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

Lozano, Gloria; Jiménez‐Aparicio, Reyes; Herrero, Santiago Montero; Martínez-Salas, Encarnación

doi:10.1261/rna.054353.115

Cited by 20 publications

(25 citation statements)

References 56 publications

(68 reference statements)

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“…In contrast, the spacer regions between domains 2, 3, 4 and 5, and the hairpin of domain 5 showed enhanced reactivity (red boxes in Figure 2C ). The observed protections in domain 3 are coincident with the binding-site of the protein PCBP2, as well as with local flexibility areas observed in vitro within this domain ( 52 – 55 ). The enhanced reactivity noticed at positions 410–450 could be interpreted as a higher flexibility of the initiation zone.…”

Section: Resultssupporting

confidence: 65%

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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Section: Resultssupporting

confidence: 65%

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

2016

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“…RNA flexibility appears to be a key feature of picornavirus IRES elements, as mentioned above for the HCV IRES. In this regard, the implementation of novel di-metallic chemical compounds able to detect flexible regions on the RNA structure in solution have allowed the identification of four-way and three-way junctions within flexible regions of the FMDV and HCV IRES conformation ( Lozano et al, 2016b ), which are consistent with experimental data obtained from independent approaches ( Figure 4 ). Noteworthy, the ability of RNA molecules to acquire distinct conformations in response to specific ligands, as well as environmental signals, determines their function.…”

Section: Internal Initiation Mechanisms: Rna Structural Motifs Involvsupporting

confidence: 76%

Insights into Structural and Mechanistic Features of Viral IRES Elements

et al. 2018

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Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of protein synthesis using cap-independent mechanisms. However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements.

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“…The construct expressing the monocistronic IRES RNA was previously described 40 . RNA was synthesized in vitro using a plasmid linearized with SphI.…”

Section: Methodsmentioning

confidence: 99%

“…Prior to treatment, in vitro synthesized RNA was folded by heating at 95 °C for 2 min, snap cooling on ice for 2 min, and subsequently incubated in a final volume of 18 μl of folding mix (100 mM HEPES pH 8.0, 100 mM NaCl, 5.25 mM MgCl 2 ) for 20 min at 37 °C 40 . Prefolded RNA (2 pmol) was incubated with 6.5 mM 1-methyl-7-nitroisatoic anhydride (1M7), 1-methyl-6-nitroisatoic anhydride (1M6) or isatoic anhydride (IA) (Thermo-Fisher) for 70 s, 3 min or 36 min, respectively 4 , 6 , 44 at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE

Lozano

Francisco‐Velilla

Martínez-Salas

2018

Sci Rep

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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.

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

Cited by 20 publications

References 56 publications

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

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

Insights into Structural and Mechanistic Features of Viral IRES Elements

Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE

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