NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila Sex-lethal protein with target RNA fragments with site-specific [3-N-15]uridine substitutions
NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila Sex-lethal protein with target RNA fragments with site-specific [3-N-15]uridine substitutions
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DOI：
10.1093/nar/25.8.1565
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发表时间：
1997-04-15
影响因子：
14.9
通讯作者：
Shimura, Y
中科院分区：
文献类型：
--
作者：
Kim, I;Muto, Y;Shimura, Y
据报道，一个183个残基的片段，由两个RNA结合结构域组成，果蝇性致死（Sri）蛋白的RBD 1-RBD 2与靶RNA序列的寡核苷酸强结合（5 '-GUUUUUUUC-3'），其调节可变剪接，并与RNA的亚氨基形成四个或五个氢键。在本研究中，我们使用定点诱变来提高Sri的双结构域片段的溶解性，并证实该突变片段以与野生型片段相同的方式与靶RNA形成氢键。该突变片段显示出比UUUUUUUC更紧密地结合同源RNA序列GUUUUUUUC和AUUUUUUC，利用[3-N-15]尿苷亚磷酰胺合成了一系列N-15标记的靶RNA，其中一个尿苷残基被[3-N-15]尿苷特异性取代。通过观察标记尿苷残基的亚氨基H-1-N-15偶联，我们将所有四个氢键亚氨基质子分别归属于U1，U2，U 5和U6，U2和U6的亚氨基质子与蛋白质的脂肪族质子具有核Overhauser效应。这些结果表明，（G/A）UUUUUUUU靶序列中的A/G、U1、U2、U 5和U6残基被Sri蛋白的两个RNA结合结构域特异性识别。
It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1-RBD2) of the Drosophila melanogster Sex-lethal (Sri) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA, In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sri, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment, The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC, By using a [3-N-15]uridine phosphoramidite, we synthesized a series of N-15-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-N-15]uridine, By observing the imino H-1-N-15 coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA, The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein, All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sri protein.