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Journal of Virology, October 2005, p. 12934-12943, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12934-12943.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Sonia Delgado, Ángel E. Martínez de Alba, Carmen Hernández, and Ricardo Flores*

Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain

Received 10 May 2005/ Accepted 26 July 2005

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.

* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. Phone: 34-96-3877861. Fax: 34-96-3877859. E-mail: rflores{at}ibmcp.upv.es.

{dagger} We dedicate this paper to the memory of our dear colleague and friend Martin Tabler.

Journal of Virology, October 2005, p. 12934-12943, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12934-12943.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rodio, M.-E., Delgado, S., De Stradis, A., Gomez, M.-D., Flores, R., Di Serio, F. (2007). A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development. Plant Cell 19: 3610-3626 [Abstract] [Full Text]  
  • Molina-Serrano, D., Suay, L., Salvador, M. L., Flores, R., Daros, J.-A. (2007). Processing of RNAs of the Family Avsunviroidae in Chlamydomonas reinhardtii Chloroplasts. J. Virol. 81: 4363-4366 [Abstract] [Full Text]  
  • Carbonell, A., De la Pena, M., Flores, R., Gago, S. (2006). Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads. Nucleic Acids Res 34: 5613-5622 [Abstract] [Full Text]  
  • Hernandez, C., Di Serio, F., Ambros, S., Daros, J.-A., Flores, R. (2006). An Element of the Tertiary Structure of Peach Latent Mosaic Viroid RNA Revealed by UV Irradiation.. J. Virol. 80: 9336-9340 [Abstract] [Full Text]  


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