Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo

[EN] While biogenesis of viroid RNAs is well-known, how they decay is restricted to data involving host RNA silencing. Here we report an alternative degradation pathway operating on potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids (family Pospiviroidae). Northern-blot hybridizations with full-and partial-length probes revealed a set of PSTVd (+) subgenomic (sg) RNAs in early-infected eggplant, some partially overlapping and reaching levels comparable to those of the genomic circular and linear forms. Part of the PSTVd (+) sgRNAs were also observed in Nicotiana benthamiana (specifically in the nuclei) and tomato, wherein they have been overlooked due to their low accumulation. Primer extensions of representative (+) sgRNAs failed to detect a common 5' terminus, excluding that they could result from aborted transcription initiated at one specific site. Supporting this view, 5'- and 3'-RACE indicated that the (+) sgRNAs have 5'-OH and 3'-P termini most likely generated by RNase-mediated endonucleolytic cleavage of longer precursors. These approaches also unveiled PSTVd (-) sgRNAs with features similar to their (+) counterparts. Our results provide a mechanistic insight on how viroid decay may proceed in vivo during replication, and suggest that synthesis and decay of PSTVd strands might be coupled as in mRNA.
Ministerio de Economia y Competitividad (MINECO) [BFU2011-28443 to R. F. laboratory]; MINECO [to S.M. and S.D.]. Funding for open access charge: MINECO (BFU2011-28443 to R. F.); Ministero dell'Economia e Finanze Italiano to the CNR (CISIA, Legge 191/2009 to B.N. and F.D.S.).