Structural Study on the RNA recognition mechanism by the Drosophila Sex-lethal protein

果蝇的性致死蛋白(SXL)通过与性别决定级联中的Tra多嘧胺束结合来调节Transformer(TrA)mRNA前体的选择性剪接。SXL蛋白有两个单链连接的RNA结合域。由于SXL蛋白的氨基末端RBD(Rbd1)与典型的RbD序列同源性很低，特别是在推测的功能残基上，很难明确定位RNP1和RNP2基序。因此，我们首先通过有限的胰酶消化确定了SXL蛋白第一个RNA结合域(Rbd1)的氨基和羧基末端边界。通过用Tyr替换Phe 166，我们构建了一个高度可溶性的突变体，它表现出与野生型相同的RNA结合特性。其次，我们确定了SXL蛋白的两个螺旋排列的RNA结合域与来自反式多嘧啶的12核苷酸单链RNA之间的复合物的晶体结构。这两个RNA结合域的β-折叠平台相互面对，形成V形裂隙。RNA在这个裂隙中特异性地延伸和结合，其中UGUUUUUU序列被蛋白质特异性识别。这一结果首次揭示了蛋白质与没有分子内碱基对的同源RNA特异结合的机制。

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) mRNA precursor by binding to the tra polypyrimidine tract in the sex determination cascade. The Sxl protein has two tandemly-linked RNA binding domains. As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, paticularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, at first, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe 166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, andelucidated the secondary and tertiary structures of the Sxl RBD1 in solution.Second, we have determined the crystal structure of the complex between the two tandemly-arranged RNA binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNa derived from the tra polypyrimidine tract. The two RNA-binding domains have their beta-sheet platforms facing each other, to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognised by the protein. This result provides the first insight into the mechanism by which a protein binds specifically to a cognate RNA that has no intramolecular base pairs.