An Element of the Tertiary Structure of Peach Latent Mosaic Viroid RNA Revealed by UV Irradiation

Abstract

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3 termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.Elements of tertiary structure are crucial for RNA function (12,18). Such elements exert their physiological role by promoting specific local RNA folding and/or facilitating binding of certain proteins. In viroids, which are small, non-protein-encoding, single-stranded, circular RNAs, able to replicate autonomously in some higher plants (11,14,29), several elements of tertiary structure have been reported. In vitro UV irradiation and enzymatic and chemical probing of purified preparations of Potato spindle tuber viroid (PSTVd) (10, 19)-the type species of the family of nuclear viroids (Pospiviroidae) characterized by the presence of a central conserved region (CCR) and the absence of hammerhead ribozymes (15)-have revealed that its CCR contains an element of local tertiary structure with high sequence and structural similarity to loop E of eukaryotic 5S rRNA (4, 17). There is evidence supporting the involvement of loop E in PSTVd replication (3), symptom expression (26), and host specificity (30).In Peach latent mosaic viroid (PLMVd) (20)-which belongs to the family of chloroplastic viroids (Avsunviroidae) lacking a CCR but endowed with self-cleavage through hammerhead ribozymes embedded in the strands of both polarities (15)-in vitro nuclease mapping and oligonucleotide binding shift assays indicate the existence of a pseudoknot-like interaction between two hairpin loops of the proposed branched conformation (6). Moreover, inoculations with natural and artificial variants of another member of the family Avsunviroidae, Chrysanthemum chlorotic mottle viroid (24), to assess their biological properties and analysis of the genetic stability in the resulting progenies support the existence of a similar kissing-loop interaction in this viroid that is critical for its in vitro folding and in vivo viability (16). Also in the family Avsunviroidae, recent data show that modifications of loops 1 and 2 of natural hammerheads induce a severe reduction in their catalytic activity, indicating that these periphe...

Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identified by High-Throughput Sequencing

“…The positions of circular forms of CEVd and ASBVd (CEVdc and ASBVdc), visible in the gel stained with ethidium bromide, are reported on the left; the positions of circular and linear forms of FHVd-LR detected by the specific probes for the (+) and (−) polarity strands [panels (A,B) , respectively] are indicated on the right. Bands between the circular and linear forms could correspond to artifacts generated by UV-induced cross-links during the visualization of the first non-denaturing gel of the double PAGE, as observed previously for some viroids ( Hernández et al, 2006 ; Serra et al, 2018 ).…”

“…(A,B), respectively] are indicated on the right. Bands between the circular and linear forms could correspond to artifacts generated by UV-induced cross-links during the visualization of the first non-denaturing gel of the double PAGE, as observed previously for some viroids (Hernández et al, 2006;Serra et al, 2018).…”

The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens

“…In addition, UVC radiation generates reactive oxygen species that may produce more than 35 different base modifications [28]. The UVC daily dose used in this experiment was in the same order of magnitude as that required for inducing in vitro and in vivo intramolecular cross-linking in viroids from both families [29,30], while still minimizing its effect on the growth of the host plants.…”

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

“…UV cross-linking has been used for detecting tertiary interactions in vitro in a wide range of RNAs, for example, hepatitis delta virus (5,7,9), tRNAs (4), small nuclear RNAs (32), hairpin ribozymes (8), ribosomal RNAs (22), and Peach latent mosaic viroid (16). As shown here, this approach may be extended to investigate the in vivo existence of these tertiary interactions.…”