Insight into Mammalian Selenocysteine Insertion: Domain Structure and Ribosome Binding Properties of Sec Insertion Sequence Binding Protein 2

There are many examples of translational regulation that involve RNA binding proteins interacting with specific sequences in the 3′ untranslated regions (UTRs) of various mRNAs (10, 18). A specialized case of this is found in the incorporation of selenocysteine (Sec) into a select group of eukaryotic proteins where a sequence specific 3′ UTR binding protein is required for Sec insertion at its cognate UGA codon (6). The Sec-containing proteins that have been characterized perform myriad biological functions including oxidant defense and hormone maturation (reviewed in references 9 and 20). While mice deficient in the selenoprotein glutathione peroxidase are viable (11), one or more selenoproteins are apparently required for early development, as elimination of the tRNASec gene in mice causes early embryo lethality (3). While the incorporation of Sec into the Escherichia coli formate dehydrogenase isozymes is fairly well characterized (2), the system which governs mammalian Sec insertion has only been partially elucidated. Several essential components of what we here term the Sec insertion complex (SIC) have been identified. These include the UGA codon that encodes Sec (13), the Sec insertion sequence (SECIS) element found in all selenoprotein 3′ UTRs (1), and the recently identified SECIS binding protein (SBP2), which we demonstrated to be required for Sec insertion in vitro (6). A fourth component of the SIC that is also likely to be required for Sec incorporation is the Sec-specific elongation factor (eEFsec) that has been recently shown to be functional in transfected cells (7, 21). Because Sec is encoded by what is ordinarily a stop codon, the mechanism of Sec insertion is likely to involve direct competition with translation termination. The SIC may therefore interact with or regulate the interactions of the release factors required for translation termination. This work focuses on the RNA binding protein SBP2, a novel 94-kDa protein that shares a 32-amino acid motif with several ribosomal proteins and eukaryotic translation termination release factor 1 (eRF-1). eRF-1 functionally and structurally mimics a tRNA molecule that is specific for stop codons and in this fashion binds the ribosomal A site and terminates chain elongation (19). The fact that SBP2 and eRF-1 share this sequence suggests that they also share a common class of targets, which may shed light on the molecular basis for the competition between Sec insertion and termination. This conserved sequence has been proposed to be a novel RNA binding motif (12) and has recently been demonstrated to be involved in the binding of ribosomal protein L30 to its own mRNA (15). In this report, we establish that this motif, which we will refer to as the L30 RNA binding domain, is required for SBP2 RNA binding activity and function as measured by the ability to incorporate Sec in vitro. In addition, we have identified a nonoverlapping functional domain that is required for Sec insertion but not RNA binding. Further analysis indicates that SBP2 is stably associated with the ribosomal fraction in transfected cells and in vitro, and that this interaction may be mediated by 28S rRNA. We hypothesize that SBP2 may be involved in selecting a subset of ribosomes which are competent for Sec insertion.