Incorporation of the 21st amino acid, selenocysteine, into proteins is specified 
in all three domains of life by dynamic translational redefinition of UGA 
codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting 
selenocysteine insertion sequence (SECIS) usually located in the 3'UTR of 
selenoprotein mRNAs. Here we present comparative sequence analysis and 
experimental data supporting the presence of a second stop codon redefinition 
element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal 
gene, SEPN1. This element is sufficient to stimulate high-level (6%) 
translational redefinition of the SEPN1 UGA codon in human cells. Readthrough 
levels further increased to 12% when tested in the presence of the SEPN1 3'UTR 
SECIS. Directed mutagenesis and phylogeny of the sequence context strongly 
supports the importance of a stem loop starting six nucleotides 3' of the UGA 
codon. Sequences capable of forming strong RNA structures were also identified 
3' adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH, 
SelO, and SelT selenoprotein genes.