Secondary Structure Probing of Potato Spindle Tuber Viroid (PSTVd) and Sequence Comparison with Other Small Pathogenic RNA Replicons Provides Evidence for Central Non-canonical Base-pairs, Large A-rich Loops, and a Terminal Branch

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

doi:10.1006/jmbi.1996.0543

Cited by 77 publications

(81 citation statements)

References 67 publications

(174 reference statements)

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“…It is localized in the nucleus (Schumacher et al 1983), where it is thought to replicate via the DNA-dependent RNA polymerase II (Rackwitz et al 1981;Schindler & Muhlbach 1992) via the rolling circle mechanism (Branch and Robertson 1984). The partial self-complementarity that occurs along the 359 bases of the PSTVd genome gives rise to the secondary structure of an interrupted double helix with a branch at the left terminus (Gast et al 1996). The viroid molecule appears under the microscope shaped as a rod (Sanger et al 1976).…”

Section: Introductionmentioning

confidence: 99%

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

Maniataki¹,

Tabler²,

Tsagris³

2003

RNA

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Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.

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Section: Introductionmentioning

confidence: 99%

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

Maniataki¹,

Tabler²,

Tsagris³

2003

RNA

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“…This conformation, however, differed significantly in the distribution and size of some loops and double-stranded segments when compared with others reported previously [11,17,20,21]. For the mc ASBVd (À) RNA the agreement was even better, with the three software programs predicting the same rod-shaped secondary structure.…”

Section: Discussionmentioning

confidence: 38%

“…The mc ASBVd (À) strand, although considerably less prevalent than its (+) counterpart, still reaches in planta a titre high enough to obtain the amount needed for dissection in vitro with several approaches. Previous thermodynamics-based predictions of the secondary structures of the mc ASBVd (+) and (À) RNAs, usually performed with the Mfold software program [30], have led to rod-like conformations that in some cases include a short bifurcation in one of the terminal domains [11,17,20,21,31]. Here we have reassessed this issue with recent versions of the same software program, and two others, considering that the high A+U content of the mc ASBVd (+) and (À) forms should result in secondary structures of minimal free energy less stable and more difficult to predict than those typical of other viroids.…”

Section: Resultsmentioning

confidence: 99%

“…The secondary structure of the mc ASBVd (+) and (À) RNAs has been analysed in silico by searching those of minimal free energy [11,17,[20][21][22]. While this thermodynamics-based methodology predicts rod-like or quasi-rod-like conformations for both strands, in vitro examination of the ml ASBVd (+) and (À) RNAs by selective 2¢-hydroxyl acylation analysed by primer extension (SHAPE), complemented by gel electrophoresis and other physical approaches [23][24][25], has led to secondary structures exhibiting significant differences.…”

Section: Introductionmentioning

confidence: 99%

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The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

López-Carrasco

Flores

2017

Journal of General Virology

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Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (À) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2¢-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (À) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

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“…Uno de los más interesantes es el llamado bucle E, identificado inicialmente en el 5S rRNA y posteriormente detectado in vitro en el PSTVd mediante radiación UV (Branch et al, 1985) y modificación con dimetilsulfato (Gast et al, 1996). Es un motivo de RNA mantenido por interacciones no canónicas, al que podría unirse alguna proteína involucrada en la replicación o el transporte intranuclear .…”

Section: Tcr Ccrunclassified

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

Olivares¹

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Secondary Structure Probing of Potato Spindle Tuber Viroid (PSTVd) and Sequence Comparison with Other Small Pathogenic RNA Replicons Provides Evidence for Central Non-canonical Base-pairs, Large A-rich Loops, and a Terminal Branch

Cited by 77 publications

References 67 publications

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

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