3S-DB Identifies an RNA Repository Facilitating Stop Codon Readthrough for Selenocysteine Insertion and Selenoproteome Expansion

Selenoproteins are proteins that contain selenocysteine (Sec, U), the 21st amino acid, whose highly reactive selenol group makes these proteins essential for redox regulation. Sec is incorporated via a stop codon readthrough mechanism—a widespread, functionally essential, and tightly regulated mode of gene expression. This process primarily involves the UGA stop codon, the SECIS RNA element, and its binding protein SBP2. Currently, the 25 known human selenoproteins were largely discovered through predictions of stem-looped SECIS structures. However, the exploration of yet-to-be-defined selenoproteins and SECIS elements has been hindered by the accuracy and depth of these predictions. In this study, we focus on SBP2, the core SECIS-binding protein, to analyze all its binding RNAs using RNA immunoprecipitation sequencing (RIP-Seq) technology. Based on this data, we have constructed a robust selenoprotein RNA repository known as the 3S-DB (SBP2-bound RNA sequencing-supported selenoprotein database, SSS-DB or 3S-DB). This 3S-DB contains 1,333 RNA sequences, including all those known selenoprotein RNAs, that can bind to SBP2 with potential SECIS function enabling UGA stop codon readthrough. Importantly, we validate that the 3' UTRs of PDF and ATP5MJ exhibit SECIS activity. In summary, we established the 3S-DB, which highlights mRNAs that can be directly bound by SBP2, potentially facilitating the recruitment of the entire Sec insertion machinery for the synthesis of new selenoproteins. Our results provide a valuable resource for the discovery of novel selenoproteins and previously unrecognized SECIS elements, with the potential to redefine the selenoproteome landscape and deepen our understanding of their roles in redox biology and beyond. Furthermore, this work offers new insights into non-canonical stop codon readthrough and the broader mechanisms governing translational regulation of the genetic code.