We investigated the requirements for selenocysteine insertion at single or 
multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements 
were identified in the 3' untranslated region of the rat selenoprotein P mRNA, 
with predicted stem-loops and critical nucleotides similar to those in the SECIS 
elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione 
peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS 
elements confirmed that conserved nucleotides in the loop and in unpaired 
regions of the stem are critical for activity. This indicates that multiple 
contact sites are required for SECIS function. Stop codon function at any of 
five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements 
from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of 
SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in 
UGA codon position.