Abstract Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC–MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m 6 A per RNA molecule. ONT sequencing predicted several m 6 A positions, which were, however, not confirmed by RNase T1/A treatment and LC–MS, likely because of low methylation level at each potential position. Our results suggest that m 6 A is not in one specific position and is distributed in CEVd sequence at low stoichiometries presumably in the predicted sites. Abstract Figure

Exploring RNA modifications in infectious non-coding circular RNAs