SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy

Selenocysteine incorporation at UGA codons requires cia-acting mRNA seconda ry structures and several specialized trans-acting factors. The latter incl ude a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation facto r to the selenoprotein mRNA, Overexpression of selenoprotein mRNAs in trans fected cells results in inefficient selenocysteine incorporation due to lim itation of one or more of these factors. Using a transfection-based competi tion assay employing overexpression of selenoprotein mRNAs to compete for s elenoprotein synthesis, we investigated the ability of the trans-acting fac tors to overcome competition and restore selenocysteine incorporation. We r eport that co-expression of SBP2 overcomes the limitation produced by selen oprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocyst eine-specific elongation factor do not. Competition studies indicate that o nce bound to SECIS elements, SBP2 does not readily exchange between them. F inally, we show that SBP2 preferentially stimulates incorporation directed by the selenoprotein P and phospholipid hydroperoxide glutathione peroxidas e SECIS elements over those of other selenoproteins. The mechanistic implic ations of these findings for the hierarchy of selenoprotein synthesis and n onsense-mediated decay are discussed.