Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis
Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis
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DOI：
10.1074/jbc.274.36.25447
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发表时间：
1999-09-03
影响因子：
4.8
通讯作者：
Driscoll, DM
中科院分区：
文献类型：
--
作者：
Copeland, PR;Driscoll, DM
在哺乳动物硒蛋白mRNA中，高度结构化的3' UTR含有硒代半胱氨酸插入序列（SECIS）元件，其是识别UGA作为硒代半胱氨酸密码子所需的。我们先前的工作证明了密码子特异性翻译通读与120-kDa RNA结合蛋白的活性之间的紧密相关性，该蛋白与磷脂氢过氧化物谷胱甘肽过氧化物酶mRNA中的SECIS元件特异性相互作用，本研究报道了SECIS结合蛋白2（SBP 2）的RNA结合和生物化学性质。我们用阴离子交换和RNA亲和层析法从肝、肝癌细胞和睾丸提取物中纯化了SBP 2，该方案使我们能够确定一个120 kDa的多肽，共洗脱与SBP 2结合活性从野生型，但不是突变体RNA亲和柱。SBP 2生物化学性质的表征揭示，SBP 2结合对氧化敏感，并且肝素、rRNA和poly（G）的存在下，SBP 2活性在凝胶过滤色谱期间以类似于500 kDa的分子量洗脱，表明存在大的功能复合物。直接交联和竞争实验表明，最小的磷脂氢过氧化物谷胱甘肽过氧化物酶3' UTR结合位点在82和102个核苷酸之间，这与翻译通读所需的最小序列相关。SBP 2还与另一种硒蛋白mRNA脱碘酶1的最小功能3' UTR特异性相互作用。
In mammalian selenoprotein mRNAs, the highly structured 3' UTR contains selenocysteine insertion sequence (SECIS) elements that are required for the recognition of UGA as the selenocysteine codon, Our previous work demonstrated a tight correlation between codon-specific translational read-through and the activity of a 120-kDa RNA-binding protein that interacted specifically with the SECIS element in the phospholipid hydroperoxide glutathione peroxidase mRNA, This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2), We detected SBP2 binding activity in liver, hepatoma cell, and testis extracts from which SBP2 has been purified by anion exchange and RNA affinity chromatography, This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 binding activity from wild-type but not mutant RNA affinity columns. A characterization of SBP2 biochemical properties reveals that SBP2 binding is sensitive to oxidation and the presence of heparin, rRNA, and poly(G), SBP2 activity elutes with a molecular mass of similar to 500 kDa during gel filtration chromatography, suggesting the existence of a large functional complex. Direct cross-linking and competition experiments demonstrate that the minimal phospholipid hydroperoxide glutathione peroxidase 3' UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequence necessary for translational read-through, SBP2 also interacts specifically with the minimally functional 3' UTR of another selenoprotein mRNA, deiodinase 1.