Abstract
Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro, and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.
Highlights
The function of RNA molecules depends on their three-dimensional (3D) structure[1] and on their ability to acquire distinct conformations on its own and/or in response to specific signals[2]
Aiming at understanding the role of RNA structural domains on ribosome recruitment, we studied the local flexibility of the foot-and-mouth disease virus (FMDV) internal ribosome entry sites (IRES) element incubated with ribosomal fractions
On the free RNA, nucleotides reaching the final conformation on long timescales are placed on domains 4–5 of the IRES region upstream of the start codon
Summary
The function of RNA molecules depends on their three-dimensional (3D) structure[1] and on their ability to acquire distinct conformations on its own and/or in response to specific signals[2]. The RNA reactivity toward these compounds provides information on nucleotides that undergo local conformational changes on long timescales (IA) and those involved in tertiary interactions (1M6)[5,8], time-dependent RNA-ligand interactions. The vast majority of mRNAs initiates translation by a cap-dependent mechanism that depends on the recognition of the m7G(5′) ppp(5′)N structure (designated cap) placed at the 5′ end of most mRNAs9 This process begins with the binding of the translation initiation factor (eIF)-4F complex to the cap. This complex recruits the 40S ribosomal subunit www.nature.com/scientificreports/. Different IRES elements perform the same function despite lacking conservation of primary sequence, secondary RNA structure, and host factor requirement to recruit the ribosomal subunits[12]. Whereas the IGR assembles a complex with 80S ribosomes in the absence of eIFs15,16, the HCV IRES, and those of picornaviruses, require different combinations of eIFs to assemble 48S complexes in vitro[17,18]
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