Essential roles for the splicing regulator nSR100/SRRM4 during nervous system development
Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing.
Proper development and function of the mammalian nervous system depend on the tight coordination of multiple layers of gene regulation. During development, neurons progress through maturation stages to acquire their subtype-specific functions (Gao et al. 2013). For example, neurons born in the subventricular zone (SVZ) of the embryonic cortex use endocrine and exocrine cues while migrating dorsally to establish and populate specific cortical layers. Similarly, neuronal projections in the brain and periphery rely on successive adjustments of intrinsic to extrinsic factors to arrive at their targets. Considerable remodeling of the cytoskeleton, vesicular transport, and other subcellular processes allows neurons to achieve their designated morphologies and functions. While similar repertoires of genes are associated with these processes, it is becoming apparent that extensive variation at the level of post-transcriptional regulation generates the remarkable transcriptomic and proteomic diversity required for establishing biological complexity during vertebrate nervous system development (Li et al. 2007; Norris and Calarco 2012; Lipscombe et al. 2013a; Zheng and Black 2013).
Alternative splicing (AS) is the process by which different pairs of splice sites are selected to produce multiple transcripts from a single gene. It is controlled by the concerted action of multiple cis-acting motifs and cognate trans-acting factors that promote or repress the assembly of productive splicing complexes (spliceosomes) at splice sites (Chen and Manley 2009; Braunschweig et al. 2013). Widely expressed and tissue-restricted RNA-binding proteins combine to regulate AS decisions via positive- and negative-acting cis motifs located in exons or flanking introns, referred to as splicing enhancers and silencers, respectively. AS represents a major source of biological diversity that likely afforded the evolution of complexity associated with the development and function of the vertebrate nervous system (Barbosa-Morais et al. 2012; Merkin et al. 2012). Indeed, AS patterns are more complex in the brain than other tissues, and many of these events happen in genes implicated in complex neuronal processes, such as the control of synaptic plasticity associated with cognition (Lipscombe 2005; Ule and Darnell 2006). While most tissue differential splicing patterns are species-specific in vertebrates, there is a higher frequency of conserved alternative cassette exon inclusion events in vertebrate brains than in other tissue types (Barbosa-Morais et al. 2012; Merkin et al. 2012). This suggests the existence of a core set of conserved functions for AS across vertebrate species in addition to roles for AS underlying species-specific neurodevelopmental and behavioral characteristics. However, little is known about the in vivo functions of the protein factors that are responsible for establishing AS complexity in the nervous system or the functions of the individual AS events that are controlled by these factors.
Neural-enriched splicing regulators, including the Nova, Rbfox, and Ptbp proteins, have been characterized using mouse models. Nova proteins, which were originally identified as the autoantigens in patients with paraneoplastic opsoclonus myoclonus ataxia (Buckanovich et al. 1993; Yang et al. 1998), control the inhibitory synapse, and their knockout results in cortical migration (Yano et al. 2010) and neuromuscular junction (NMJ) defects (Ruggiu et al. 2009). Rbfox1 and Rbfox2 mutant mice are susceptible to seizures and display disrupted cerebellar development (Gehman et al. 2012). Depending on the strain background, Ptbp2 knockout mice die at birth or else exhibit cortical degeneration and lethality during the first few postnatal weeks (Licatalosi et al. 2012; Li et al. 2014). Additional studies using Nova knockout mice have revealed functions for specific Nova-regulated splice variants (including alternative exons in the Dab1 gene) that facilitate the proper migration of newly born cortical neurons (Yano et al. 2010) and exons in the Agrin gene that are important for the formation of NMJs (Ruggiu et al. 2009). However, aside from these examples, few other neuronal genes have been characterized at isoform resolution in vivo (Norris and Calarco 2012; Lipscombe et al. 2013b; Zheng and Black 2013).
