroblasts was carried out by ADAR1. IFN treatment increased editing in both WT and Adar2(-/-) cells but not in either Adar1(-/-) or Adar1(-/-) (p150) cells or Stat1(-/-) or Stat2(-/-) cells. Hyper-edited sites found in predicted duplex structures showed strand bias of editing for some RNAs. These results implicate ADAR1 p150 as the major A-to-I editor in mouse embryo fibroblasts, acting as a feedback suppressor of innate immune responses otherwise triggered by self-RNAs possessing regions of double-stranded character.

Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

Identification of human RNA editing sites: A historical perspective.

A-to-I RNA editing is an essential gene regulatory mechanism. Once thought to be a rare phenomenon only occurring in a few transcripts, the emergence of high-throughput RNA sequencing has facilitated the identification of over 2 million RNA editing sites in the human transcriptome. In this review, we survey the current RNA-seq based methods as well as historical methods used to identify RNA editing sites.

Reliable identification of genomic variants from RNA-seq data.

Identifying genomic variation is a crucial step for unraveling the relationship between genotype and phenotype and can yield important insights into human diseases. Prevailing methods rely on cost-intensive whole-genome sequencing (WGS) or whole-exome sequencing (WES) approaches while the identification of genomic variants from often existing RNA sequencing (RNA-seq) data remains a challenge because of the intrinsic complexity in the transcriptome. Here, we present a highly accurate approach termed SNPiR to identify SNPs in RNA-seq data. We applied SNPiR to RNA-seq data of samples for which WGS and WES data are also available and achieved high specificity and sensitivity. Of the SNPs called from the RNA-seq data, >98% were also identified by WGS or WES. Over 70% of all expressed coding variants were identified from RNA-seq, and comparable numbers of exonic variants were identified in RNA-seq and WES. Despite our method's limitation in detecting variants in expressed regions only, our results demonstrate that SNPiR outperforms current state-of-the-art approaches for variant detection from RNA-seq data and offers a cost-effective and reliable alternative for SNP discovery.

Identifying RNA editing sites using RNA sequencing data alone.

We show that RNA editing sites can be called with high confidence using RNA sequencing data from multiple samples across either individuals or species, without the need for matched genomic DNA sequence. We identified many previously unidentified editing sites in both humans and Drosophila; our results nearly double the known number of human protein recoding events. We also found that human genes harboring conserved editing sites within Alu repeats are enriched for neuronal functions.

RADAR: a rigorously annotated database of A-to-I RNA editing.

We present RADAR--a rigorously annotated database of A-to-I RNA editing (available at http://RNAedit.com). The identification of A-to-I RNA editing sites has been dramatically accelerated in the past few years by high-throughput RNA sequencing studies. RADAR includes a comprehensive collection of A-to-I RNA editing sites identified in humans (Homo sapiens), mice (Mus musculus) and flies (Drosophila melanogaster), together with extensive manually curated annotations for each editing site. RADAR also includes an expandable listing of tissue-specific editing levels for each editing site, which will facilitate the assignment of biological functions to specific editing sites.

Accurate identification of human Alu and non-Alu RNA editing sites.

We developed a computational framework to robustly identify RNA editing sites using transcriptome and genome deep-sequencing data from the same individual. As compared with previous methods, our approach identified a large number of Alu and non-Alu RNA editing sites with high specificity. We also found that editing of non-Alu sites appears to be dependent on nearby edited Alu sites, possibly through the locally formed double-stranded RNA structure.