Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

Abstract

Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue-or cell typespecific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue-or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.new alternative exon | brain transcriptome | RNA-Seq | nonsense-mediated decay | chromatin regulator M olecular diversity derived from alternative splicing (AS) is believed to be critical for the creation of different cell types and tissues with distinct physiological properties and functions (1). This is particularly relevant to the central nervous system (CNS), which requires a large protein repertoire to generate its intricate and complex neural circuits (2). Therefore, a comprehensive catalog of AS events and identification of those with potential functional significance are important steps toward understanding the complexity of the nervous system.Over the past two decades, discovery and characterization of AS events using different technologies have provided important insights into the evolution and regulation of AS (3, 4). Earlier expressed sequence tag (EST)-based studies revealed the prevalence of AS in mammals (5). Investigation of these AS events, especially comparison of AS patterns in different species, led to an important observation that AS is rapidly evolving in mammals, with many alternative exons created after the split of primates and rodents (6). Evolutionarily recent exons in general have low level of inclusion and frequently result in frame shift and premature...

Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand

“…Fourth, the Nrxn1 gene expresses from a third promoter, Nrxn1γ, a developmentally and spatially regulated short isoform whose gene product was previously predicted computationally (27) (Fig. 5).…”

“…Deep sequencing of RNA from mouse brain recently identified a novel, very short Nrxn1 transcript that lacks all extracellular Nrxn1 sequences except for the stalk region (27). Thus, this isoform-referred to as Nrxn1γ-is predicted to bind to CA10 but not to other known neurexin ligands.…”

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

“…The presence of poison exons encoding translational stop codons reported here and by other groups (Yan et al, 2015) suggests a potential mechanism of human disease that to our knowledge has not been previously described: mutations that disrupt normal suppression of a poison exon would inactivate protein expression in a cell that normally skips the exon. We tested this model by studying FLNA , since heterozygous null mutations in females typically cause periventricular heterotopia (PVNH), where neurons form nodules along the VZ, reflecting failure of normal migration (Fox et al, 1998).…”

“…Transcriptional profiling of mammalian forebrain has revealed dynamic AS changes between different brain regions (Johnson et al, 2009), cortical layers (Belgard et al, 2011) or developmental stages (Dillman et al, 2013; Yan et al, 2015). Dysregulation of AS in human brain by RBFOX1 mutations or disturbed nSR100 levels has been associated with intellectual disability and autism spectrum disorders (ASD) (Bhalla et al, 2004; Irimia et al, 2014; Sebat et al, 2007).…”

Building an RNA Sequencing Transcriptome of the Central Nervous System

“…Notably, a group of NMD exons reside in chromatin regulator genes. Thus, NMD can modulate chromatin regulators, thereby coupling AS- and epigenetics-based mechanisms (Yan and others 2015). …”