Identification of Determinants that Govern Processive and Efficient Selenocysteine (Sec) Incorporation

Selenoproteins include essential proteins and are unique as they incorporate selenocysteine (Sec), the 21st amino acid, in response to an stop codon (UGA). Thus selenoprotein expression is a challenge as it requires stop codon redefinition. To date, five essential factors for Sec incorporation have been identified. This includes the mRNA with an inframe UGA codon, a stem loop structure referred to as SECIS element located in the 3′UTR, a dedicated elongation factor (eEFSec), a SECIS binding protein (SBP2) and a unique Sec tRNA Sec‐tRNA Sec . However the mechanistic details of Sec incorporation still remains unclear. Among the known selenoproteins, Selenoprotein P (SEPP1) is unique mainly as it contains multiple UGA codons and possesses two SECIS elements in the 3′UTR. Thus, full length SEPP1 synthesis requires processive Sec incorporation which in vivo appears to be efficient. The focus of this research is to identify core determinants that govern processive and efficient Sec incorporation. Using 75 Se labeling in vitro we demonstrate that selenium supplementation stimulates processive Sec incorporation by 4‐fold thus linking active Sec‐tRNA Sec synthesis with processive Sec incorporation. We also establish that while the SEPP1 mRNA is inherently processive for Sec incorporation, there exists an interplay between the coding region and the 3′UTR that governs the degree of processivity. Furthermore cellular transfection studies indicate RNA stability as a crucial determinant of efficiency in mammalian SEPP1 synthesis. Thus translation regulation primarily governs processive and efficient Sec incorporation via channeling of Sec incorporation components and also by recruiting novel cis elements.