Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.
Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.
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鉴定进化保守的外显子是发育中剪接激活剂TRA2β的调节靶标。
DOI：
10.1371/journal.pgen.1002390
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发表时间：
2011-12
期刊：
影响因子：
4.5
通讯作者：
Elliott DJ
中科院分区：
文献类型：
--
作者：
Grellscheid S;Dalgliesh C;Storbeck M;Best A;Liu Y;Jakubik M;Mende Y;Ehrmann I;Curk T;Rossbach K;Bourgeois CF;Stévenin J;Grellscheid D;Jackson MS;Wirth B;Elliott DJ
选择性剪接放大了基因组的信息含量，从单个基因中产生多种mRNA亚型。进化上保守的剪接激活因子Tra 2 β（Sfrs 10）是小鼠胚胎发生所必需的，并与精子发生有关。在此我们发现Tra 2 β在含有有丝分裂干细胞的雄性生殖细胞群分化为减数分裂和减数分裂后细胞时上调。使用CLIP结合深度测序，我们发现Tra 2 β结合高频率的外显子，并将特定的富含G/A的基序鉴定为常见的靶点。值得注意的是，我们第一次通过产生条件性神经元特异性Sfrs 10基因敲除小鼠（Sfrs 10 fl/fl; Nestin-Sfrs 10 g/+）分析了Sfrs 10缺失的体内剪接效应。该小鼠在脑发育中存在缺陷，并允许真正的生理Tra 2 β调节外显子的相关性。这些属于一种新的类别，其比平均大小更长，并且重要的是需要多个协同Tra 2 β结合位点来进行有效的剪接激活，从而解释了在敲除小鼠中观察到的剪接缺陷。受调控的外显子包括一个盒式外显子，该外显子产生Nasp组蛋白伴侣的减数分裂同种型，有助于监测DNA双链断裂。我们还发现了一个以前未表征的毒物外显子，识别了脊椎动物Tra 2蛋白之间的反馈控制新途径。Nasp-T和Tra 2a毒素外显子在进化上是保守的，这表明它们可能控制基本的发育过程。缺乏RRM的Tra 2 β蛋白亚型能够激活特定的靶外显子，表明作为剪接共激活因子的额外功能作用。值得注意的是，苍蝇和人类之间保守的N末端RS 1结构域对于Tra 2 β的剪接激活剂功能至关重要。缺乏该N-末端RS 1结构域的Tra 2 β版本有效抑制由全长Tra 2 β蛋白激活的相同靶外显子。选择性剪接放大了基因组的信息内容，从单个基因中产生多种mRNA亚型。Tra 2蛋白结合并激活替代外显子，在小鼠中Tra 2 β通过未知的靶RNA对胚胎发育至关重要。在这里，我们报告了第一个目标外显子，在发育中的小鼠生理调节Tra 2 β。这些受调节的外显子的正常激活依赖于多个Tra 2 β结合位点，当Tra 2 β被去除时，在小鼠发育期间观察到这些外显子的显著错误调节。正如预期的那样，Tra 2 β通过其RNA识别基序通过直接RNA结合激活一些靶外显子的剪接。令人惊讶的是，对于一些外显子，Tra 2 β也可以通过富含精氨酸和丝氨酸残基的两个结构域（称为RS结构域）激活剪接，而不依赖于直接RNA结合。Tra 2 β的N端RS 1结构域对于生理靶外显子的剪接激活是绝对必要的，这解释了为什么该结构域在脊椎动物和无脊椎动物之间是保守的。令人惊讶的是，没有RS 1的Tra 2 β蛋白作为剪接阻遏物起作用，这表明内源性Tra 2 β蛋白亚型可能差异调节相同的靶外显子。
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. Alternative splicing amplifies the informational content of the genome, making multiple mRNA isoforms from single genes. Tra2 proteins bind and activate alternative exons, and in mice Tra2β is essential for embryonic development through unknown target RNAs. Here we report the first target exons that are physiologically regulated by Tra2β in developing mice. Normal activation of these regulated exons depends on multiple Tra2β binding sites, and significant mis-regulation of these exons is observed during mouse development when Tra2β is removed. As expected, Tra2β activates splicing of some target exons through direct RNA binding via its RNA Recognition Motif. Surprisingly, for some exons Tra2β can also activate splicing independent of direct RNA binding through two domains enriched in arginine and serine residues (called RS domains). The N-terminal RS1 domain of Tra2β is absolutely essential for splicing activation of physiological target exons, explaining why this domain is conserved between vertebrates and invertebrates. Surprisingly, Tra2β proteins without RS1 operate as splicing repressors, suggesting the possibility that endogenous Tra2β protein isoforms may differentially regulate the same target exons.
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影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
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Tuschl T
影响因子：
4.8
作者：
Jiang, ZH;Tang, H;Wu, JY
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Wu, JY
影响因子：
5.3
作者：
Grellscheid, SN;Smith, CWJ
通讯作者：
Smith, CWJ
影响因子：
30.8
作者：
Cartegni, L;Krainer, AR
通讯作者：
Krainer, AR
影响因子：
12.3
作者：
Langmead B;Trapnell C;Pop M;Salzberg SL
通讯作者：
Salzberg SL