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

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.可变剪接（Alternative splicing）可扩增基因组的信息容量，能够从单个基因生成多种mRNA异构体。进化保守的剪接激活因子Tra2β（Sfrs10）对小鼠胚胎发生至关重要，且与精子发生过程相关。本研究发现，在雄性生殖细胞中，包含有丝分裂干细胞的群体向减数分裂及减数分裂后细胞分化时，Tra2β的表达会上调。我们采用结合高通量测序的紫外交联免疫沉淀（CLIP）技术，发现Tra2β可结合大量外显子，并鉴定出富含G/A的特定基序为其常见结合靶点。值得注意的是，我们首次通过构建条件性神经元特异性Sfrs10敲除小鼠（Sfrs10fl/fl; Nestin-Cretg/+），在活体水平分析了Sfrs10缺失所带来的剪接效应。该小鼠存在大脑发育缺陷，这使得我们能够将真实生理状态下受Tra2β调控的外显子与该发育缺陷建立关联。这些受调控的外显子属于一类新的亚型：它们的长度长于平均水平，且至关重要的是，需要多个协同作用的Tra2β结合位点才能实现高效的剪接激活，这也解释了敲除小鼠中观察到的剪接缺陷。受调控的外显子包含一个盒式外显子，该外显子可编码Nasp组蛋白伴侣的减数分裂异构体，而Nasp组蛋白伴侣有助于监测DNA双链断裂。我们还发现了一个此前未被表征的有毒外显子（poison exon），这揭示了脊椎动物Tra2蛋白之间一种全新的反馈调控通路。Nasp-T与Tra2a有毒外显子均在进化上保守，这提示它们可能调控着核心发育过程。缺失RNA识别模体（RNA Recognition Motif, RRM）的Tra2β蛋白异构体仍可激活特定的靶外显子，这表明Tra2β作为剪接共激活因子存在额外的功能角色。尤为关键的是，果蝇与人类之间保守的N端RS1结构域，是Tra2β发挥剪接激活功能所必需的。而缺失该N端RS1结构域的Tra2β突变体，则会强效抑制全长Tra2β蛋白所激活的同一批靶外显子。