Efficient Incorporation of Multiple Selenocysteines Involves an Inefficient Decoding Step Serving as a Potential Translational Checkpoint and RibosomeBottleneck

Abstract

Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3 untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.Selenoprotein P is an enigma of genetic recoding. It is the only known protein in which multiple potential stop codons are recoded to function as sense codons. In selenoprotein mRNAs, UGA codons, which would normally signal the termination of protein synthesis, are decoded as the amino acid selenocysteine. This process involves a unique tRNA with an anticodon complementary to UGA and specialized secondary structures located in the 3Ј untranslated regions (UTRs) of selenoprotein mRNAs, termed selenocysteine incorporation sequence (SECIS) elements (3). For selenocysteine incorporation to occur, the SECIS element must recruit a SECIS binding protein, SBP2 (6). SBP2, in turn, recruits a dedicated elongation factor, EFsec (7, 26), complexed with selenocysteyl-tRNA (2, 31). Most selenoprotein mRNAs contain a single UGA codon and a single SECIS element. Selenoprotein P mRNAs contain 10 to 18 UGA codons, depending on the species, and two SECIS elements differing in secondary structure. The majority of studies to date on the mechanism of selenoprotein synthesis have focused on selenoprotein mRNAs containing a single UGA codon and a single SECIS element. Thus, little is known about the mechanism for incorporating multiple selenocysteines.The incorporation of selenium into selenoprotein P serves several important biological functions: it allows the accumulation of this essential but potentially toxic trace element in a biologically stable and nontoxic form, it plays a critical role in the transport of selenium from the liver via the circulation to target organs (13,25), it is thought to function in sequestering and thereby detoxifying heavy metals in plasma...

Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2

“…We found SECIS 1 to be a dominant regulator of Sec incorporation with SECIS 2 seeming largely dispensable. Overall, these results are congruous with the finding from zebrafish Sel P where mutating SECIS 1 was more detrimental to Sec incorporation than mutating SECIS 2 but the latter was required for processive Sec incorporation (13).…”

“…The role of the dual SECIS elements has only recently been studied in detail. In 2006 Stoytcheva et al (13) determined that the second SECIS element of zebrafish Sel P was required for readthrough of the first UGA and the first SECIS element was necessary for readthrough of subsequent UGAs and consequently, production of full-length protein (13). However, the mechanistic basis for this distinction of function remains elusive.…”

A novel insight into the mechanism of mammalian selenoprotein synthesis

“…While most, if not all, of the numerous molecular actors involved have been identified ), the mechanistic issues are still shrouded in mystery. The question is even more acute when considering the case of the selenoprotein P mRNA, which contains 10-17 UGA Sec codons and two SECIS elements (Stoytcheva et al 2006). In an attempt to increase our knowledge, in the work described here, we designed for the first time a minimal capped selenoprotein mRNA bearing photoreactive groups in the SECIS element.…”

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“…This raises the intriguing question of how the translation machinery decides between termination and translation at these UGA sites. A potential model was recently presented, suggesting that the two SECIS elements play different roles in the synthesis of SelP (292). It appears that SECIS 1 promotes the readthrough of the second and subsequent UGA codons directing synthesis of fulllength SelP, whereas SECIS 2 plays a more substantial role in decoding the first UGA codon, potentially controlling translation of the truncated isoforms.…”

“…It appears that SECIS 1 promotes the readthrough of the second and subsequent UGA codons directing synthesis of fulllength SelP, whereas SECIS 2 plays a more substantial role in decoding the first UGA codon, potentially controlling translation of the truncated isoforms. Hence, the interplay between the SECIS elements and distinct UGA codons could serve both as a translational checkpoint to determine the fate of the mRNA, depending on intracellular selenium status, and a bottleneck for ribosome loading on the mRNA to decode multiple UGA codons (292 (150,191). One of the studies found that cancer carriers were twice as frequently homozygotes for the A than the G allele (150).…”