title: The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.
abstract: Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5' ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (-) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.
authors.affiliation: a Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas , Valencia , Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: López-Carrasco
authors.affiliation: b Department of Molecular Signal Processing , Leibniz Institute of Plant Biochemistry , Halle ( Saale ), Germany.
authors.forename: Selma
authors.initials: S
authors.lastname: Gago-Zachert
authors.affiliation: a Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas , Valencia , Spain.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Mileti
authors.affiliation: a Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas , Valencia , Spain.
authors.forename: Sofia
authors.initials: S
authors.lastname: Minoia
authors.affiliation: a Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas , Valencia , Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Flores
authors.affiliation: a Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas , Valencia , Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Delgado
mesh_terms: D003198:Computer Simulation; D014644:Genetic Variation; D008958:Models, Molecular; D018087:Plastids / Q000235:genetics; D012330:RNA, Double-Stranded / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D032323:Solanum melongena / Q000821:virology*; D024363:Transcription Initiation Site; D014772:Viroids / Q000145:classification / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral
keywords: Catalytic RNAs; RNA secondary structure; SHAPE; hammerhead ribozymes; non-coding RNAs