An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations

Kuhlmann, Micki M.; Chattopadhyay, Maitreyi; Stupina, Vera A.; Gao, Feng; Simon, Anne

doi:10.1128/jvi.01129-16

Cited by 29 publications

(41 citation statements)

References 50 publications

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“…For readthrough, disruption of the upstream hairpin present in TCV did not affect readthrough in vitro; however, genome accumulation was reduced in infections to 28% that of wt (12). Our results with Pre-RTSL in TNV-D are similar to those for TCV in that infectivity was reduced (ϳ48% of wt), but, in contrast, readthrough levels were also negatively affected, albeit to a lesser degree (79 to 88% of wt) (Fig.…”

Section: Structural Features Of the Upper Portion Of The Rtsl Moderatsupporting

confidence: 72%

“…Comparable RNA hairpins are also present in other members of the genus Betanecrovirus ( Fig. 2A), and similarly positioned stem-loops are predicted in all tombusvirids (12). Silent mutations designed to disrupt the stem of Pre-RTSL in mutants Pre1 through Pre3, (Fig.…”

Section: Structural Features Of the Upper Portion Of The Rtsl Moderatmentioning

confidence: 78%

“…Among these, the RNA elements that modulate translational readthrough in plusstrand RNA viruses have been the focus of many studies (1,9).Members of family Tombusviridae are plus-sense RNA viruses that express their RNA-dependent RNA polymerase (RdRp) by either Ϫ1 frameshifting or readthrough (17). In both cases, there is an extended stem-loop (SL) RNA structure located immediately downstream of the slippery sequence or stop codon that is required for efficient recoding (5,6,12,18,19). In addition, for maximal recoding to occur, a bulge in this RNA structure must base pair, via a long-range RNA-RNA interaction, with a sequence located 3= proximally in the viral genome (5,6,18,19).…”

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confidence: 99%

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Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Newburn

White

2017

J Virol

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Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus and family The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3' untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3' to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed. The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3' proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

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Section: Structural Features Of the Upper Portion Of The Rtsl Moderatsupporting

confidence: 72%

Section: Structural Features Of the Upper Portion Of The Rtsl Moderatmentioning

confidence: 78%

mentioning

confidence: 99%

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Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Newburn

White

2017

J Virol

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“…This correlation was supported by subsequent work extending SMFS studies of −1 PRF to other pseudoknots (30), different types of stimulatory structures such as hairpins (31), and the effects of antiframeshifting ligands (32). Evidence supporting the importance of conformational plasticity has also been found from single-molecule fluorescence experiments of ribosomes translocating through pseudoknots (33) and ensemble structural studies of stimulatory structures using such methods as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) (21,34). However, structures stimulating extremely high levels of frameshifting, which should pose the most stringent test of any hypotheses relating stimulatory structure properties and −1 PRF efficiency, have not yet been studied to probe their mechanical properties or dynamics.…”

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confidence: 80%

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure

Halma

Ritchie

Cappellano

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates −1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during −1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of −1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.programmed ribosomal frameshifting | RNA folding | pseudoknots | force spectroscopy | West Nile virus

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“…Two additional bands of moderate intensity were observed between 35 and 40 kDa. The larger product, migrating at 38 kDa, corresponded to the CP (29), and the product at 35 kDa corresponded to translation initiating within the p88 ORF, at position 1393 (determined by altering individual AUG start codons [data not shown]). The identity of CP was confirmed by introducing an in-frame UGA stop codon after 5 amino acids within the CP ORF that abolished CP synthesis (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus

May

Johnson

Saleem

et al. 2017

J Virol

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To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5= cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo. An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo. Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCE Cap-independent translation is a common strategy among positivesense, single-stranded RNA viruses for bypassing the host cell requirement of a 5= cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.KEYWORDS IRES, translation, carmovirus, cap-independent translation, Turnip crinkle virus, unstructured RNA, internal ribosome entry site, translational control E ukaryotic mRNAs utilize a cap-dependent translation where the 40S ribosomal subunit is recruited to a 5= 7-methylguanosine (m 7 G) cap with the assistance of eukaryotic initiation factors (eIFs). Translation initiation is largely governed by eIF4F, which consists of eIF4E and eIF4G in plants. eIF4E is responsible for binding to the 5= cap structure, while eIF4G is a scaffolding protein that interacts with eIF4E and poly(A) binding protein (PABP) bound to the 3= poly(A) tail, thus circularizing the mRNA (1). The 43S preinitiation complex, composed of the ribosomal 40S subunit bound to eIF3/eIF5 and eIF2-Met-tRNAi Met , is attracted to the 5= cap via binding of eIF3 to eIF4G and then

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An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations

Cited by 29 publications

References 50 publications

Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus

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