Purification, Redox Sensitivity, and RNA Binding Properties of SECIS-binding Protein 2, a Protein Involved in Selenoprotein Biosynthesis

Abstract

In mammalian selenoprotein mRNAs, the highly structured 3 UTR contains selenocysteine insertion sequence (SECIS) elements that are required for the recognition of UGA as the selenocysteine codon. Our previous work demonstrated a tight correlation between codon-specific translational read-through and the activity of a 120-kDa RNA-binding protein that interacted specifically with the SECIS element in the phospholipid hydroperoxide glutathione peroxidase mRNA. This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2). We detected SBP2 binding activity in liver, hepatoma cell, and testis extracts from which SBP2 has been purified by anion exchange and RNA affinity chromatography. This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 binding activity from wild-type but not mutant RNA affinity columns. A characterization of SBP2 biochemical properties reveals that SBP2 binding is sensitive to oxidation and the presence of heparin, rRNA, and poly(G). SBP2 activity elutes with a molecular mass of ϳ500 kDa during gel filtration chromatography, suggesting the existence of a large functional complex. Direct cross-linking and competition experiments demonstrate that the minimal phospholipid hydroperoxide glutathione peroxidase 3 UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequence necessary for translational read-through. SBP2 also interacts specifically with the minimally functional 3 UTR of another selenoprotein mRNA, deiodinase 1.

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

“…Pretreatment with oxidizing reagents diamide or N-ethylmaleimide also eliminated binding activity. 40 These observations suggest that free cysteines influence the RNAbinding activity of SBP2. Therefore, we decided to examine the contribution of cysteine residues in the RNA-binding domain of SBP2 to its SECIS-binding activity.…”

“…The SECIS-binding activity of SBP2-CT was previously shown to be sensitive to reducing conditions. 40 In the complete absence of DTT, little or no binding activity was observed. Pretreatment with oxidizing reagents diamide or N-ethylmaleimide also eliminated binding activity.…”

“…The initial characterization of SBP2 also showed redox effects on its function as its SECIS-binding activity required the addition of DTT to the reaction. 40 Additional experimental support was provided by Papp et al,29 who demonstrated the presence of reversible disulfide bonds as well as a free thiol in the C-terminal part of SBP2 (aa584-854 of human SBP2). They also observed a change in subcellular localization in response to oxidative stress, although our results suggest that this is not uniformly true across all cell types.…”

Regulation of gene expression by stop codon recoding: selenocysteine

“…Alternatively, SBP2 could form a complex in the absence of a SECIS or ribosome. There are several pieces of evidence that support SBP2 multimerization: SBP2 was shown to sediment in a ≥500 kDa complex during gel filtration of a partially purified SBP2 preparation (Copeland and Driscoll, 1999), glycerol gradient sedimentation of purified, recombinant SBP2 showed the formation of a salt-sensitive complex , and in mobility shift assays, increasing concentrations of SBP2 yielded both high and low-mobility complexes (Fletcher et al, 2001;Lescure et al, 2002). An attractive model, therefore, would be that SBP2 is bound to the ribosome as a dimer with one RNA binding domain interacting with 28S rRNA and the other with an incoming SECIS element (see Fig.…”

RNA affinity chromatography

“…While all strategies can be used to successfully isolate RNA-binding proteins, they all present distinct challenges that need to be considered when designing the experiments. Cyanogen bromide activated sepharose and adipic acid dihyrazide agarose are widely used to covalently immobilize RNA (Kaminski et al, 1995;Caputi et al, 1999;Copeland & Driscoll, 1999;Sela-Brown et al, 2000;Hovhannisyan & Carstens, 2009). In the first approach, random sites of RNA attach to the matrix and therefore not all RNA molecules may maintain a homogeneous conformation which limits the number of accessible protein binding sites.…”