Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis.
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 approximately 500 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.
Copeland
P R
PR
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Driscoll
D M
DM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D011506:Proteins; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D051140:Selenoproteins
D011506:Proteins; D016601:RNA-Binding Proteins; C498488:Secisbp2 protein, rat; D051140:Selenoproteins; C119984:selenocysteine insertion sequence binding protein, mammalian; D012313:RNA