A novel RNA sensor responds to stress and regulates selenium distribution in mammals

许多环境毒素通过产生活性氧物种而导致肝脏损伤。虽然对肝毒性的转录反应已被绘制出来，但转录后反应的机制仍未得到充分研究。我们最近发现，在应激过程中，肝细胞因子硒蛋白SELENOP的产生受到转录后调控。SELENOP有10个硒半胱氨酸(SEC)残基，具有向外周输送硒的功能，这是男性生育和正常大脑功能所必需的过程。先前的工作已经证实，SELENOP以两种主要的亚型存在，它们的硒含量要么低，要么高(分别是短形式和长形式)，这是由于第二个SEC密码子提前终止造成的。这两种形式与不同的功能相关，短形式具有肝素结合和硫氧还蛋白样活性，而长形式增加了硒的运输功能。这项提案的首要目标是确定环境毒素产生的细胞压力如何改变SELENOP异构体的产生。在这里，我们提供了初步的数据，SELENOP 3‘非编码区中的特定序列是在应激条件下在肝细胞中有效地产生长形蛋白所必需的。该序列不同于特征良好的SEC插入序列(SECIS)，其中SELENOP有两个，在特定的UGA密码子上掺入SEC所需的。此外，我们先前的工作表明，补充硒在体外通过一种未知的机制使长形SELENOP的量增加4倍。我们现在知道下游的SELENOP SECIS元件(SECIS-2)对于这种硒反应是必要的，但不是充分的。总而言之，这些数据表明，肝细胞使用SELENOP mRNA中基于RNA的传感器，该传感器对多种环境线索做出反应。基于这些初步数据和先前的工作证明，SELENOP编码区序列是有效的SEC掺入所必需的，我们预测这些传感器不是离散序列，而是形成调节核糖体进程或SEC掺入效率或两者兼而有之的RNA结构的复杂元件集。因此，它们可能代表真正的哺乳动物核糖开关，但只有一种核糖开关已被报道。这一调控机制的预期功能结果是，即使在氧化应激和亚最佳硒浓度期间，SELENOP仍会继续生成长形的SELENOP，从而保持硒在肝外组织的分布。我们提出了三个高度集中的目标来确定SELENOP mRNA如何对环境线索做出反应：1)我们将确定在表达转基因SELENOP mRNA的细胞中改变长：短SELENOP比率的环境毒素的类型；2)我们将确定SECIS-2依赖的长形SELENOP产生的机制；以及3)我们将使用选择性2‘OH酰化分析的引物延伸(SHAPE)来确定SELENOP mRNA中应激和硒传感的结构基础。

Many environmental toxins cause liver damage by generating reactive oxygen species. While transcriptional responses to hepatotoxicity have been mapped, mechanisms of post-transcriptional responses remain understudied. We have recently found that production of the hepatokine selenoprotein, SELENOP, is post-transcriptionally regulated during stress. SELENOP, which has 10 selenocysteine (Sec) residues functions to deliver selenium to the periphery, a process that is essential for male fertility and normal brain function. Prior work has established that SELENOP exists as two major isoforms with either low or high selenium content (short and long forms, respectively) resulting from premature termination at the second Sec codon. These two forms are associated with distinct functions, the short form possessing a heparin binding and thioredoxin-like activity, with the long form adding the selenium transport function. The overarching goal of this proposal is to determine how SELENOP isoform production is altered by cellular stress generated by environmental toxins. Here we present preliminary data that a specific sequence in the SELENOP 3' UTR is required for efficient production of the long form under stress conditions in liver cells. This sequence is distinct from the well characterized Sec Insertion Sequence (SECIS), of which SELENOP has two, that is required for the incorporation of Sec at specific UGA codons. Additionally, our prior work showed that supplemental selenium increased the amount of long form SELENOP by 4-fold in vitro through an unknown mechanism. We now know that the downstream SELENOP SECIS element (SECIS-2) is necessary but not sufficient for this selenium response. Together these data indicate that hepatocytes use an RNA based sensor in the SELENOP mRNA that responds to multiple environmental cues. Based on this preliminary data and prior work demonstrating that the SELENOP coding region sequence is required for efficient Sec incorporation, we predict that these sensors are not discrete sequences but rather complex sets of elements forming RNA structures that regulate ribosome progression or Sec incorporation efficiency or both. As such, they may represent bona fide mammalian riboswitches of which only one other has been reported. The predicted functional consequence of this regulatory mechanism is that the long form of SELENOP continues to be made even during oxidative stress and suboptimal selenium concentrations, thus preserving selenium distribution to extrahepatic tissues. We propose three highly focused aims to determine how the SELENOP mRNA is responding to environmental cues: 1) We will identify the types of environmental toxins that alter the long:short SELENOP ratio in cells expressing genetically modified SELENOP mRNA; 2) We will determine the mechanism of SECIS-2 dependent long form SELENOP production; and 3) We will use selective 2' OH acylation analyzed by primer extension (SHAPE) to determine the structural basis for stress and selenium sensing in the SELENOP mRNA.