RNA–protein interaction methods to study viral IRES elements

Francisco‐Velilla, Rosario; Fernandez-Chamorro, Javier; Lozano, Gloria; Díaz-Toledano, Rosa; Martínez‐Salas, Encarnación

doi:10.1016/j.ymeth.2015.06.023

Cited by 25 publications

(28 citation statements)

References 67 publications

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“…This process includes preprocessing of traces, signal alignment, sequence alignment, and reactivity estimation by Gaussian peak integration. The reactivity values obtained for the untreated RNA (DMSO) were subtracted from the treated (OPW-Ru or NMIA) samples to obtain the net reactivity for each nucleotide (Francisco-Velilla et al 2015). Quantitative reactivity for individual data sets were normalized to a scale spanning 0 to 2, in which 0 indicates an unreactive nucleotide and the average intensity at highly reactive residues is set to 1.0.…”

Section: Discussionmentioning

confidence: 99%

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

Lozano

Jiménez‐Aparicio

Herrero

et al. 2016

RNA

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RNA function is determined by its structural organization. The RNA structure consists of the combination of distinct secondary structure motifs connected by junctions that play an essential role in RNA folding. Selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) probing is an established methodology to analyze the secondary structure of long RNA molecules in solution, which provides accurate data about unpaired nucleotides. However, the residues located at the junctions of RNA structures usually remain undetected. Here we report an RNA probing method based on the use of a novel open-paddlewheel diruthenium (OPW-Ru) compound [Ru 2 Cl 2 (µ-DPhF) 3 (DMSO)] (DPhF = N,N ′ -diphenylformamidinate). This compound has four potential coordination sites in a singular disposition to establish covalent bonds with substrates. As a proof of concept, we have analyzed the reactivity of OPW-Ru toward RNA using two viral internal ribosome entry site (IRES) elements whose function depends on the structural organization of the molecule. Our study suggests that the compound OPW-Ru preferentially attacks at positions located one or two nucleotides away from junctions or bulges of the RNA structure. The OPW-Ru fingerprinting data differ from that obtained by other chemical reagents and provides new information about RNA structure features.

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Section: Discussionmentioning

confidence: 99%

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

Lozano

Jiménez‐Aparicio

Herrero

et al. 2016

RNA

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“…For SHAPE data processing obtained with P 32 -labeled primers, the intensity of RT-stops was quantified as described (45). The reactivity values observed in the untreated RNA were subtracted from all the NAI-treated RNAs.…”

Section: Methodsmentioning

confidence: 99%

In-cell SHAPE uncovers dynamic interactions between the untranslated regions of the foot-and-mouth disease virus RNA

2016

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The genome of RNA viruses folds into 3D structures that include long-range RNA–RNA interactions relevant to control critical steps of the viral cycle. In particular, initiation of translation driven by the IRES element of foot-and-mouth disease virus is stimulated by the 3΄UTR. Here we sought to investigate the RNA local flexibility of the IRES element and the 3΄UTR in living cells. The SHAPE reactivity observed in vivo showed statistically significant differences compared to the free RNA, revealing protected or exposed positions within the IRES and the 3΄UTR. Importantly, the IRES local flexibility was modified in the presence of the 3΄UTR, showing significant protections at residues upstream from the functional start codon. Conversely, presence of the IRES element in cis altered the 3΄UTR local flexibility leading to an overall enhanced reactivity. Unlike the reactivity changes observed in the IRES element, the SHAPE differences of the 3΄UTR were large but not statistically significant, suggesting multiple dynamic RNA interactions. These results were supported by covariation analysis, which predicted IRES-3΄UTR conserved helices in agreement with the protections observed by SHAPE probing. Mutational analysis suggested that disruption of one of these interactions could be compensated by alternative base pairings, providing direct evidences for dynamic long-range interactions between these distant elements of the viral genome.

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“…The proteins of interest were prepared as GST-fusions as described [46]. For binding, the GST-fusion protein (4 lg) bound to the glutathione resin (GE HealthCare) was incubated with HIS-eIF4B (100, 200, 500 ng) or His-PTB proteins (100, 200 ng), in five volumes of binding buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM DTT, 2 mM MgCl 2 , 0.5% Igepal CA-630, 10% glycerol) 2 h at 4°C in a rotating wheel.…”

Section: Gst-pull Down Assaymentioning

confidence: 99%

“…All reactivity values were normalized to this average value to generate the corresponding SHAPE reactivity profiles [46]. In all cases, formation of full-length product at the top of the gel was verified.…”

Section: Rna-protein Footprintingmentioning

confidence: 99%

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G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

et al. 2017

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RNA-protein interactions play a pivotal role in the function of picornavirus internal ribosome entry site (IRES) elements. Here we analysed the impact of Ras GTPase SH3 domain binding protein 1 (G3BP1) in the IRES activity of foot-and-mouth disease virus (FMDV). We found that G3BP1 interacts directly with three distinct sequences of the IRES element using RNA electrophoretic mobility-shift assays. Analysis of the interaction with domain 5 indicated that the G3BP1 binding-site is placed at the single-stranded region although it allows large sequence heterogeneity and the hairpin located upstream of this region enhances retarded complex formation. In addition, G3BP1 interacts directly with the polypyrimidine tract-binding protein and the translation initiation factor 4B (eIF4B) through the C-terminal region. Moreover, G3BP1 is cleaved during FMDV infection yielding two fragments, Ct-G3BP1 and Nt-G3BP1. Both fragments inhibit cap- and IRES-dependent translation, but the Ct-G3BP1 fragment shows a stronger effect on IRES-dependent translation. Assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein. Our results highlight the IRES-binding capacity of G3BP1 and illustrate its function as a translation inhibitor.

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RNA–protein interaction methods to study viral IRES elements

Cited by 25 publications

References 67 publications

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

In-cell SHAPE uncovers dynamic interactions between the untranslated regions of the foot-and-mouth disease virus RNA

G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

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