The translation of mammalian selenoprotein mRNAs
requires the 3′ untranslated region that contains
a selenocysteine insertion sequence (SECIS) element necessary
for decoding an in-frame UGA codon as selenocysteine (Sec).
Selenoprotein biosynthesis is inefficient, which may be
due to competition between Sec insertion and termination
at the UGA/Sec codon. We analyzed the polysome distribution
of phospholipid hydroperoxide glutathione peroxidase (PHGPx)
mRNA, a member of the glutathione peroxidase family of
selenoproteins, in rat hepatoma cell and mouse liver extracts.
In linear sucrose gradients, the sedimentation velocity
of PHGPx mRNA was impeded compared to CuZn superoxide dismutase
(SOD) mRNA, which has a coding region of similar size.
Selenium supplementation increased the loading of ribosomes
onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether
the slow sedimentation velocity of PHGPx mRNA is due to
a block in elongation, we analyzed the polysome distribution
of wild-type and mutant mRNAs translated in vitro. Mutation
of the UGA/Sec codon to UGU/cysteine increased ribosome
loading and protein synthesis. When UGA/Sec was replaced
with UAA or when the SECIS element core was deleted, the
distribution of the mutant mRNAs was similar to the wild-type
mRNA. Addition of SECIS-binding protein SBP2, which is
essential for Sec insertion, increased ribosome loading
and translation of wild-type PHGPx mRNA, but had no effect
on the mutant mRNAs. These results suggest that elongation
is impeded at UGA/Sec, and that selenium and SBP2 alleviate
this block by promoting Sec incorporation instead of termination.