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 sel enocysteine insertion sequence (SECIS) elements that are required for the r ecognition of UGA as the selenocysteine codon, Our previous work demonstrat ed a tight correlation between codon-specific translational read-through an d the activity of a 120-kDa RNA-binding protein that interacted specificall y with the SECIS element in the phospholipid hydroperoxide glutathione pero xidase mRNA, This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2), We detected SBP2 binding a ctivity in liver, hepatoma cell, and testis extracts from which SBP2 has be en purified by anion exchange and RNA affinity chromatography, This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 b inding activity from wild-type but not mutant RNA affinity columns. A chara cterization of SBP2 biochemical properties reveals that SBP2 binding is sen sitive to oxidation and the presence of heparin, rRNA, and poly(G), SBP2 ac tivity elutes with a molecular mass of similar to 500 kDa during gel filtra tion chromatography, suggesting the existence of a large functional complex . Direct cross-linking and competition experiments demonstrate that the min imal phospholipid hydroperoxide glutathione peroxidase 3' UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequen ce necessary for translational read-through, SBP2 also interacts specifical ly with the minimally functional 3' UTR of another selenoprotein mRNA, deio dinase 1.