Apical Loop−Internal Loop Interactions:  A New RNA−RNA Recognition Motif Identified through in Vitro Selection against RNA Hairpins of the Hepatitis C Virus mRNA

Aldaz-Carroll, Lydia; Tallet, Béatrice; Dausse, Eric; Yurchenko, Ludmila; Toulmé, Jean‐Jacques

doi:10.1021/bi0121508

Cited by 66 publications

(56 citation statements)

References 37 publications

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“…ALIL complexes are a type of kissing complex widely distributed in nature, and play important roles in many biological processes (Brunel et al 2002). In vitro-selected RNA aptamers also exploit this strategy to efficiently associate with their targets (Aldaz-Carroll et al 2002;Da Rocha Gomes et al 2004). In all cases, the presence of a short stretch of sequence complementarity located in the apical and internal loops of the interacting domains is sufficient to stabilize the resulting complex.…”

Section: Discussionmentioning

confidence: 99%

“…In all cases, the presence of a short stretch of sequence complementarity located in the apical and internal loops of the interacting domains is sufficient to stabilize the resulting complex. A peculiarity shown by all these systems is that the G-C rich stems flank the internal interacting loop (Aldaz-Carroll et al 2002;Brunel et al 2002;Da Rocha Gomes et al 2004). Interestingly, this feature is also shown by 5BSL3.2, further supporting the ALIL interaction model.…”

Section: Discussionmentioning

confidence: 99%

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A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

Romero-López¹,

Berzal‐Herranz²

2009

RNA

112

146

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The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 59 UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 39 UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 39 end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 39 end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA-RNA interaction between the 59 and 39 ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 59-39 end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

Romero-López¹,

Berzal‐Herranz²

2009

RNA

112

146

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“…For treatment carried out under denaturing conditions, RNAs were incubated in 10 mM Tris-HCl pH 8, 1 mM EDTA (Branch et al 1989). Similarly, the desired RNAs were treated with 5 × 10 −6 mg/mL of RNase A (Aldaz-Carroll et al 2002) (Boehringer Manheim) under the same buffer conditions used for T1; when required digested RNA was analyzed by primer extension. RNase V1 (Ambion) partial digestions were performed as described using 0.2 U per assay, followed of incubation during 10 min at 37°in buffer N, but then analyzed by primer extension.…”

Section: Ribonuclease Accessibilitymentioning

confidence: 99%

“…For RNA-RNA interactions, [␥-32 P]-ATP labeled oligoribonucleotide (GUAA, UCCG, or GUAG) or the indicated FMDV IRES domains were incubated independently at 80°C for 1 min, chilled on ice (Aldaz-Carroll et al 2002), and then mixed in 50 mM sodium cacodylate pH 7.5, 300 mM KCl, 10 mM MgCl 2 (Ferrandon et al 1997). RNA-RNA complexes were allowed to form for 90 min at 20°C and inmediately analyzed by electrophoresis in native acrylamide gels supplemented with 2.5 mM MgCl 2 (Fedor and Uhlenbeck 1990;Paillart et al 1996).…”

Section: Rna-rna and Rna-protein Interactionmentioning

confidence: 99%

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

Fernández-Miragall

Martínez‐Salas²

2003

RNA

117

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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.

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“…SPR is widely used to investigate protein-protein, protein-small molecules, or protein-DNA interactions (for review, see Rich and Myszka 2011) but rarely for investigating RNA-RNA complexes, likely due to their susceptibility to hydrolysis. In previous studies, we have successfully investigated interactions between RNA oligonucleotides with structured RNA of human viruses or cells (Ducongé et al 2000;Aldaz-Carroll et al 2002;Lebars et al 2008;Dausse et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Palau

Masante

Ventura

et al. 2013

RNA

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Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3 ′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

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Apical Loop−Internal Loop Interactions: A New RNA−RNA Recognition Motif Identified through in Vitro Selection against RNA Hairpins of the Hepatitis C Virus mRNA

Cited by 66 publications

References 37 publications

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

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

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

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