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

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.

Keywords: C5-methylcytosine; bisulfite sequencing; nucleotide modification; viroid RNA.

Figure 1

Comparison of RNA bisulfite sequencing by standard (St) and improved (Im) protocols. Percentage of unconverted cytosines detected in + and − strands of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) infecting their natural hosts and in the in vitro transcript of the + strand of hop stunt viroid (HSVd) used as a negative control.

Figure 2

Bisulfite sequencing of + PSTVd RNAs. Bisulfite cytosine conversion generated by the standard (St) and the improved (Im) protocols of two + PSTVd fragments amplified with the primer pairs PSTVd_met_1F_plus/PSTVd_met_2R_plus and PSTVd_met_3F_plus/PSTVd_met_4R_plus (panel (a,b), respectively). The sequences targeted by the specific primers (salmon background) and the positions of cytosines in the reference variant PSTVd-Nb (green background) are indicated on the top. Converted (T, grey background) and unconverted (C, white background) cytosines are reported for each sequenced clone, the name of which is reported on the left, together with the percentage of unconverted cytosines.