NMR elucidation of the role of Mg2+ in the structure and stability of the conserved RNA motifs of the EMCV IRES element

Abstract

The three dimensional solution structures of a highly conserved 16mer RNA, endowed with a classic 'GNRA' tetraloop motif, and a 17mer RNA containing a cytosine-rich heptaloop which is predicted to be a potential receptor for the former RNA, of the I-domain of Encephalomyocarditis virus IRES have been determined by NMR spectroscopy. As Mg(2+) plays an important role in the activity of the IRES, the corresponding NMR structures of the Mg(2+) bound RNA complexes have also been determined. These RNA NMR structures, 16mer (21 constraints per residue), 16mer RNA/Mg(2+) (21 constraints per residue), 17mer (17 constraints per residue) and 17mer RNA/Mg(2+) (16 constraints per residue), were calculated to a high degree of precision with low RMSDs and low clash scores represent, to the best our knowledge, the first structures of a type II picornavirus IRES. Conformational analysis of the average structure showed that the RNAs and their Mg(2+) complexes adopt characteristic A-form helical structures, stabilised by canonical and non-canonical interactions in both the stem and loop regions. The GCGA tetraloop of the 16mer folds into a standard GNRA conformation, with the structural role of A550 being in the form of a G547.A550 sheared base-pair made up of two hydrogen bonds. Further, the previously uncharacterised AACCCCA heptaloop present in the 17mer forms a compact tertiary loop motif, held together by strong π-π interactions. Analysis of the NMR structures demonstrates that the role of Mg(2+) is principally to confer enhanced stability to the RNAs whereby the tetra- and heptaloops can achieve optimum conformation for any RNA-RNA interactions which are crucial for understanding the structure-function relationship of the IRES.

Structural insights into viral IRES-dependent translation mechanisms

“…It is interesting to note that the local RNA flexibility of specific IRES regions, including the apical stem-loop of domain 3, the 10-nt loop of domain 4 (subdomain J), and the basal region of domain 5, are sensitive to the concentration of Mg 2+ ions in the RNA folding buffer [57 ]. Along this line of evidences, recent NMR studies carried out with synthetic oligoribonucleotides corresponding to short stem-loops of the EMCV IRES have shown that Mg 2+ ions affect the conformation adopted by the GNRA tetraloop and the C-rich loop [59].…”

Insights into Structural and Mechanistic Features of Viral IRES Elements

“…The apical region of this domain harbors essential motifs for IRES function ( Lopez de Quinto and Martinez-Salas, 1997 ; Robertson et al, 1999 ). One of the best studied is the GNRA motif, which adopts a tetraloop conformation ( Fernandez-Miragall et al, 2006 ; Dupont and Snoussi, 2009 ; Mohammed et al, 2014 ). Computational modeling of this domain generated a three-dimensional RNA structure integrating experimental evidences for tertiary contacts between distant residues of the secondary structure ( Jung and Schlick, 2013 ).…”

IRE mRNA riboregulators use metabolic iron (Fe2+) to control mRNA activity and iron chemistry in animals

“…2), which is paired to G 7 . 49 This is the same region where a number of metal complexes bind, such as 1,10-Cu 2+ -phenanthroline and [Ru(tpy)(bpy)O] 2+ . 50 Crystallographic analyses, fruitful, as they have been for structural determinations of other RNA-metal complexes, 45 cannot provide information about Fe 2+ binding to IRE-RNA.…”

“…The structure of Mg 2+ complexed to two different mRNA regulatory elements is known: (1) the Mg 2+ -Internal Ribosome mRNA Entry Site (IRES) complex 49 and (2) the Mg 2+ -catalytic RNA (ribozyme) complex, exemplified by Mg 2+ binding to a G-U wobble pair and an GNRA tetraloop. 2 For IRE-RNAs, based on changes in RNA-bound, ethidium bromide fluorescence upon Fe 2+ binding, the IRE-RNA conformation in the active form (when Fe 2+ is bound) is different from free IRE-RNA, 11 but more detail awaits future investigations.…”