Abstract
Alternative splicing (AS) is a posttranscriptional mechanism regulating gene expression that complex organisms utilize to expand proteome diversity from a comparatively limited set of genes. Recent research has increasingly associated AS with increased functional complexity in the central nervous systems in higher order mammals. This work has heavily implicated aberrant AS in several neurocognitive and neurodevelopmental disorders, including autism. Due to the strong genetic association between germline PTEN mutations and autism spectrum disorder (ASD), we hypothesized that germline PTEN mutations would alter AS patterns, contributing to the pathophysiology of ASD. In a murine model of constitutional mislocalization of Pten, recapitulating an autism-like phenotype, we found significant changes in AS patterns across the neural transcriptome by analyzing RNA-sequencing data with the program rMATS. A few hundred significant alternative splicing events (ASEs) that differentiate each m3m4 genotype were identified. These ASEs occur in genes enriched in PTEN signaling, inositol metabolism, and several other pathways relevant to the pathophysiology of ASD. In addition, we identified expression changes in several splicing factors known to be enriched in the nervous system. For instance, the master regulator of microexons, Srrm4, has decreased expression, and consequently, we found decreased inclusion of microexons in the Ptenm3m4/m3m4 cortex (~10% decrease). We also demonstrated that the m3m4 mutation disrupts the interaction between Pten and U2af2, a member of the spliceosome. In sum, our observations point to germline Pten disruption changing the landscape of alternative splicing in the brain, and these changes may be relevant to the pathogenesis and/or maintenance of PTEN-ASD phenotypes.
Highlights
Metazoans utilize alternative splicing (AS), nominally a posttranscriptional mechanism by which various exonic segments are excised from intronic segments of the geneencoded RNA message and conjoined combinatorically to form a diverse set of transcriptional messages, to exponentially expand the coding potential of the genome[1,2,3]
We considered alternative splicing events (ASEs) to meet our threshold for subsequent analysis if they met three criteria: p ≤ 0.05, false discovery rate (FDR) ≤ 0.05, and ΔΨ ≤ −0.1 or ΔΨ ≥ 0.1
Building on our previous observation that the Pten model’s neural transcriptome mimics human idiopathic autism, we show that the m3m4 mutation can disrupt the many aspects of Pten function that presumptively participate in the regulation of AS
Summary
Metazoans utilize alternative splicing (AS), nominally a posttranscriptional mechanism (though the process occurs co-transcriptionally) by which various exonic segments are excised from intronic segments of the geneencoded RNA message and conjoined combinatorically to form a diverse set of transcriptional messages, to exponentially expand the coding potential of the genome[1,2,3]. PTEN is one of the most strongly associated autism risk genes with ~23% of individuals with germline mutations receiving an ASD diagnosis and an even greater percentage with developmental delay and other neurological phenotypes[9,11,12]. The Ptenm3m4 mouse is a model of cytoplasmicpredominant Pten expression, resulting from five nucleotide substitutions distributed across the third and fourth nuclear localization-like signals of Pten in exon 713. The neural transcriptome of these mice reveals differentially expressed genes similar to those associated with idiopathic autism[14]. These organismal, molecular, and cellular phenotypes characterize the Ptenm3m4 mouse as a suitable model of idiopathic autism. RNAsequencing found that many known ASD-risk genes annotated by the Simons Foundation Autism Research
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