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 s...