The Dimerization Domain of Potato Spindle Tuber Viroid, a Possible Hallmark for Infectious RNA

Gast, Frank-Ulrich; Kempe, Dirk; Sänger, Heinz L.

doi:10.1021/bi980830d

Cited by 9 publications

(9 citation statements)

References 39 publications

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“…In contrast, examination of the hairpin I/double-stranded structure reveals some appealing features. Hairpin I is composed by a tetraloop, a 3-bp stem, an internal symmetric loop of 1–3 nt in each strand that presumably interact by non-Watson-Crick base pairs [40], and a 9–10-bp stem that can be interrupted by a 1-nt symmetric or asymmetric internal loop [18,35] (Figure 3). Remarkably, these structural features are conserved in the type species of the five genera composing the family Pospiviroidae and additionally: i) the capping tetraloop is palindromic itself, and ii) the two central positions of the tetraloop and the central base pair of the 3-bp stem are phylogenetically conserved (Figure 3) [35].…”

Section: Discussionmentioning

confidence: 99%

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

et al. 2007

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Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase.

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Section: Discussionmentioning

confidence: 99%

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

et al. 2007

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“…In contrast, examination of the hairpin I/double-stranded structure reveals some appealing features. Hairpin I is composed by a tetraloop, a 3-bp stem, an internal symmetric loop of 1–3 nt in each strand that presumably interact by non-Watson-Crick base pairs [28], and a 9–10-bp stem that can be interrupted by a 1-nt symmetric or asymmetric internal loop [22,25] (Figure 2). Remarkably, these structural features are conserved in the type species of the five genera composing the family Pospiviroidae and additionally: i) the capping tetraloop is palindromic itself, and ii) the two central positions of the tetraloop and the central base pair of the 3-bp stem are phylogenetically conserved (Figure 2) [25].…”

Section: Family Pospiviroidae: An Asymmetric Rolling-circle Mechanismmentioning

confidence: 99%

“…Characterization of the ml (+) RNAs from A. thaliana transgenically expressing dt CEVd (+) RNAs has shown that this is actually the case [32]. The adoption in vivo of the double-stranded structure with a GC-rich central region containing the cleavage sites could be promoted by hairpin I because prior work with PSTVd has mapped a dimerization domain at this hairpin [28]. This situation resembles that observed previously in retroviruses in which dimerization, a critical step of their infectious cycle, is mediated by a hairpin with a palindromic loop that can dimerize via a kissing loop interaction [33].…”

Section: Family Pospiviroidae: An Asymmetric Rolling-circle Mechanismmentioning

confidence: 99%

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Viroid Replication: Rolling-Circles, Enzymes and Ribozymes

Flores

Gas

Molina‐Serrano

et al. 2009

Viruses

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Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5′ and 3′ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.

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“…Recent work indicates that the PAL2 sequence does not appear to form the stable stem-loop structure previously assumed but is within a flexible domain that may make dimerization more favorable thermodynamically and/or kinetically. Dimerization of RNA molecules has also been documented for tRNAs (Grosjean et al 1976;Wittenhagen and Kelley 2002), a 75-nucleotide (nt) segment of the u29 viral encoded pRNA (Chen et al 2000), and potato spindle tuber viroid RNA (Gast et al 1998). In these examples, complementary base-pairing of loop-loop ''kissing complexes'' was proposed to initiate dimerization.…”

Section: Introductionmentioning

confidence: 98%

Conversion of stable RNA hairpin to a metastable dimer in frozen solution

Sun¹,

Li²,

Wartell³

2007

RNA

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Previous studies employing a 79-nucleotide (nt) RNA indicated that this RNA could form two bands in a native polyacrylamide gel while one band was observed in a denaturing gel. This report describes an investigation on the nature of the two corresponding structures and the segment responsible for forming the slower mobility band. Sedimentation equilibrium of the 79-nt RNA was consistent with the two gel bands corresponding to monomer and dimer forms. The portion of the RNA required for dimer formation was explored using a secondary structure prediction algorithm of two 79-nt RNAs linked in a head-to-tail fashion. The predicted structure suggested that the first 21-nt at the 59 end of each RNA formed a selfcomplementary duplex. A ribonuclease H assay carried out with RNA prepared as monomer (M), or a mixture of monomer and dimer (M/D), gave results consistent with the predicted M and D structures. Gel mobility experiments on 59 and 39 segments of the 79-nt RNA also indicated that dimer formation was due to the 21-nt 59 end. Studies on the 21-nt RNA molecule and sequence variants showed that this sequence can form a hairpin and a dimer complex. Unexpectedly, the hairpin to dimer conversion was shown to occur at high efficiency in frozen solution, although little or no conversion was observed above 0°C. The results indicate that a freezing environment can promote formation of intermolecular RNA complexes from stable RNA hairpins, supporting the notion that this environment could have played a role in the evolution of RNA complexity.

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The Dimerization Domain of Potato Spindle Tuber Viroid, a Possible Hallmark for Infectious RNA

Cited by 9 publications

References 39 publications

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

Viroid Replication: Rolling-Circles, Enzymes and Ribozymes

Conversion of stable RNA hairpin to a metastable dimer in frozen solution

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