Internal initiation of translation is promoted
by internal ribosome entry site (IRES) cis-acting
elements. Using transcripts that correspond to the structural
domains of the foot-and-mouth disease virus (FMDV) IRES,
we have identified RNA–RNA interactions between separated
domains (1–2, 3, 4–5, or HH) of the IRES structure.
All the assayed domains were able to interact with the
full-length IRES as well as with domain 3, although to
a different extent, with the most efficient interactions
being those occurring between domains 3 and 4–5,
and domains 3 and 1–2. RNA–RNA complexes were
stable over 1 h of incubation at 37 °C, and depended
on Mg2+ and RNA concentration. Neither the antisense
domain 1–2 nor tRNA interacted with domain 3, providing
experimental evidence of the specificity for the sense
strand of the IRES sequence. Additionally, domain 1–2
did not interact with 4–5, leading to the suggestion
that domain 3 acts as a scaffold structure where the other
domains bind. The thermal disassociation profile of these
complexes indicated different strength in these interactions.
Whereas 50% of the complexes between domains 3 and 4–5
were destabilized at 45 °C, those formed by domain
1–2 and 3 required temperatures higher than 51 °C.
Efficient self-dimerization of domains 3 and 4–5
was found in the absence of other transcripts. Formation
of domain 3 homodimer competed with formation of heterocomplexes
with other domains, and conversely, domain 3 homodimers
were competed out by the presence of the other domains.
RNA interactions were also observed at physiological concentrations
of Mg2+ and K1+. The identification
of the RNA–RNA complexes reported here provide direct
experimental evidence of tertiary interactions within IRES