The use of a combination of computer‐assisted structure prediction and SHAPE probing to elucidate the secondary structures of five viroids

Abstract

The elucidation of the structures of viroids, noncoding infectious RNA species, is paramount to obtain an understanding of the various aspects of their life cycles (including replication, transport and pathogenesis). In general, the secondary structures of viroids have been predicted using computer software programs which have been shown to possess several important limitations. Clearly, the predicted structure of a viroid needs to receive physical support prior to its use in the accurate interpretation of any mechanistic studies. Here, SHAPE probing coupled to computer-assisted structure prediction using the RNAstructure software program was employed to determine the structures of five viroids. These species belong to four genera of the Pospiviroidae family, and none have had their structure characterized in solution. In addition, several interesting questions were addressed by either studying various sequence variants or varying the SHAPE conditions. More importantly, this approach is novel in the study of viroids, and should be of significant aid in the determination of the structures of other RNA species.

Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae

“…Both of these features were also present in CVd-IV, another member of the genus Cocadviroid whose structure was elucidated previously [24]. Taking into account all three of these genera, and of the subfamily Pospiviroinae , the structures of eight species were elucidated.…”

“…Previously, structure of CVd-IV was elucidated using SHAPE [24]. In the present work, the structure of CCCVd, the type species of the genus, was elucidated.…”

In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

“…This region, consisting of double-helical segments broken intermittently by small internal loops and bulges, is reminiscent of in vitro transcribed viroid RNA [22]. The SHAPE-restrained structure is noticeably different from the minimum free energy (MFE) structure (Figure 3) predicted using RNAstructure [18].…”

Agents Resembling or Altering Virus Diseases

“…Dubé et al (2011) and Xu et al (2012) describe the prediction of viroid secondary structure using RNA-selective 2′-hydroxyl acylation analyzed by primer extension probing (Steen et al, 2010) coupled with computer-assisted structure prediction. Very small differences in structure can have a major effect on these properties.…”