Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb 27;43(4):2313-25.
doi: 10.1093/nar/gkv034. Epub 2015 Feb 6.

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

Sofia Minoia  1 , Beatriz Navarro  2 , Sonia Delgado  1 , Francesco Di Serio  3 , Ricardo Flores  4
Affiliations

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

Sofia Minoia et al. Nucleic Acids Res. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Infection of eggplant by PSTVd results in the accumulation of a series of PSTVd (+) sgRNAs accompanying the genomic mc and ml (+) forms. Total RNAs from upper non-inoculated leaves collected at 15, 20 and 25 dpi were separated by denaturing PAGE in 5% gels and revealed with ethidium bromide (A), and by northern-blot hybridization with a radiolabeled riboprobe for detecting PSTVd (+) strands (B). In each panel lane 1 corresponds to PSTVd-infected N. benthamiana, lane 2 to mock-inoculated eggplant, and lanes 3 and 4 to PSTVd-infected eggplant. Positions of RNA markers (in nt) are indicated on the left, and those of the PSTVd mc and ml forms on the right.
Figure 2.
Figure 2.
PSTVd (+) sgRNAs are not artifacts generated during in vitro extraction. Total RNAs were separated by denaturing PAGE in 5% gels and revealed with ethidium bromide (A), and by northern-blot hybridization with a radiolabeled riboprobe for detecting PSTVd (+) strands (B). Lane M, RNA markers; lanes 1, mock-inoculated eggplant; lanes 2, PSTVd-infected eggplant; lanes 3, mock-inoculated eggplant supplemented before extraction with PSTVd mc forms from infected tissue. Positions of RNA markers (in nt) are indicated on the left, and those of the mc and ml forms on the right.
Figure 3.
Figure 3.
Accumulation in three different hosts of PSTVd (+) mc, ml and sgRNAs. Total RNAs from upper non-inoculated leaves were separated by denaturing PAGE in 5% gels and revealed with ethidium bromide (A), and by northern-blot hybridization with a radiolabeled riboprobe for detecting PSTVd (+) strands (B). Lanes 1 to 3, mock-inoculated controls of eggplant, N. benthamiana and tomato, respectively. Lanes 4 to 8, PSTVd-infected eggplant (lanes 4 and 5), N. benthamiana (lanes 6 and 7) and tomato (lane 8). Loading was equalized by the intensity of the 5S RNA stained with ethidium bromide (not shown). Positions of the PSTVd (+) mc and ml forms are indicated on the right and those of RNA markers (in nt) on the left. Arrowheads mark some PSTVd (+) sgRNAs common to the three hosts. Leaves were collected at similar time intervals (20–25 dpi).
Figure 4.
Figure 4.
(A) Primer extension analysis of five PSTVd (+) sgRNAs and of the ml form reveals a complex population of 5′ termini (marked by arrowheads) mapping at different domains of the secondary structure of the genomic (+) viroid RNA predicted by Mfold (http://mfold.rna.albany.edu/?q=mfold/rna-folding-form) and supported by structural/functional analysis of a slightly different strain (48). Notice, however, that the 5′ termini of the (+) sgRNAs 1 and 3 map at the same position (U296). (B–G) Representative examples of extensions using primers RF-1242 (for sgRNAs 1 and 2, and for the ml form), RF-1194 (for sgRNAs 3 and 4) and RF-1191 (for sgRNA 5). In the six panels the sequencing ladders were obtained with the same primers and a recombinant plasmid containing a monomeric PSTVd-cDNA insert of the same variant (NB) used for inoculation. Arrows point to cDNA bands corresponding to 5′ termini.
Figure 5.
Figure 5.
RLM-RACE analysis of the 5′ termini of the PSTVd (+) sgRNAs 1 to 5, using as control a PSTVd (+) ml form with 5′-hydroxyl (G2) and 2′,3′-cyclic phosphodiester (C1) termini resulting from ribozyme-mediated self-cleavage. Prior to RLM-RACE (see Supplementary Table S3 for the RNA adaptor and primers used), aliquots of each RNA were left untreated (lanes 1 and 3) or treated with PK and ATP to phosphorylate 5′-hydroxyl groups (lanes 2 and 4), with the resulting amplification products being then separated by non-denaturing PAGE and stained with ethidium bromide. Arrowheads indicate the amplicons of expected length that were eluted, cloned and sequenced. Lane M, DNA markers.
Figure 6.
Figure 6.
Infection of eggplant by PSTVd also results in the accumulation of a series of PSTVd (−) sgRNAs. Total RNAs from upper non-inoculated leaves collected at 20 dpi were separated by denaturing PAGE in 5% gels and revealed with ethidium bromide (A), and by northern-blot hybridization with full-length radiolabeled riboprobes (equalized in acid-precipitable counts) for detecting PSTVd (+) or (−) strands (B). In each panel lanes 1 and 2 correspond to equal amounts of full-length PSTVd (+) and (−) transcripts, respectively (in both cases with 5′ and 3′ plasmid tails), lanes 3 to mock-inoculated eggplant, and lanes 4 and 5 to PSTVd-infected eggplant. Positions of the PSTVd (+) mc and ml forms are indicated with arrowheads and those of RNA markers (in nt) on the left.
Figure 7.
Figure 7.
(A) Primer extension analysis of three PSTVd (−) sgRNAs showing their 5′ termini (marked by arrowheads) mapping at different positions on the secondary structure of the genomic (−) viroid RNA predicted by Mfold and validated by temperature-gradient gel electrophoresis .
Figure 8.
Figure 8.
Schematic mapping of the PSTVd (+) sgRNAs 1 to 5 and of the (−) sgRNAs 1 to 3 on the genomic PSTVd (+) (A) and (−) (B) secondary structures, respectively. For easier comparison, the PSTVd (−) form is presented as a circular form, although this form has not been detected in infected tissue (7). The size of (+) sgRNAs 1 to 5 (242, 177, 158, 91 and 73 nt, respectively), and of the (−) sgRNAs 3 (184 nt), has been accurately determined by primer extension and 5′- and 3′-RACE; the size of the (−) sgRNAs 1 and 2 (285 and 280 nt) has been inferred from primer extension and mobility on denaturing PAGE, and is only approximate. Notice that the 5′ termini of the (+) sgRNAs 1 and 3 are coincidental, and that cleavage of the former at a specific site would generate the latter. Numbering in the (−) polarity is the same as in the (+) polarity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Diener T.O. Discovering viroids—a personal perspective. Nat. Rev. Microbiol. 2003;1:75–80. - PubMed
    1. Tabler M., Tsagris M. Viroids: petite RNA pathogens with distinguished talents. Trends Plant Sci. 2004;9:339–348. - PubMed
    1. Flores R., Hernández C., Martínez de Alba A.E., Daròs J.A., Di Serio F. Viroids and viroid-host interactions. Annu. Rev. Phytopathol. 2005;43:117–139. - PubMed
    1. Ding B. The biology of viroid-host interactions. Annu. Rev. Phytopathol. 2009;47:105–131. - PubMed
    1. Ishikawa M., Meshi T., Ohno T., Okada Y., Sano T., Ueda I., Shikata E. A revised replication cycle for viroids: the role of longer than unit RNA in viroid replication. Mol. Gen. Gen. 1984;196:421–428. - PubMed

Publication types