Structural analyses of Avocado sunblotch viroid reveal differences in the folding of plus and minus RNA strands.
Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.
Sorbonne Universités, Université Pierre et Marie Curie UPMC Univ Paris 06, Centre National de la Recherche Scientifique, FRE3207, Laboratoire ANBIOPHY, Fonctions et Interactions des Acides Nucléiques, 75005 Paris, France. clementine.delanforino@gmail.com.
Clémentine
C
Delan-Forino
Centre National de la Recherche Scientifique, Unité Mixte de Recherche UMR8015, Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France. jules.deforges@gmail.com.
Jules
J
Deforges
Centre National de la Recherche Scientifique, Unité Mixte de Recherche UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France. lionel.benard@ibpc.fr.
Lionel
L
Benard
Centre National de la Recherche Scientifique, Unité Mixte de Recherche UMR8015, Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France. bruno.sargueil@parisdescartes.fr.
Bruno
B
Sargueil
Sorbonne Universités, Université Pierre et Marie Curie UPMC Univ Paris 06, Centre National de la Recherche Scientifique, FRE3207, Laboratoire ANBIOPHY, Fonctions et Interactions des Acides Nucléiques, 75005 Paris, France. marie-christine.maurel@upmc.fr.
Marie-Christine
MC
Maurel
Sorbonne Universités, Université Pierre et Marie Curie UPMC Univ Paris 06, Centre National de la Recherche Scientifique, FRE3207, Laboratoire ANBIOPHY, Fonctions et Interactions des Acides Nucléiques, 75005 Paris, France. claire.torchet_devaux@upmc.fr.
Claire
C
Torchet
D020029:Base Pairing; D024202:Electrophoretic Mobility Shift Assay; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D027421:Persea / Q000821:virology*; D012367:RNA, Viral / Q000737:chemistry*; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification*
D012367:RNA, Viral