Viroids are single‐stranded circular noncoding RNAs that mainly infect crops. Upon infection, nuclear‐replicating viroids engage host DNA‐dependent RNA polymerase II for RNA‐templated transcription, which is facilitated by a host protein TFIIIA‐7ZF. The sense‐strand and minus‐strand RNA intermediates are differentially localised to the nucleolus and nucleoplasm regions, respectively. The factors and function underlying the differential localisation of viroid RNAs have not been fully elucidated. The sense‐strand RNA intermediates are cleaved into linear monomers by a yet‐to‐be‐identified RNase III‐type enzyme and ligated to form circular RNA progeny by DNA ligase I (LIG1). The subcellular compartment for the ligation reaction has not been characterised. Here, we show that LIG1 and potato spindle tuber viroid (PSTVd) colocalise near the nucleolar region in
The prevailing view of intracellular RNA trafficking in eukaryotic cells is that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope, entering the cytoplasm for function. However, emerging evidence illustrates that numerous functional RNAs move in the reverse direction, from the cytoplasm to the nucleus. The mechanism underlying RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using Nicotiana benthamiana, tomato (Solanum lycopersicum), and nuclear-replicating viroids as a model, we showed that cellular IMPORTIN ALPHA-4 (IMPa-4) is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (viroid RNA-binding protein 1 [VIRP1]) that binds both IMPa-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs VIRP1 binding, viroid nuclear accumulation, and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on VIRP1 for nuclear import. These results advance our understanding of subviral RNA infection and the regulation of RNA nuclear import.
more » « less- Award ID(s):
- 2145967
- PAR ID:
- 10372679
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- The Plant Cell
- Volume:
- 34
- Issue:
- 10
- ISSN:
- 1040-4651
- Page Range / eLocation ID:
- p. 3543-3556
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Daròs, José-Antonio (Ed.)Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.more » « less
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