Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

Abstract

Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase.

Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase

“…The results reported here using a combination of in vitro and in vivo approaches show that the host DNA ligase 1 is the long-sought enzyme involved in circularizing the monomeric PSTVd RNA during replication. Moreover, because the cleavage mechanism of the oligomeric RNA intermediates appears conserved in the family Pospiviroidae (14), the ligation mechanism that we propose for PSTVd most likely also operates for the rest of the viroid species in this family (Fig. 2).…”

“…Importantly, in this assay we used as substrate an in vitro synthesized monomeric linear PSTVd RNA opened between positions G95 and G96 and containing 5′-phosphomonoester and 3′-hydroxyl termini (Fig. 1B); our previous results indicated that this is the physiological precursor of the circular forms (14,15). Reaction products were separated by denaturing PAGE and visualized by Northern blot hybridization with a complementary radioactive RNA probe.…”

“…The situation became even more complicated when the monomeric linear precursor was shown to have 5′-phosphomonoester and 3′-hydroxyl ends (14,15), because the only RNA ligase described so far in plants, the tRNA ligase, specifically requires 5′-hydroxyl and 2′,3′-cyclic phosphodiester termini (20). This observation led to the prediction (15) that higher plants must contain an RNA ligase activity similar to T4 bacteriophage RNA ligases, with the ability to catalyze joining of 5′-phosphomonoester and 3′-hydroxyl termini (21,22).…”

“…Additional support for this view was obtained with Citrus exocortis viroid, closely related to PSTVd, by immunoprecipitation studies with an antibody against the largest subunit of RNA polymerase II (13). In addition, site-directed mutagenesis analyses suggest that a palindromic quasi-double-stranded structure-formed by the interaction of the upper CCR strand of two consecutive viroid units in the oligomeric intermediates-is cleaved at two symmetric sites by a host type-III RNase, whereas both CCR strands promote ligation by forming a rod-like structure containing a loop E (14,15) (Fig. 1A).…”

Current status of viroid taxonomy

“…A functional role has been proposed for some of these domains in certain members of this family: for example, nucleotide changes in the P domain have been correlated with strain virulence in citrus exocortis viroid (CEVd) and PSTVd [41,54,55]. The upper CCR strand may assume an alternative conformation (hairpin I) with major role(s) in replication [25], and the TL domain may contain either a terminal conserved region (TCR) or a terminal conserved hairpin (TCH). Because the TCR and TCH occupy the same relative position in several members of the family, they are assumed to play some as yet unknown functional role(s).…”