Identification of the Selenoprotein S Positive UGA Recoding (SPUR) element and its position-dependent activity

Selenoproteins are a unique class of proteins that contain the 21^st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3′ UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3′ UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3′ UTR reveals a more complex regulation of Sec incorporation than previously realized.

硒蛋白（Selenoproteins）是一类独特的蛋白质，其含有第21种氨基酸——硒半胱氨酸（selenocysteine, Sec）。将Sec掺入蛋白质需通过对UGA终止密码子的重编码实现。所有25种哺乳动物硒蛋白的mRNA均拥有3′非翻译区（3′ untranslated region, 3′ UTR）的茎环结构，即硒半胱氨酸插入序列（Selenocysteine Insertion Sequence, SECIS），该结构是Sec掺入所必需的。学界普遍认为SECIS是调控Sec掺入的主要RNA元件，但本实验室近期针对硒蛋白S（Selenoprotein S, SelS）的研究结果却与之相悖。本研究报道，SelS 3′ UTR的前91个核苷酸包含一个近端茎环（proximal stem loop, PSL）以及一段被命名为SelS UGA重编码正向元件（SelS Positive UGA Recoding, SPUR）的保守序列。我们开发了一种用于检测UGA重编码的SelS-V5/UGA替代测定法，并通过质谱验证了该方法可准确反映细胞内的Sec掺入水平。利用该测定法，我们发现SPUR元件中的点突变会显著降低报告基因的重编码效率，因此SPUR是实现UGA-Sec密码子通读所必需的。与之相反，删除PSL则会提升Sec掺入水平；当用其他茎环结构或无结构化序列替换PSL后，该提升效应会被逆转，这表明PSL并未在Sec掺入过程中发挥主动调控作用。进一步研究显示，SPUR相对于UGA-Sec密码子的位置对实现最优UGA重编码至关重要。我们对SelS 3′ UTR中SPUR元件的鉴定，揭示了比此前认知更为复杂的Sec掺入调控机制。