3S-DB identifies an RNA repository facilitating stop codon readthrough for selenocysteine insertion and selenoproteome expansion

Selenoproteins contain the selenocysteine (Sec, U), which is essential for redox regulation due to its reactive selenol group. The current set of 25 human selenoproteins was defined by the presence of SECIS elements in the 3' UTR coupled with in-frame UGA codons through a stop codon readthrough mechanism. However, the discovery of novel selenoproteins and SECIS elements remains limited by the constraints of SECIS prediction methods. In this study, we focus on SECISBP2, the core SECIS-binding protein, to analyze its binding RNAs using RNA immunoprecipitation sequencing (RIP-Seq) technology. We constructed the 3S-DB, a database of 1,333 SECISBP2-bound RNAs with potential SECIS functions for UGA recoding, including most known selenoprotein transcripts. Importantly, we validate that the 3' UTRs of PDF and ATP5MJ exhibit SECIS activity using luciferase assays and by fusing them to known selenoprotein RNAs. In summary, our results provide a valuable resource of mRNAs with potential for UGA recoding and previously unrecognized SECIS elements, with the potential to expand the known selenoproteome and advance 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.