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

一种新型 RNA 传感器对压力做出反应并调节哺乳动物体内硒的分布

• 批准号: 10380881
• 负责人: PAUL R COPELAND
• 金额: $ 19.63万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380881
• 关键词: 3' Untranslated Regions; Acylation; Amino Acids; Aortic Aneurysm; Biology; Brain; Cells; Cellular Stress; Code; Codon Nucleotides; Complex; Cues; DNA Insertion Elements; Data; Dietary Selenium; Disease; Elements; Ensure; Environmental Impact; Extrahepatic; Genetic Polymorphism; Genetic Transcription; Goals; Health; HepG2; Heparin Binding; Hepatocyte; Hepatotoxicity; Human; Hydroxyl Radical; IS Elements; In Vitro; Life; Malignant Neoplasms; Mammals; Maps; Messenger RNA; Molecular; Molecular Conformation; Nutritional; Obesity; Oxidative Stress; Pathway interactions; Patients; Prevention; Primer Extension; Process; Production; Protein Isoforms; Public Health; RNA; Reactive Oxygen Species; Reporting; Ribosomes; Selenium; Selenocysteine; Stress; Structure; TXN gene; Testing; Tissues; Toxic Environmental Substances; Toxin; Trace Elements; Translating; Work; base; cell injury; experience; high risk; liver injury; male fertility; mutant; novel; premature; preservation; response; selenoprotein; sensor

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.

许多环境毒素通过产生活性氧引起肝脏损伤。虽然对肝毒性的转录反应已被绘制，但转录后反应的机制仍未得到充分研究。我们最近发现肝因子硒蛋白（SELENOP）的产生在应激过程中受到转录后调控。SELENOP含有10个硒半胱氨酸（Sec）残基，其功能是将硒输送到外周，这一过程对男性生育能力和正常脑功能至关重要。先前的研究已经确定SELENOP存在两种主要的同工异构体，分别是低或高硒含量（短形式和长形式），这是由于在第2秒密码子处过早终止而导致的。这两种形式具有不同的功能，短形式具有肝素结合和硫氧还蛋白样活性，长形式具有硒转运功能。本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异构体的产生。在这里，我们提供的初步数据表明，在应激条件下，肝细胞需要SELENOP 3' UTR中的特定序列才能有效地产生长形式。该序列不同于特征良好的Sec插入序列（SECIS）， SELENOP有两个SECIS， SECIS是将Sec插入特定的UGA密码子所必需的。此外，我们之前的研究表明，添加硒可以通过一种未知的机制使体外长形SELENOP的数量增加4倍。我们现在知道下游SELENOP SECIS元件（SECIS-2）对于硒响应是必要的，但不是充分的。综上所述，这些数据表明肝细胞在SELENOP mRNA中使用一种基于RNA的传感器来响应多种环境信号。基于这些初步数据和先前的工作表明，SELENOP编码区序列是Sec有效整合所必需的，我们预测这些传感器不是离散序列，而是形成RNA结构的复杂元件集，这些RNA结构调节核糖体的进展或Sec整合效率，或两者都有。因此，它们可能代表了真正的哺乳动物核开关，其中只有一种被报道过。这种调节机制的预期功能结果是，即使在氧化应激和硒浓度不理想的情况下，长形式的SELENOP也会继续产生，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是如何对环境线索做出反应的：1)我们将确定环境毒素的类型，这些毒素会改变表达转基因SELENOP mRNA的细胞中的长/短SELENOP比率；2)确定SECIS-2依赖性长形式SELENOP生成的机理；3)我们将利用引物延伸（SHAPE）分析的选择性2' OH酰化来确定SELENOP mRNA中应激和硒感知的结构基础。