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Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level

Istituto per la Protezione Sostenibile delle Piante (CNR), 70126 Bari, Italy
Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), 46022 Valencia, Spain
Authors to whom correspondence should be addressed.
Viruses 2019, 11(4), 357;
Received: 26 March 2019 / Revised: 12 April 2019 / Accepted: 16 April 2019 / Published: 18 April 2019
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m5C), also play a role has not been deeply investigated so far. Here, the possible existence of m5C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m5C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m5C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology. View Full-Text
Keywords: viroid RNA; bisulfite sequencing; nucleotide modification; C5-methylcytosine viroid RNA; bisulfite sequencing; nucleotide modification; C5-methylcytosine

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Di Serio, F.; Torchetti, E.M.; Daròs, J.-A.; Navarro, B. Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level. Viruses 2019, 11, 357.

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