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Title: Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop

Abstract

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [2]
  1. Institut fuer Physikalische Biologie, Heinrich-Heine-Universitaet Duesseldorf, Geb. 26.12.U1, Universitaetsstr.1, D-40225 Duesseldorf (Germany)
  2. Institut fuer Physikalische Biologie, Heinrich-Heine-Universitaet Duesseldorf, Geb. 26.12.U1, Universitaetsstr.1, D-40225 Duesseldorf (Germany). E-mail: riesner@biophys.uni-duesseldorf.de
Publication Date:
OSTI Identifier:
20779481
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 347; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2005.11.039; PII: S0042-6822(05)00789-0; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CELL CULTURES; CHROMATOGRAPHY; DNA; HOST; INFECTIVITY; MUTANTS; POTATOES; PROTEINS; RNA; TRANSCRIPTION

Citation Formats

Kolonko, Nadine, Bannach, Oliver, Aschermann, Katja, Hu, Kang-Hong, Moors, Michaela, Schmitz, Michael, Steger, Gerhard, and Riesner, Detlev. Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop. United States: N. p., 2006. Web. doi:10.1016/J.VIROL.2005.1.
Kolonko, Nadine, Bannach, Oliver, Aschermann, Katja, Hu, Kang-Hong, Moors, Michaela, Schmitz, Michael, Steger, Gerhard, & Riesner, Detlev. Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop. United States. doi:10.1016/J.VIROL.2005.1.
Kolonko, Nadine, Bannach, Oliver, Aschermann, Katja, Hu, Kang-Hong, Moors, Michaela, Schmitz, Michael, Steger, Gerhard, and Riesner, Detlev. Mon . "Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop". United States. doi:10.1016/J.VIROL.2005.1.
@article{osti_20779481,
title = {Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop},
author = {Kolonko, Nadine and Bannach, Oliver and Aschermann, Katja and Hu, Kang-Hong and Moors, Michaela and Schmitz, Michael and Steger, Gerhard and Riesner, Detlev},
abstractNote = {Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.},
doi = {10.1016/J.VIROL.2005.1},
journal = {Virology},
number = 2,
volume = 347,
place = {United States},
year = {Mon Apr 10 00:00:00 EDT 2006},
month = {Mon Apr 10 00:00:00 EDT 2006}
}
  • The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA fingerprinting and secondary analysis. Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (i) both crosslinks occur in an identical eight-base context, with the sequence 5 GGGAA 3 on one side and the sequence 5 UAC 3 on the other; (ii) both crosslinks connectmore » bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as single-stranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules.« less
  • The structure and size of potato spindle tuber viroid (PSTV) were investigated by electron microscopy. PSTV appears to be composed of two single-stranded RNA components of different sizes, one linear and one circular. The linear component is about four times more common than the circular. Both components exist in collapsed, highly base-paired structures under nondenaturing conditions. Both components are sensitive to digestion by pancreatic ribonuclease but are resistant to snake venom phosphodiesterase. From length measurements of formylated and nonformylated PSTV RNA, the estimated molecular weight for the linear molecule is 110,000, and for the circular, it is 137,000.
  • Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequentmore » GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.« less
  • The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities ofmore » HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.« less