Abstract
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.
Highlights
One of the most surprising and fascinating traits emerging in the last decades in molecular biology is the central role of RNA as regulatory molecule in many and very different biological processes [1].Primary, secondary and tertiary structural elements have been involved in RNA functionality.In this context, modified nucleotides have been reported providing functional roles to several cellularRNAs, including ribosomal RNAs, transfer RNAs, messenger RNAs, small nuclear RNAs, long non-coding RNAs and others [2,3]
Functional roles of RNA methylation are still unclear, these findings showed that the previous reports on the limited presence of methylated nucleotides in cellular RNAs other than ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) were mainly due to technical limitation [2]
Leaf samples (10 g) were collected from Nicotiana benthamiana and avocado (Persea americana) plants infected with potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd), respectively, and total nucleic acid were extracted with phenol and enriched in highly structured RNAs, including tRNAs and viroid RNAs, by a chromatography on CF11 cellulose as reported previously [33]
Summary
One of the most surprising and fascinating traits emerging in the last decades in molecular biology is the central role of RNA as regulatory molecule in many and very different biological processes [1].Primary, secondary and tertiary structural elements have been involved in RNA functionality.In this context, modified nucleotides have been reported providing functional roles to several cellularRNAs, including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), long non-coding RNAs (lncRNAs) and others [2,3]. Secondary and tertiary structural elements have been involved in RNA functionality. In this context, modified nucleotides have been reported providing functional roles to several cellular. Nucleotide modifications were thought to be mainly restricted to the stable and highly abundant tRNAs and rRNAs. Initially, nucleotide modifications were thought to be mainly restricted to the stable and highly abundant tRNAs and rRNAs In these cellular RNAs, the modified nucleotides, including m5 C, were mapped in technically demanding studies that support their possible structural and/or functional roles [9,10,11,12]. The improvement of bisulfite sequencing protocols for RNA [17,18], originally developed for identifying m5 C in DNA, allowed to overcome the main limitation to the extension of this technique to RNA analysis, unveiling the Viruses 2019, 11, 357; doi:10.3390/v11040357 www.mdpi.com/journal/viruses
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