UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins

A A Turanov,A V Lobanov,V N Gladyshev,D L Hatfield

doi:10.1093/nar/gkt409

Abstract

It is thought that the SelenoCysteine Insertion Sequence (SECIS) element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3′-UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3′-UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3′-UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoprotein-specific manner, and that this process is controlled by the SECIS element in the 3′-UTR.

Highlights

Mammalian selenoproteins are a diverse group of proteins, which contain selenocysteine (Sec), which is known as the 21st genetically encoded amino acid [1,2]
These findings suggested that Sec insertion may be limited to designated positions within the thioredoxin reductase 1 (TR1) ORF, and to the natural Sec position and its close proximity
A quantitative PCR (qPCR) analysis showed no significant differences in the corresponding mRNA levels of EGFP-hTR1 expression constructs, suggesting that the SelenoCysteine Insertion Sequence (SECIS)-dependent Sec insertion was regulated at the translational level (Supplementary Figure S19)

Summary

Introduction

Mammalian selenoproteins are a diverse group of proteins, which contain selenocysteine (Sec), which is known as the 21st genetically encoded amino acid [1,2]. Incorporation of selenium as Sec into selenoproteins occurs via a specific mechanism that recodes the UGA codon from its normal translation termination function. Sec biosynthesis and incorporation depends on several proteins, including selenophosphate synthetase 2, which converts selenide to monoselenophosphate and is itself a selenoprotein, phosphoseryl-tRNA(Ser)Sec kinase that phosphorylates the seryl moiety on seryltRNA(Ser)Sec, Sec synthase that synthesizes Sec on its tRNA, a Sec-specific elongation factor, SelenoCysteine Insertion Sequence (SECIS)-binding protein 2 (SBP2), ribosomal protein L30 and possibly several other factors [4,5]. The selenoprotein synthesis machinery uses a specific structure in the 30-UTRs of selenoprotein mRNAs, termed the SECIS element that recruits protein factors and Sec tRNA to synthesize Sec and incorporate it into nascent polypeptides in response to UGA codons [6,7]. Despite great progress in our understanding of Sec biosynthesis and incorporation into proteins, many of the detailed mechanisms that regulate Sec insertion remain to be investigated

Methods

Results

Conclusion