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

黑腹果蝇的性致死性蛋白（Sxl）通过在性别决定级联中结合多聚嘧啶通道来调节变形（tra） mRNA前体的选择性剪接。Sxl蛋白有两个串联的RNA结合域。由于Sxl蛋白的氨基末端RBD （RBD1）与典型RBD的序列同源性较低，特别是在假定的功能残基上，因此很难明确定位RNP1和RNP2基序。因此，首先，我们通过有限的胰蛋白酶消化确定了Sxl蛋白的第一个rna结合域（RBD1）的氨基端和羧基端边界。我们用Tyr取代Phe 166，构建了一个高可溶性突变体，具有与野生型相同的rna结合特性。利用该突变蛋白，我们进行了核磁共振测量，并在溶液中阐明了Sxl RBD1的二级和三级结构。其次，我们确定了Sxl蛋白的两个串联排列的RNA结合域和来自多聚嘧啶通道的12核苷酸单链RNA之间复合物的晶体结构。这两个rna结合域的β -薄片平台彼此相对，形成一个v形的裂缝。RNA特征性地延伸并结合在该裂缝中，其中UGUUUUUUU序列被蛋白质特异性识别。这一结果首次揭示了蛋白质与同源RNA特异性结合的机制，同源RNA没有分子内碱基对。

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.