Picornavirus internal ribosome entry sites (IRESs) are
∼450 nt. RNA elements that direct internal initiation
of translation, such that when placed between the two cistrons
of a dicistronic construct, they drive independent translation
of the downstream cistron. Consequently they have been
widely used for coordinated expression of two or more proteins.
All picornavirus IRESs have an AUG triplet at the very
3′ end, which is thought to be the actual site of
internal ribosome entry. However with some IRESs, such
as foot-and-mouth disease virus, and especially poliovirus,
the majority of ribosomes do not initiate translation at
this putative entry site AUG, but at the next AUG further
downstream, which is thought to be accessed by a process
of linear ribosome scanning from the entry site. If this
is so, then it should be possible to regulate IRES-dependent
translation by inserting an iron responsive element (IRE)
between the putative entry site AUG and the main functional
initiation site. This should make IRES-dependent translation
sensitive to the concentration of iron regulatory protein
(IRP), the protein that specifically binds to the IRE.
This has been attempted with both the foot-and-mouth disease
virus and poliovirus IRESs, and was successful in so far
as an inhibition specifically of IRES-dependent translation
was observed that was strictly dependent on both the presence
of IRP and of a functional IRE motif inserted in the sense
orientation. However, the range over which expression could
be varied was rather limited (three- to fourfold maximum),
because some IRES-dependent translation remained completely
refractory to inhibition by even very high IRP concentrations.
In contrast, with a cap-proximal IRE in the 5′ untranslated
region of an mRNA translated by the scanning mechanism,
addition of sufficient IRP results in complete inhibition.
These results support the model of IRES-promoted ribosome
entry at an upstream site followed by strictly linear scanning
to the main functional initiation site for the majority
of internal initiation events, but imply that some ribosomes
must access the functional initiation site by another route,
possibly a nonlinear shunting-like mechanism.