A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription

Abstract

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.

“Pathomorphogenic” Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco

“…CBCVd has a wide host range and can be replicated in some Solanacenaceae species [9], including N. tabacum, where it can replicate but cannot move; that is, its propagation can be mediated only through plant transformations using infectious plant vectors [13] and its infection is, in general, without morphological symptoms. Recent results and our previous study [14] also suggest that CBCVd probably adopted, as other nuclear-replicating viroids, a splicing variant of the transcription factor TFIIIA, TFIIIA-7ZF with only seven zinc fingers, for their own replication by DNA-dependent polymerase II [15,16].…”

Viroid Replication, Movement, and the Host Factors Involved

“…This implies that viroids possess the ability to autonomously regulate their replication and impact specific regulatory factors, leading to modifications in the splicing of a restricted set of genes [ 17 ]. Furthermore, analysis through nano-liquid chromatography–tandem mass spectrometry utilizing a purified Pol II complex on RNA templates has unveiled modified Pol II lacking Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9 [ 18 ]. This contrasts with the conventional 12-subunit Pol II or the 10-subunit Pol II cores observed on DNA templates.…”

“…This adapted Pol II exhibits transcriptional activity with the assistance of TFIIIA-7ZF and does not seem to require other typical general transcription factors. This observation suggests the presence of a unique mechanism or machinery specifically for viroid RNA-templated transcription [ 18 , 19 ]. Pol II-mediated transcription results in the generation of linear (-)-strand concatemers, acting as replicative intermediates [ 20 , 21 , 22 , 23 ].…”

Heterologous expression of circular RNAs in Escherichia coli for analyzing the ligation process of chloroplastic viroids and producing double-stranded RNAs with insecticidal activity

“…Viroids of this family hijack the host DNA-dependent RNA polymerase II (Pol II) Flores and Semancik, 1982;Yoshikawa and Takahashi, 1986), an ability apparently shared with the complementary (-) strand (Warrilow and Symons, 1999). The Pol II involved in this process, however, has a remodeled architecture with reduced number of components in contrast to the polymerase complex on DNA templates (Dissanayaka Mudiyanselage et al, 2022). In the case of PSTVd, the transcription factor TFIIIA-7ZF is also required (Wang et al, 2016b), being essential for the polymerase to use RNA as a template, while other canonical factors of general transcription do not participate, suggesting a different transcription machinery (Dissanayaka Mudiyanselage et al, 2022).…”

“…The Pol II involved in this process, however, has a remodeled architecture with reduced number of components in contrast to the polymerase complex on DNA templates (Dissanayaka Mudiyanselage et al, 2022). In the case of PSTVd, the transcription factor TFIIIA-7ZF is also required (Wang et al, 2016b), being essential for the polymerase to use RNA as a template, while other canonical factors of general transcription do not participate, suggesting a different transcription machinery (Dissanayaka Mudiyanselage et al, 2022). The binding sites of Pol II and TFIIIA-7ZF are found in the left terminal region of the PSTVd (+), where the transcription start site is consequently found (nucleotide U359 or C1) (Kolonko et al, 2006).…”