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

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 (m(5)C), also play a role has not been deeply investigated so far. Here, the possible existence of m(5)C 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 m(5)C 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 m(5)C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.