Summary The RNA-binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here we performed HITS-CLIP for all three Rbfox family members to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We found that the two guanines in the Rbfox-binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites combined with additional datasets defined 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with down-regulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing- regulatory network.

Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.

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...

The relative importance of regulatory versus structural evolution for the evolution of different biological systems is a subject of controversy. The primacy of regulatory evolution in the diversification of morphological traits has been promoted by many evolutionary developmental biologists. For physiological traits, however, the role of regulatory evolution has received less attention or has been considered to be relatively unimportant. To address this issue for electrophysiological systems, we examined the importance of regulatory and structural evolution in the evolution of the electrophysiological function of cardiac myocytes in mammals. In particular, two related phenomena were studied: the change in action potential morphology in small mammals and the scaling of action potential duration across mammalian phylogeny. In general, the functional properties of the ion channels involved in ventricular action potential repolarization were found to be relatively invariant. In contrast, there were large changes in the expression levels of multiple ion channel and transporter genes. For the Kv2.1 and Kv4.2 potassium channel genes, which are primary determinants of the action potential morphology in small mammals, the functional properties of the proximal promoter regions were found to vary in concordance with species-dependent differences in mRNA expression, suggesting that evolution of cis-regulatory elements is the primary determinant of this trait. Scaling of action potential duration was found to be a complex phenomenon, involving changes in the expression of a large number of channels and transporters. In this case, it is concluded that regulatory evolution is the predominant mechanism by which the scaling is achieved.

Alternative splicing (AS) is a crucial step of gene expression that must be tightly controlled, but the precise timing of dynamic splicing switches during neural development and the underlying regulatory mechanisms are poorly understood. Here we systematically analyzed the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis revealed early-and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the "immature" splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.