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簇引起肝脏损伤。虽然肝毒性的转录反应已被映射，转录后反应的机制仍然研究不足。我们最近发现，生产的肝细胞因子硒蛋白，硒，是转录后调节过程中的压力。具有10个硒代半胱氨酸（Sec）残基的SELENOP功能是将硒传递到外周，这是男性生育力和正常脑功能所必需的过程。先前的工作已经确定，由于在第二个Sec密码子处提前终止，因此SELENOP以两种主要同种型存在，具有低或高硒含量（分别为短型和长型）。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，长形式增加了硒转运功能。该提案的总体目标是确定环境毒素产生的细胞应激如何改变SELENOP亚型的生产。在这里，我们提供了初步的数据，在肝细胞中的应激条件下，需要在SELENOP 3' UTR中的特定序列来有效地产生长形式。该序列与充分表征的Sec插入序列（SECIS）不同，其中SELENOP具有两个Sec插入序列，这是在特定UGA密码子处掺入Sec所需的。此外，我们之前的工作表明，补充硒通过未知的机制使体外长型SELENP的量增加了4倍。我们现在知道，下游的SELENOPSECIS元件（SECIS-2）对于这种硒响应是必要的，但还不够。这些数据共同表明，肝细胞在响应多种环境线索的SELENOPmRNA中使用基于RNA的传感器。基于这一初步数据和先前的工作表明，SELENOP编码区序列是需要有效的Sec掺入，我们预测，这些传感器不是离散的序列，而是复杂的一套元件形成RNA结构，调节核糖体的进展或Sec掺入效率或两者兼而有之。因此，它们可能代表真正的哺乳动物核糖开关，其中只有一个其他已被报道。这种调节机制的预测功能结果是，即使在氧化应激和亚最佳硒浓度期间，也会继续生成长型的SELENP，从而保持硒在肝外组织中的分布。我们提出了三个高度集中的目标来确定SELENOPmRNA如何响应环境线索：1）我们将鉴定在表达遗传修饰的SELENOPmRNA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依赖性长形式SELENOPmRNA产生的机制;我们将使用引物延伸（SHAPE）分析的选择性2' OH酰化来确定SELENOPmRNA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.