A Short Double-Stranded RNA Motif of Peach Latent Mosaic Viroid Contains the Initiation and the Self-Cleavage Sites of Both Polarity Strands

Abstract

The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5 termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels.Viroids represent the lowest step of the biological scale. Their genome is composed exclusively by a small (246 to 401 nucleotides [nt]) circular RNA without any apparent proteincoding capacity (16,20,21,49), a key aspect that sets a demarcating difference with virus genomes, which do code for one or more proteins of their own. Despite their structural simplicity, viroids are able to replicate autonomously in their host plants and to elicit symptoms in most instances. Therefore, viroids offer unique opportunities for studying RNA replication without interference with the concurrent transcription and translation accompanying the replication of viral RNAs.Viroids replicate through a rolling-circle mechanism, with two alternative pathways mediated by three catalytic activities: RNA polymerase, RNase, and RNA ligase (6). The most abundant viroid circular RNA, arbitrarily considered as having a plus polarity, is transcribed into oligomeric minus strands that, by themselves (asymmetric pathway) or after processing to their monomeric circular counterparts (symmetric pathway), serve as templates for a second RNA-RNA transcription round leading to oligomeric plus strands that are finally cleaved and ligated to the monomeric plus circular forms. There is empirical evidence supporting the asymmetric pathway for Potato spindl...

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

“…Using this strategy, initiation of (+) and (-) strands in ASBVd has been mapped at the right terminal A+U-rich loops of their proposed quasi-rod-like secondary structures, 50 and in PLMVd at short double-stranded RNA motifs that also contain the self-cleavage sites. 51 Therefore, two structurally distinct elements, hairpinloops and double-stranded RNA motifs, serve the same purpose, with their adjacent regions being also possibly involved in recruiting both NEP and auxiliary transcription factors.…”

Respuesta De Distintos Genotipos De Cítricos Y Géneros Afines a La Infección Con Viroides

“…Tabla 1. Clasificación de los viroides caracterizados molecularmente según la ICTV (adaptada de Flores et al, 2005). Los viroides no tienen capacidad de codificar proteínas y lógicamente sus funciones biológicas dependen de su propia secuencia que determina la conformación del RNA y/o de la adopción de determinadas estructuras secundarias.…”

“…El núcleo central está flanqueado por tres hélices, I, II y III. El sitio de auto-corte está indicado por una flecha (adaptado de Flores et al, 2005y Góra-Sochacka, 2004.…”

“…Esta afirmación se estableció tras ensayos realizados con distintos inhibidores de polimerasas en PSTVd (Mühlbach y Sänger, 1979;Schnidler y Mühlbach, 1992), así como de HSVd y CEVd (revisado por Ding e Itaya, 2007). Los trabajos realizados con viroides de la familia Avsunviroidae mostraron que éstos se replican mediante una RNA polimerasa dependiente de DNA y diferente a la RNA polimerasa II, y se ha propuesto que una polimerasa tipo NEP (Nuclear-encoded polymerase) que es una enzima codificada en el núcleo pero con actividad en el cloroplasto sea la responsable de la transcripción de estos viroides (Delgado et al, 2005). Sin embargo, no puede descártarse la posibilidad que sean las polimerasas codificadas en el cloroplasto (PEP) las implicadas en la replicación de viroides de esta familia.…”

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

“…Por lo (Branch et al, 1988). Por el contrario, la detección en tejidos infectados por los miembros de la familia Avsunviroidae de los monómeros circulares de polaridad negativa (Hutchins et al, 1985;Bussière et al, 1999;Delgado et al, 2005), así como la observación de que los RNAs de ambas polaridades son capaces de autocortarse in vitro Hernández y Flores, 1992;Navarro y Flores, 1997;Fadda et al, 2003a), indican que estos viroides se replican siguiendo la variante simétrica del modelo.…”