The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: Identification of SBP2 amino acids important to SECIS RNA binding
The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: Identification of SBP2 amino acids important to SECIS RNA binding
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DOI：
10.1017/s1355838202020034
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发表时间：
2002-10-01
期刊：
影响因子：
4.5
通讯作者：
Krol, A
中科院分区：
文献类型：
--
作者：
Allmang, C;Carbon, P;Krol, A
真核生物合成硒蛋白需要硒半胱氨酸插入序列（SECIS） RNA，这是硒蛋白mrna 3'非翻译区中的一个发夹。SECIS RNA被SECIS结合蛋白2 （SBP2）识别，而SBP2在这个特殊的翻译机制中起着关键作用。这项工作的目的是获得SBP2-SECIS RNA复合物的结构洞察力。多个序列比对结果显示，SBP2与U4 snrna结合蛋白15.5 kD/Snu13p具有相同的L7A/L30家族的RNA结合结构域，box H/ACA snoRNP蛋白Nhp2p和几种核糖体蛋白也存在相同的RNA结合结构域。因此，我们在SECIS和U4 rna中检测到类似的二级结构基序。结合15.5 kD- u4 snRNA复合物的晶体结构数据，以及SBP2/15.5 kD序列相似性，我们设计了一种结构指导策略，预测了12个SBP2氨基酸，这些氨基酸对SECIS RNA结合至关重要。对这些氨基酸进行丙氨酸替代，然后对SBP2突变蛋白进行凝胶转移分析，发现其中四个残基的突变严重削弱或消除了SECIS RNA结合，其他八个引起了中间下调效应。除了确定SBP2识别SECIS的关键氨基酸外，我们的研究结果还提出了SBP2-SECIS RNA复合体中控制15.5 kD-U4 snRNA相互作用的一些识别原则必须相似。
Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3' untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished-or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.