title: Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES).
abstract: 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 degrees 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 degrees C, those formed by domain 1-2 and 3 required temperatures higher than 51 degrees 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 elements.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Ramos
authors.forename: E
authors.initials: E
authors.lastname: Martínez-Salas
mesh_terms: D000818:Animals; D005537:Aphthovirus / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002412:Cations; D019281:Dimerization; D007477:Ions; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013696:Temperature
chemical_list: D002412:Cations; D007477:Ions; D012367:RNA, Viral