Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development

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.

Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2β (Tra2β)

“…The functional importance of RS1 domain in spatial organization is supported by a recent finding that removal of the RS1 domain completely disabled Tra2␤-mediated splicing activation of the physiological target exons (47). In addition, we observed that GFP-Tra2␤ and GFP-RS1RRM were concentrated in nuclear speckles with a diffusive distribution in the nearby nucleoplasm (Fig.…”

Splicing-factor alterations in cancers

“…Up-regulation of TRA2β promotes several splicing switches, including in CD44, which were previously associated with tumor progression and metastasis (Watermann et al 2006). Another example of a TRA2β splicing target is the nuclear autoantigenic sperm protein NASP, which is important for cell survival (Grellscheid et al 2011). TRA2β also regulates splicing of the estrogen receptor α, decreasing the expression of the ERaΔ7 isoform, which correlates with a better outcome in endometrial cancer (Hirschfeld et al 2015).…”

3′ UTR lengthening as a novel mechanism in regulating cellular senescence

“…4D; Grellscheid et al 2011). Considering that "AGAA" shared 1 nt with the downstream PAS "ATTAAA" in the alternative 3 ′ UTR of Rras2, we mutated the first 3 nt to avoid disruption of the PAS ("AGAA" to "CCCA") (designated Mut3 in Fig.…”