Howard Michael T, Moyle Mark W, Aggarwal Gaurav, Carlson Bradley A, Anderson Christine B
Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA.
RNA. 2007 Jun;13(6):912-20. doi: 10.1261/rna.473907. Epub 2007 Apr 24.
Selenocysteine insertion during decoding of eukaryotic selenoprotein mRNA requires several trans-acting factors and a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3' UTR. A second cis-acting selenocysteine codon redefinition element (SRE) has recently been described that resides near the UGA-Sec codon of selenoprotein N (SEPN1). Similar phylogenetically conserved elements can be predicted in a subset of eukaryotic selenoprotein mRNAs. Previous experimental analysis of the SEPN1 SRE revealed it to have a stimulatory effect on readthrough of the UGA-Sec codon, which was not dependent upon the presence of a SECIS element in the 3' UTR; although, as expected, readthrough efficiency was further elevated by inclusion of a SECIS. In order to examine the nature of the redefinition event stimulated by the SEPN1 SRE, we have modified an experimentally tractable in vitro translation system that recapitulates efficient selenocysteine insertion. The results presented here illustrate that the SRE element has a stimulatory effect on decoding of the UGA-Sec codon by both the methylated and unmethylated isoforms of Sec tRNA([Ser]Sec), and confirm that efficient selenocysteine insertion is dependent on the presence of a 3'-UTR SECIS. The variation in recoding elements predicted near UGA-Sec codons implies that these elements may play a differential role in determining the amount of selenoprotein produced by acting as controllers of UGA decoding efficiency.
真核生物硒蛋白mRNA解码过程中的硒代半胱氨酸插入需要几种反式作用因子和顺式作用的硒代半胱氨酸插入序列（SECIS），该序列通常位于3'UTR中。最近描述了第二种顺式作用的硒代半胱氨酸密码子重新定义元件（SRE），它位于硒蛋白N（SEPN1）的UGA-Sec密码子附近。在一部分真核生物硒蛋白mRNA中可以预测到类似的系统发育保守元件。先前对SEPN1 SRE的实验分析表明，它对UGA-Sec密码子的通读有刺激作用，这并不依赖于3'UTR中SECIS元件的存在；尽管正如预期的那样，通过包含SECIS，通读效率进一步提高。为了研究SEPN1 SRE刺激的重新定义事件的性质，我们改进了一个易于实验操作的体外翻译系统，该系统概括了有效的硒代半胱氨酸插入。此处给出的结果表明，SRE元件对Sec tRNA（[Ser]Sec）的甲基化和未甲基化异构体对UGA-Sec密码子的解码均有刺激作用，并证实有效的硒代半胱氨酸插入依赖于3'-UTR SECIS的存在。在UGA-Sec密码子附近预测的重新编码元件的变化意味着这些元件可能通过作为UGA解码效率的控制器，在确定产生的硒蛋白量方面发挥不同的作用。
