Structural organization of a viral IRES depends on the integrity of the GNRA motif

Abstract

Little is known about the tertiary structure of internal ribosome entry site (IRES) elements. The central domain of foot-andmouth disease (FMDV) IRES, named 3 or I, contains a conserved GNRA motif, essential for IRES activity. We have combined functional analysis with RNA probing to define its structural organization. We have found that a UNCG motif does not functionally substitute the GNRA motif; moreover, binding of synthetic GNRA stem-loops to domain 3 was significantly reduced in RNAs bearing UCCG or GUAG substitutions. The apical region of domain 3 consists of a four-way junction where residues of the GNRA tetraloop are responsible for the organization of the adjacent stem-loops, as deduced from ribonucleases and dimethyl sulfate accessibility. A single A-to-G substitution in the fourth position of this motif led to a strong RNA reorganization, affecting several nucleotides away in the secondary structure of domain 3. The study of mutants bearing UNCG or GUAG tetraloops revealed lack of protection to chemical attack in native RNA at specific nucleotides relative to the parental GUAA, suggesting that the GNRA motif dictates the organization and stability of domain 3. This effect is likely mediated by the interaction with distant residues. Therefore, the GNRA motif plays a crucial role in the organization of IRES structure with important consequences on activity.

Picornavirus IRES elements: RNA structure and host protein interactions

“…Interestingly, the GNRA motif and the C-rich loop are conserved in IRES elements classified as type I or II (Fernandez et al, 2011a;Yu et al, 2011b). The GNRA motif, which is essential for IRES activity in both FMDV and EMCV (Lopez de Quinto and Martinez-Salas, 1997; Robertson et al, 1999), adopts a tetraloop conformation (Fernandez-Miragall and Martinez-Salas, 2003;Phelan et al, 2004), and determines long-range interactions (Fernandez-Miragall et al, 2006;Ramos and Martinez-Salas, 1999). Domain 4 consists of a Yshape RNA structure (corresponding to subdomains J and K) and provides the binding-site for eIF4G (Bassili et al, 2004;Kolupaeva et al, 1998;Lopez de Quinto and Martinez-Salas, 2000;Lozano et al, 2014).…”

Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

“…The first 85 nt on the 59 end encompasses domain 2 (also termed H); it consists of a stem-loop that contains a binding site for the polypyrimidine tract-binding protein (PTB) (Luz and Beck 1991). Second, the central domain (termed 3 or I, encompassing nucleotides 86-299) is a selffolding region that adopts a cruciform RNA structure (Fernandez-Miragall and Martinez-Salas 2003) including internal RNA-RNA interactions (Fernandez-Miragall et al 2006). Third, on the 39 end of the IRES, domains 4 and 5 (or J-K and L, encompassing nucleotides 300-462) determine RNA-protein interactions involving translation initiation factors, eIF4G, eIF4B, eIF3, and auxiliary proteins as PTB (Lopez de Quinto and Martinez-Salas 2000; Pilipenko et al 2000;Lopez de Quinto et al 2001).…”

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

“…This hypothesis is in agreement with earlier proposals in which nucleotides belonging to the GNRA motif, the RAAA stem-loop and G 240 within the C-rich bulge are linked in the three-dimensional RNA structure. 12,57 As demonstrated by mutational and RNA probing studies, domain 3 plays a fundamental role in dictating the formation of a constrained structure likely providing the correct orientation to recruit the ribosome subunits to the initiation site. 10 Therefore, the conformational changes induced by IRAB would result in a misfolded IRES structure leading to diminished internal translation initiation, as observed in the translation assays reported here.…”

“…[5][6] Functional and structural analysis have shown that the FMDV IRES is organized in 5 structural domains of which domain 2 includes a polypyrimidine tract that provides the polypyrimidine tract-binding protein (PTB) binding site. 11 Domain 3 consists of 210 nucleotides, the most apical region of which is arranged as a cruciform structure 12 that harbors conserved motifs. Disruption of these motifs abrogates IRES activity, in addition to altering distant RNA-RNA interactions.…”