We previously identified and characterized the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) (Calarco et al. 2009; Raj et al. 2011, 2014). Knockdown and overexpression experiments performed in cell culture revealed that nSR100 promotes the inclusion of 30%–50% of the conserved human and mouse cassette alternative exons that display brain-specific inclusion patterns in transcriptome profiling data (Raj et al. 2014). Knockdown of nSR100 in Neuro2a cells and developing zebrafish was shown to impair neurite outgrowth and branching of trigeminal ganglia, respectively (Calarco et al. 2009), and in utero knockdown of nSR100 in mice prevented differentiation of neuronal progenitors in the cortex (Raj et al. 2011). Recently, the Bronx waltzer (bv) mouse mutation was mapped to the nSR100 gene (Nakano et al. 2012). bv homozygotes display hearing and balance defects attributed to degeneration of inner ear hair cells. The apparent limited phenotypic consequences of the bv mutation are likely because this mutation eliminates only the terminal exon and part of the 3′ untranslated region (UTR) of nSR100 transcripts, leaving the majority of the nSR100 protein intact.
nSR100-regulated exons were found to be concentrated in genes that function in various aspects of neuronal development and function (Calarco et al. 2009; Raj et al. 2011, 2014; Nakano et al. 2012). These and other neural-regulated exons that are >27 nucleotides (nt) in length are highly concentrated in surface-accessible disordered regions of proteins and function in the regulation of protein–protein interactions (Buljan et al. 2012; Ellis et al. 2012). Furthermore, in a very recent study, we showed that nSR100 strongly promotes the inclusion of very short, 3- to 27-nt, neuronal “microexons” (Irimia et al. 2014). The corresponding microexon residues are concentrated within—or immediately adjacent to—protein–protein or protein–lipid interaction domains. Most of these exons display striking increases in inclusion during neuronal maturation, coincident with increased expression of nSR100. Notably, they also show significant decreases in inclusion—coincident with reduced expression of nSR100—in the cortices of individuals with autism spectrum disorder (ASD) (Irimia et al. 2014). A key function of nSR100 thus appears to be the widespread regulation of protein interactions required for the maturation and proper function of neurons. However, the scope of the in vivo functions of nSR100 during nervous system development has not been previously addressed.
To investigate the functions of nSR100 in vivo, we generated mice carrying a conditional exon deletion in the nSR100 (Srrm4) gene that results in widespread loss of the full-length protein. We observed that nSR100 is essential for early postnatal survival of a large majority of mutant animals, with the few surviving animals displaying balance defects similar to those seen in bv/bv mice but also exhibiting persistent tremors. Additionally, loss of nSR100 in mice results in impaired neurite outgrowth in the diaphragm, early neuronal commitment of neural progenitors leading to defective cortical layering, and a failure of callosal axons to cross the midline in the forebrain. Using RNA sequencing (RNA-seq) profiling, we defined all classes of AS (including alternative microexons and longer alternative cassette exons, 5′ and 3′ splice sites, and retained introns) that are controlled by nSR100 in vivo, a great majority of which were not previously reported. A large fraction of alternative cassette exons and microexons positively regulated by nSR100 are neurally enriched, which is not the case for other classes of nSR100-dependent splicing events. Moreover, a higher proportion of neural microexons is affected by disruption of nSR100 than are other neural-regulated AS events. These include highly conserved exons with the potential to insert only one or two amino acids in proteins of key functional relevance to neuronal maturation. Remarkably, an nSR100-regulated 6-nt microexon in the Unc13b gene promotes neurite growth in mouse primary neurons. Cortical neurons from nSR100 mice display a neuritogenesis defect, and expression of Unc13b transcripts including the microexon, but not transcripts lacking the microexon, is sufficient to rescue the mutant phenotype. Collectively, our results define critical new in vivo functions of nSR100 during mouse development and further link these functions to the disruption of a conserved program of nSR100-dependent neuronal microexons.Click here to view.
We thank Marina Gertsenstein, Sandra Tondat, Monica Pereira, Christina Dalrymple, Jorge Cabezas, Jessica Raponi, and Amanda Leonelli at the Toronto Centre for Phenogenomics for assistance with the generation and maintenance of mutant mouse strains. Dax Torti and Danica Leung of the Donnelly Sequencing Centre are gratefully acknowledged for sequencing samples. We also thank Jonathan Ellis for generating constructs used in primary neuronal cultures, and members of the Blencowe and Cordes laboratories for helpful discussions and comments on the manuscript. This research was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP-67011, MOP-14609, and MOP-111199) to B.J.B. and S.P.C. M.I. was supported by a long-term Fellowship from the Human Frontiers Science Program Organization. M.Q.-V. was supported by CIHR Banting and Best Scholarship and Ontario Graduate Scholarship. B.J.B. holds the Banbury Chair of Medical Research at the University of Toronto.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.256115.114.