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 (Sec(188)) residue participating in a selenosulfide bond with 
cysteine (Cys(174)). 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.