SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy

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Abstract
Selenocysteine incorporation at UGA codons requires cis ‐acting mRNA secondary structures and several specialized trans ‐acting factors. The latter include a selenocysteine‐specific tRNA, an elongation factor specific for this tRNA and a SECIS‐binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection‐based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans ‐acting factors to overcome competition and restore selenocysteine incorporation. We report that co‐expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl‐tRNA and the selenocysteine‐specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense‐mediated decay are discussed.