Abstract
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
Highlights
Almost all transcripts from genes encoding multiple exons are alternatively spliced, and correct patterns of alternative splicing are important for health and normal development [1,2,3]
A larger Sfrs10 RT-PCR product made from mRNAs including poison exon 2 was detected at high levels in just two tissues, testis and muscle, indicating that expression of Tra2b is tightly controlled in these tissues [22]
Identification is based on the criteria of in vivo cross-linking of endogenous RNAs and proteins, in cellulo experiments using transfected minigenes and proteins, RNAprotein interaction assays and genetic analysis using a newly derived conditional mouse strain which does not express Tra2b protein in neurons and has significant abnormalities in brain development
Summary
Almost all transcripts from genes encoding multiple exons are alternatively spliced, and correct patterns of alternative splicing are important for health and normal development [1,2,3]. Alternative splicing introduces new coding information into mRNAs, thereby increasing genome capacity to encode an expanded number of mRNAs and proteins from a finite number of genes [3]. Alternative splicing amplifies the informational content of the genome, making multiple mRNA isoforms from single genes. Tra proteins bind and activate alternative exons, and in mice Tra2b is essential for embryonic development through unknown target RNAs. Here we report the first target exons that are physiologically regulated by Tra2b in developing mice. Tra2b activates splicing of some target exons through direct RNA binding via its RNA Recognition Motif. For some exons Tra2b can activate splicing independent of direct RNA binding through two domains enriched in arginine and serine residues (called RS domains). Tra2b proteins without RS1 operate as splicing repressors, suggesting the possibility that endogenous Tra2b protein isoforms may differentially regulate the same target exons
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