The selenocysteine incorporation machinery: Interactions between the SECISRNA and the SECIS-binding protein SBP2

The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprot ein mRNAs requires a selenocysteine insertion sequence (SECIS) element in t he 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion m achinery is SECIS-binding protein 2 (SBP2). In this study, we define the bi nding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylatio n binding interference. We show that SBP2 binds to a variety of mammalian S ECIS elements with similar affinity and that the SBP2 binding site is conse rved across species. Based on footprinting studies, SBP2 protects the proxi mal part of the hairpin and both strands of the lower half of the upper hel ix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for t he binding of SBP2. Modification of phosphates by ethylnitrosourea along bo th strands, of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented b inding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to th e 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for t he hierarchy of seleno-protein expression that is observed in vivo.