Sex-lethal splicing autoregulation in vivo:: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping
Sex-lethal splicing autoregulation in vivo:: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping
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DOI：
10.1242/dev.00274
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发表时间：
2003-02-01
期刊：
影响因子：
4.6
通讯作者：
Salz, HK
中科院分区：
文献类型：
--
作者：
Nagengast, AA;Stitzinger, SM;Salz, HK
性致死前体mRNA的选择性剪接长期以来一直是受调控剪接事件的典型例子，但雌性特异性致死RNA结合蛋白阻止包含性致死终止雄性外显子的机制尚不清楚。到目前为止，唯一的一般剪接因子，其中有在体内的证据表明，在导致男性外显子跳跃的途径中的调节作用是sans-fille（snf），剪接体U1和U2 snRNP的蛋白质组分。然而，它的作用仍然是个谜，因为SNF是作为完整snRNP的一部分还是作为游离蛋白的一部分。我们提供的证据表明，性致死与SANSFILLE在U1 snRNP的背景下，通过表征点突变，干扰组装成U1 snRNP和复杂的形成与性致死。此外，我们发现，性致死协会与其他积分U1 snRNP组件，我们提供的遗传证据，以支持这些物理相互作用的生物相关性。类似的遗传和生物化学方法也将性致死与异二聚体剪接因子U2 AF联系起来。这些研究特别指出了性致死通过与受调控的男性外显子两端的这些关键剪接因子相互作用来抑制剪接的机制。此外，由于U2 AF和U1 snRNP在剪接体组装过程中仅与前mRNA短暂相关，因此我们的研究难以与目前提出的性致死蛋白在第二催化步骤阻断剪接的模型相一致，而是认为性致死蛋白在剪接位点识别后但在催化开始之前起作用。
Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANSFILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.