Alternative Transcripts and 3′UTR Elements Govern the Incorporation of Selenocysteine into Selenoprotein S

Abstract

Selenoprotein S (SelS) is a 189 amino acid trans-membrane protein that plays an important yet undefined role in the unfolded protein response. It has been proposed that SelS may function as a reductase, with the penultimate selenocysteine (Sec188) residue participating in a selenosulfide bond with cysteine (Cys174). Cotranslational incorporation of Sec into SelS depends on the recoding of the UGA codon, which requires a Selenocysteine Insertion Sequence (SECIS) element in the 3′UTR of the transcript. Here we identify multiple mechanisms that regulate the expression of SelS. The human SelS gene encodes two transcripts (variants 1 and 2), which differ in their 3′UTR sequences due to an alternative splicing event that removes the SECIS element from the variant 1 transcript. Both transcripts are widely expressed in human cell lines, with the SECIS-containing variant 2 mRNA being more abundant. In vitro experiments demonstrate that the variant 1 3′UTR does not allow readthrough of the UGA/Sec codon. Thus, this transcript would produce a truncated protein that does not contain Sec and cannot make the selenosulfide bond. While the variant 2 3′UTR does support Sec insertion, its activity is weak. Bioinformatic analysis revealed two highly conserved stem-loop structures, one in the proximal part of the variant 2 3′UTR and the other immediately downstream of the SECIS element. The proximal stem-loop promotes Sec insertion in the native context but not when positioned far from the UGA/Sec codon in a heterologous mRNA. In contrast, the 140 nucleotides downstream of the SECIS element inhibit Sec insertion. We also show that endogenous SelS is enriched at perinuclear speckles, in addition to its known localization in the endoplasmic reticulum. Our results suggest the expression of endogenous SelS is more complex than previously appreciated, which has implications for past and future studies on the function of this protein.

Selenocysteine incorporation: A trump card in the game of mRNA decay

“…This is true especially in the case of selenoprotein N, wherein a cis-element located in the coding region known as the Sec codon redefinition element (SRE) stimulates Sec incorporation efficiency, and a point mutation in the SRE greatly affects RNA stability (Maiti et al, 2008; Howard et al, 2007). Very recently, the 3′ UTR of selenoprotein S was also shown to contain sequences that regulated Sec incorporation efficiency both upstream and downstream of the SECIS in the 3′ UTR (Bubenik et al, 2013). Thus the varying UGA codon environment among selenoprotein transcripts and potential structural elements in the coding and untranslated region may modulate UPF1 interaction and thus NMD activation.…”

Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements

“…We did not experimentally address whether presumptive regulation of SelS may be linked to some condition such as selenium availability. Future work will need to address potential functional significance of these two forms and extend analysis of the 3′ variant lacking the sole SECIS studied previously [21] and also here.…”

“…This shorter form of human Sepp1 mRNA that lacks SECIS 2 is present in lower abundance than the canonical form, and interestingly no difference in the ratio in different tissues has been detected. The only prior report of a eukaryotic selenoprotein mRNA lacking a SECIS was selenoprotein S [21] and in that case it was the sole SECIS element. With polyA-Seq data [59], we confirmed the presence of distal polyA sites consistent with the SelS SECIS-lacking variant mRNA (electronic supplementary material, figure S2A).…”

“…Alternative splicing of selenoprotein S mRNA 8 nt from the start of the 3′ UTR yields a minority of transcripts that lack the sole SECIS element. Translation of these transcripts results in the penultimate codon, the sole UGA, mediating termination instead of selenocysteine specification [21]. The efficiency of selenocysteine specification and likely splicing are influenced by an mRNA structural element, known as SRE, 3′ adjacent to the UGA that in SECIS-containing transcripts specifies selenocysteine [21].…”

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization

“…In fact, translational regulation is known to play an important role for many selenoproteins (Howard et al 2013). In at least one case (selenoprotein S, VIMP gene), this is achieved by the regulated inclusion/exclusion of the SECIS element in the mature transcripts through alternative splicing (Bubenik et al 2013). In such SECIS-lacking truncated transcripts, no Sec insertion will take place, and termination of translation at the UGA codon is the most likely outcome.…”