3S-DB: SBP2-Bound RNA Sequencing for the Discovery of Novel Functional SECIS Elements

Selenoproteins are defined as proteins that contain selenocysteine (Sec, U), playing crucial roles in living organisms. The incorporation of Sec involves a unique mechanism that primarily includes the UGA codon, the SECIS 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 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. Importantly, we validate that the 3' UTRs of PDF and ATP5MJ exhibit SECIS activity. In summary, the established 3S-DB highlights the mRNA that SBP2 can directly bind to, potentially recruiting the entire Sec insertion machinery to generate new selenoproteins. Our results provide a crucial resource for uncovering novel selenoproteins and previously unidentified SECIS elements. Overall design: RNA immunoprecipitation sequencing (RIP-Seq) was performed using Flag antibody in HEK 293T cell lysates overexpressing the Flag vector, Flag-SBP2, and Flag-SBP2 with concomitant sodium selenite treatment. The experimental design included three groups, each with three biological replicates, and each group comprised input and IP samples.