Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae

Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia), Valencia, Spain.
Journal of Virology (Impact Factor: 4.44). 05/2012; 86(15):8269-76. DOI: 10.1128/JVI.00629-12
Source: PubMed


Avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (ELVd), the four recognized members of the family Avsunviroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5'-hydroxyl and 2',3'-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic isoform of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(+)] and minus [(-)] monomeric linear replication intermediates from the four Avsunviroidae. We also show that while this RNA ligase specifically recognizes the genuine monomeric linear (+) ELVd replication intermediate, it does not do so with five other monomeric linear (+) ELVd RNAs with their ends mapping at different sites along the molecule, despite containing the same 5'-hydroxyl and 2',3'-phosphodiester terminal groups. Moreover, experiments involving transient expression of a dimeric (+) ELVd transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing of the plant endogenous tRNA ligase show a significant reduction of ELVd circularization. In contrast, circularization of a viroid replicating in the nucleus occurring through a different pathway is unaffected. Together, these results support the conclusion that the chloroplastic isoform of the plant tRNA ligase is the host enzyme mediating circularization of both (+) and (-) monomeric linear intermediates during replication of the viroids belonging to the family Avsunviroidae.

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    • "They could also show that it was able to circularize the linear (À) polarity monomeric RNA but also the (þ) and (À ) RNA of other chloroplast-replicating viroids efficiently (Nohales et al., 2012b). Interestingly, it could not circularize any other artificially cleaved linear monomeric viroid RNA except the physiological substrate, indicating impressive selectivity (Nohales et al., 2012b). "
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    ABSTRACT: Since the discovery of non-coding, small, highly structured, satellite RNAs (satRNAs) and viroids as subviral pathogens of plants , have been of great interest to molecular biologists as possible living fossils of pre-cellular evolution in an RNA world. Despite extensive studies performed in the last four decades, there is still mystery surrounding the origin and evolutionary relationship between these subviral pathogens. Recent technical advances revealed some commonly shared replication features between these two subviral pathogens. In this review, we discuss our current perception of replication and evolutionary origin of these petite RNA pathogens. Copyright © 2015 Elsevier Inc. All rights reserved.
    Virology 02/2015; 479-480. DOI:10.1016/j.virol.2015.02.018 · 3.32 Impact Factor
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    • "How members of both families have evolved the same strategy remains an intriguing conundrum considering that pol II and NEP differ significantly in their structural complexity: the former is a multisubunit complex, the latter is a single-subunit enzyme. The third replication step, circularization, is catalyzed by an isoform of the tRNA ligase, which like NEP, is encoded in the nucleus and then translocated into the chloroplast via a signal peptide (Nohales et al., 2012b). The possibility that circularization might proceed via selfligation without the involvement of enzymes (Côté et al., 2001), appears less likely (Flores et al., 2009). "
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    ABSTRACT: The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously -and to incite diseases in certain plants- without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features -they are circular molecules that fold upon themselves adopting compact secondary conformations- and then to how they manipulate their hosts to be propagated. Replication of viroids -in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands- dealt three surprises: i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world. Copyright © 2015. Published by Elsevier B.V.
    Virus Research 01/2015; 209:136-145. DOI:10.1016/j.virusres.2015.02.027 · 2.32 Impact Factor
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    • "Analysis of ELVd RNAs expressed in chloroplasts of Chlamydomonas reinhardtii has shown that deletion mutants able to self-cleave efficiently display ligation defects, indicating that additional nucleotides – apart those forming the hammerhead – are involved in the conformation promoting ligation (Martínez et al., 2009). Moreover, this conformation should favor the physical proximity and positioning of the 5′-hydroxyl and 2′,3′ cyclic phosphodiester termini (resulting from self-cleavage) for their joining most likely catalyzed by a chloroplastic tRNA ligase (Nohales et al., 2012). "
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    ABSTRACT: As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.
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