A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells
A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells
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DOI：
10.1073/pnas.0610683104
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发表时间：
2007-05-08
影响因子：
11.1
通讯作者：
Gladyshev, Vadim N.
中科院分区：
文献类型：
--
作者：
Novoselov, Sergey V.;Lobanov, Alexey V.;Gladyshev, Vadim N.
硒蛋白是一类含有硒代半胱氨酸（Sec）的蛋白质，其密码子为UGA。在真核生物中，Sec的掺入需要Sec插入序列（SECIS）元件，即位于硒蛋白mRNA的3 '非翻译区的茎环结构。在这里，我们报告的SECIS元件的非典型形式的弓形虫和新孢子虫犬，人类和家畜的单细胞顶复形寄生虫的鉴定。该SECIS在SBP2结合位点中具有GGGA序列，代替先前认为不变的AUGA。使用计算和分子技术相结合，我们表明，弓形虫和新孢子虫具有典型和非典型的SECIS元素。GGGA型SECIS元件支持哺乳动物HEK 293和NIH 3T3细胞中的Sec插入，并且比所测试的天然哺乳动物SECIS元件更有效。此外，突变成GGGA形式的哺乳动物I型和II型SECIS元件是功能性的，但表现出降低的Sec插入效率。我们进行了计算搜索弓形虫的SECIS元件的AUGA和GGGA形式，并检测到5个硒蛋白基因，包括一个编码以前未描述的硒蛋白，指定SeIQ，和两个包含GGGA形式的SECIS元件。与此相反，GGGA型SECIS元件在哺乳动物和线虫中未检测到。作为一个实际的研究成果，我们开发了pSelenoproteins 1，一个载体，方便表达硒蛋白在哺乳动物细胞。它包含一个SBP2基因和最有效的测试SECIS元件：GGGA型弓形虫SeIT结构的AUGA突变体。
Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem-loop structure located in the 3'-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SeIQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SeIT structure.