Exploring the presence of C5-methylcytosine in viroid RNAs

Nucleotide modifications in RNAs were initially thought to be mainly restricted to the stable and highly abundant transfer and ribosomal RNAs (tRNAs and rRNAs). In these RNAs, modifications have been mapped by technically demanding studies that support their possible structural and/or functional roles. More recently, the improvement of RNA bisulfite sequencing protocols, originally developed for identifying C5-methylcytosine (m5C) in DNA, unveiled the widespread occurrence of m5C in many cellular coding and non-coding RNAs, thus highlighting that the previous reports on the limited presence of this modified nucleotide in cellular RNAs other than rRNAs and tRNAs were mainly due to technical limitation. Information on whether viroid RNAs contain m5C is poor and dates back to early RNase fingerprints analyses of bisulfite-treated viroid RNA preparations (Domdey et al., 1978, Nucleic Acids Res. 5, 1221-1236). These studies were performed mainly to determine the secondary structure of potato spindle tuber viroid (PSTVd) genomic RNA. Therefore, they did neither provide information at single-nucleotide resolution nor on the presence of m5C in viroid replication intermediates, particularly viroid RNAs of the complementary (-) polarity. To conclusively answer the question regarding the presence of m5C in either polarity strand, a protocol based on RNA bisulfite sequencing was developed to test at single-nucleotide resolution the viroid highly-structured RNAs, whose base-paired cytidines are particularly resistant to bisulfite conversion. This protocol was then applied to explore the presence of m5C in the (+) and (-) strands of PSTVd and avocado sunblotch viroid (ASBVd), which are members of the type-species of the families Pospiviroidae and Avsunviroidae, respectively. Results of these analyses support the absence of m5C in both strands of PSTVd and ASBVd.