INTERACTION OF THE SEX-LETHAL RNA-BINDING DOMAINS WITH RNA
INTERACTION OF THE SEX-LETHAL RNA-BINDING DOMAINS WITH RNA
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DOI：
10.1002/j.1460-2075.1995.tb00132.x
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发表时间：
1995-09-15
期刊：
影响因子：
11.4
通讯作者：
RIO, DC
中科院分区：
文献类型：
--
作者：
KANAAR, R;LEE, AL;RIO, DC
果蝇的性别决定和X染色体剂量补偿是由性致死（Sxl）蛋白指导的。Sxl通过与3'剪接位点多聚嘧啶区结合来调节Transformer前体mRNA的剪接，从而部分地发挥作用。多聚嘧啶束对于后生动物前体mRNA的剪接是必需的。为了阐明多聚嘧啶束的剪接调控机制，我们分析了Sxl与RNA的相互作用。将Sxl的RNA结合活性定位于蛋白质的两个核糖核蛋白共有序列结构域。定量结合表明，这两个RNA结合域（RBD）所需的顺式位点特异性RNA结合。单个RBD与RNA的相互作用更弱，并且失去了区分野生型和突变型Transformer多嘧啶束的能力。使用核磁共振技术鉴定了可能与聚嘧啶道相互作用的RBD之一中的结构元件。此外，我们的数据表明，多个亚氨基质子的Transformer聚嘧啶道参与氢键。有趣的是，在体外Sxl以相等的亲和力结合到前体mRNA的多聚嘧啶束，其在体内不调节。我们讨论了这一发现的影响，通过该机制，SXL可以获得特定的多聚嘧啶道在体内的选择性。
Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein, In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo.