Reorganizing the RNA polymerase II complex for replication of an infectious noncoding RNA in vivo

Jie Hao; Zhuan Qin; Junfei Ma; Jie Qu; Yunhan Wang; Svetlana Y. Folimonova; Bin Liu; Wenwei Li; Ying Wang

doi:10.1371/journal.ppat.1014200

Abstract

DNA-dependent RNA polymerases (DdRPs) recognize not only DNA but also RNA templates. This RNA-dependent RNA polymerase (RdRP) activity is exploited by bacterial 6S RNA, mammalian B2 RNA, viroids in plants, and hepatitis delta virus in human. A major knowledge gap exists regarding the molecular basis conferring this RdRP activity. Here, we provide evidence supporting the reorganization of the 12-subunit polymerase II (Pol II) to 7-subunit in vivo for PSTVd transcription. Rpb4/5/6/7/9 are not involved in PSTVd transcription in planta. A splicing variant of transcription factor IIIA with seven zinc finger domains (TFIIIA-7ZF) aids the remodeled Pol II in transcribing PSTVd. Using AlphaFold3, the structure of the remodeled Pol II with PSTVd RNA and TFIIIA-7ZF was predicted. The predicted structure and experimental data both show that the N-terminus of TFIIIA-7ZF binds to the left terminal domain of PSTVd, while the C-terminus interacts with Rpb2. Interestingly, AlphaFold3 also predicts the bending at PSTVd loop 8 in the TFIIIA-7ZF/PSTVd complex. Replacing this loop 8 with a rigid double-stranded conformation impairs the TFIIIA-7ZF/PSTVd interaction. Altogether, our demonstrate the heterogenous organization of the Pol II enzyme on RNA template in vivo and provide structural insights into the organization of Pol II transcription complex on RNA template.

Author summary

DNA-dependent RNA polymerases (DdRPs) are well-known for copying DNA information into RNA. However, these DdRPs can also copy information from RNA templates, a process that has not been well understood. In this study, we showed that DdRPII (Pol II) reorganizes its organization and only uses 7-subunit instead of the conventional 12-subunit to catalyze viroid RNA transcription in plants. Based on this new information, we also used the latest AlphaFold3 tool to predict the complex organization of Pol II and a transcription factor on viroid RNA templates. The prediction results also suggested a previously unknown structure that bends RNA templates during transcription initiation. In general, experimental data support the prediction results. Therefore, this study illustrates a unique regulation of Pol II activity, which may help develop anti-viroid chemicals for agricultural activities in the future.

Citation: Hao J, Qin Z, Ma J, Qu J, Wang Y, Folimonova SY, et al. (2026) Reorganizing the RNA polymerase II complex for replication of an infectious noncoding RNA in vivo. PLoS Pathog 22(4): e1014200. https://doi.org/10.1371/journal.ppat.1014200

Editor: Aiming Wang, Agriculture and Agri-Food Canada, CANADA

Received: January 20, 2026; Accepted: April 25, 2026; Published: April 30, 2026

Copyright: © 2026 Hao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data are available in the main text or the supplementary materials.

Funding: US National Science Foundation MCB-2350392 and IOS-2410009 to Ying W., US National Institutes of Health 1R15GM135893 to Ying W., and US National Institutes of Health R01AI194920-01 to W.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Transcription is a fundamental process that mediates the interplay between genetic information and phenotypes and, thus, is vital for organismal growth and responses to environmental cues [1–4]. Transcription is catalyzed by DNA-dependent RNA polymerases (DdRPs) that generally polymerize RNA molecules based on DNA templates [5–7]. Interestingly, it has long been known that some DdRPs can recognize both DNA and RNA templates for transcription [8]. The RNA-dependent RNA polymerase (RdRP) activity of DdRPs regulates gene expression in diverse organisms across kingdoms. For instance, under nutrient-deficient conditions, the noncoding 6S RNA of Escherichia coli binds to the bacterial DdRP and prevents it from transcribing DNA templates. In response to nutrient availability, the DdRP transcribes a short RNA using the 6S RNA template that leads to the de-sequestration of the DdRP. This step, in turn, enables binding to DNA promoters and the synthesis of mRNAs [9]. RNA polymerase II (Pol II) in mammalian cells binds to a hairpin within the noncoding B2 RNA and uses the longer strand of the hairpin sequence as the template to extend the short strand by transcription. This extension subsequently destabilizes the B2 RNA, representing a novel post-transcriptional mechanism to regulate RNA stability [10]. RNA-based pathogens, such as viroids and the human hepatitis delta virus (HDV), also rely on this RdRP activity for replication in corresponding hosts [11]. Notably, both (+) and (-) strands of potato spindle tuber viroid (PSTVd, the type species of the Pospiviroidae family, new species name as Pospiviroid fusituberis) are transcribed by Pol II [12–14]. Despite the importance, however, the underlying regulatory mechanism of this RdRP activity of DdRPs remains elusive.

Taking Pol II on DNA templates as an example, this 12-subunit polymerase relies on the coordination of multiple general transcription factors (i.e., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) during transcription initiation around DNA promoter regions [15–18]. In general, a minimal set of five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) is required for the promoter-driven transcription catalyzed by Pol II [19,20]. Transcription elongation, particularly in the cellular environment, requires additional factors, including TFIIS, SPT6, etc [21–23]. By contrast, the organization of the transcription complex on RNA templates was poorly defined. Interestingly, emerging evidence implies that Pol II complex on RNA templates may be organized in a distinct fashion.

In a protoplast cell-based replication system, we found that a TFIIS loss-of-function mutant did not impair the propagation of PSTVd [24], which replicates in the nucleoplasm and relies on Pol II for de novo transcription [12–14,24,25]. Moreover, we found that PSTVd replication in protoplasts yielded from Arabidopsis thaliana nrpb2–3 hypermorphic mutant was not affected, but this mutant affects transcription from DNA templates (S1 Fig) [26]. Both observations inspired us to reason that distinct regulatory mechanisms are employed by Pol II to function on DNA and RNA templates. Indeed, an alternative splicing product of the transcription factor IIIA with seven zinc finger domains (TFIIIA-7ZF), which does not possess any known DNA-binding ability [27], is essential to engage the remodeled Pol II to PSTVd RNA templates for transcription [13,14,24]. Previous studies showed that TFIIIA-7ZF interacts with Pol II as well as with (+) and (-) PSTVd RNAs in vivo. Overexpression of TFIIIA-7ZF led to the elevated PSTVd titer, while silencing of TFIIIA-7ZF reduced PSTVd accumulation [13]. Furthermore, TFIIIA-7ZF is required in a Pol II-based in vitro transcription assay for generating longer-than-unit-length PSTVd RNA intermediates, resembling the replication process in vivo [13,14,24]. This is akin to the replication of HDV that requires a virus-encoded HDAg-S protein to ensure robust processivity in generating long RNA products [28]. Hence, both PSTVd and HDV RNA templates require unique transcription factors distinct from transcription using DNA templates.

More recently, we found that a “remodeled” Pol II actively catalyzes the RNA polymerization using PSTVd RNA templates in a Pol II-based in vitro transcription system [14]. This “remodeled” Pol is composed of Rpb1/2/3/8/10/11 as confirmed by nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS) and remains catalytically active and competent. Rpb12 was not confirmed by nLC-MS/MS, which is possibly due to the technical limitation [14]. The heterogeneous organization of Pol II echoes previously published experimental evidence. First of all, the absence of Rpb9 in the “remodeled” Pol II explains the higher mutation rates of PSTVd during replication [29], when compared to the Pol II fidelity on DNA templates [30]. Furthermore, distinct Pol II subunits appear to control the transcription of different sets of genes according to degron-based transiently degradation of each subunit in mammalian cell lines [31]. Taken together, evidence supports a possible distinctive organization of the transcription complex on viroid RNA templates. In spite of the supporting evidence, whether such remodeled Pol II exists in vivo and functions in any natural process remains to be determined. Moreover, the molecular basis remains elusive regarding how TFIIIA-7ZF interacts with the remodeled Pol II for RNA-templated transcription.

To gain insights into the organization of the Pol II transcription complex on RNA template, we tested whether Rpb4/5/6/7/9 and Rpb12 are involved in binding PSTVd RNA templates in vivo. These subunits, except for Rpb12 that could not be detected due to the small size, were absent in active PSTVd transcription based on in vitro data [14]. Interestingly, our new data below showed that Rpb12 is involved in RNA template binding, while the rest five subunits (Rpb4/5/6/7/9) do not interact with PSTVd RNA template in vivo. Furthermore, we performed co-immunoprecipitation analysis to test the interaction between TFIIIA-7ZF and subunits like Rpb3/4/5/6/7/9/12. TFIIIA-7ZF could only interact with Rpb3 and Rpb12 in infected samples. Notably, the interactions between TFIIIA-7ZF and Rpb3/Rpb12 were undetectable in mock-infected samples. Therefore, our analyses provide compelling evidence supporting the re-organization of Pol II for transcribing viroid RNA templates in plants.

Based on this information, we predicted the structure of the remodeled Pol II with seven subunits (Rpb1/2/3/8/10/11/12) as well as TFIIIA-7ZF on the (+) PSTVd RNA template. The predicted model provides the opportunity to understand the structural mechanism of an RNA-specific transcription factor (i.e., TFIIIA-7ZF) in RNA-templated transcription. We experimentally tested the model to validate the interaction and structural geometry of RNA templates, TFIIIA-7ZF, and the subunits in the remodeled Pol II. Furthermore, we predicted the complex of TFIIIA-7ZF and (+) PSTVd RNA that pinpoint zinc finger domains (ZF) 2, 3, 6 as possible RNA binding domains. Experimental data clearly support the critical role of ZF3 and ZF6 in RNA binding. Moreover, the empirical data suggested that the RNA bending at the loop 8 region may be important for TFIIIA-7ZF and PSTVd interaction in the absence of Pol II.

Altogether, our data support the in vivo presence of the remodeled Pol II and illustrate how TFIIIA-7ZF bridges the RNA template and the remodeled Pol II, which provides structural insights into the novel regulation of transcription by using an altered organization of polymerase complex.

Results

The composition of in vivo Pol II complex on RNA templates

Pol II has been well documented for catalyzing the replication of nuclear-replicating viroids and human hepatitis delta virus [32–34]. Our recent findings demonstrate that Pol II can catalyze RNA-templated transcription in vitro without a complete set of 12 subunits [14]. We termed this distinct organization the “remodeled” Pol II. Those subunits are essential for the cell survival, so we cannot assess the model in vivo by using loss-of-function mutants. To corroborate the remodeled Pol II organization in vivo, we performed RNA immunoprecipitation (RIP) analyses to test the interaction between PSTVd RNA and the Pol II subunits absent from the remodeled Pol II (i.e., Rpb4/5/6/7/9). Previous work has shown that Rpb1 binds PSTVd RNA in vivo tested by RIP [13]. Here, we used Rpb3 as positive control for additional testing. We could not detect Rpb12 on RNA template in vitro in our previous work [14], but it was suspected that the small size of Rpb12 rendered it being filtered out by size cut-off columns during sample preparation as reported before [23]. Therefore, we also included Rpb12 in this test. Since most of these subunits have multiple gene copies in plants, we selected the gene copies that are expressed and incorporated into Pol II based on published data [35]. These subunits were expressed in the PSTVd-infected N. benthamiana plants as fusions with a GFP-tag for RIP analyses. Plants infiltrated with agrobacteria without the expression vector served as negative control.

As shown in Fig 1A, GFP-trap successfully precipitated all the target subunits. We then used RT-qPCR to detect PSTVd RNA templates enriched in the immunoprecipitated fraction. 5.8S rRNA was used as negative control for normalizing RT-qPCR data. As shown in Fig 1A, PSTVd RNA was significantly co-precipitated in samples expressing subunits Rpb3 and Rpb12, confirming that Rpb3 and Rpb12 are part of the remodeled Pol II in vivo. By contrast, PSTVd RNA was not significantly enriched in samples expressing subunits Rpb4, Rpb5, Rpb6, Rpb7, or Rpb9 as compared with the negative control samples.

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Fig 1. Remodeled Pol II in vivo and its predicted structure.

(A) RNA-immunoprecipitation using Pol II subunits. GFP-tagged Rpb3, Rpb4, Rpb5, Rpb6, Rpb7, Rpb9, and Rpb12 were subject to RNA immunoprecipitation (left panel) to test their in vivo interaction with PSTVd RNA templates. RNAs from input and immunoprocipitated (IP) fraction were used for RT-qPCR analysis (right panel). 5.8S rRNA was used as negative control to normalize PSTVd enrichment. Data from three biological replicates were used for t-test analysis. NC, leaves infiltrated with agrobacterium without any GFP-expressing plasmid. (B) Co-immunoprecipitation using Pol II subunits. GFP-tagged Rpb3, Rpb4, Rpb5, Rpb6, Rpb7, Rpb9, and Rpb12 were subject to co-immunoprecipitation to test their in vivo interaction with NbTFIIIA-7ZF. (PSTVd)-infected and mock-(treated) samples were used for comparison. Three biological replicates were performed. (C) Predicted structures of Pol II with DNA template and remodeled Pol II with TFIIIA-7ZF and RNA template. The catalytic subunits (Rpb1 and Rpb2) as well as the distinct subunits (Rpb4/5/6/7/9) and TFIIIA-7ZF are color coded. The stalk region is highlighted in a dash-line circle.

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Previously, co-immunoprecipitation (coIP) analysis has demonstrated the in vivo interaction between Rpb1 and TFIIIA-7ZF [13], which served as a positive control here (S2 Fig). Here, we also confirmed the interaction between Rpb2 and TFIIIA-7ZF using coIP (S2 Fig). Since TFIIIA-7ZF is a key transcription factor bridging the Pol II and RNA template interaction, we reason that TFIIIA-7ZF will not interact with subunits that are not in the remodeled Pol II. To test this, we used co-immunoprecipitation to test the interactions between TFIIIA-7ZF and subunits like Rpb3, Rpb4, Rpb5, Rpb6, Rpb7, Rpb9, and Rpb12. As shown in Fig 1B, GFP-tagged Rpb3 and Rpb12 can interact with TFIIIA-7ZF in PSTVd-infected samples. By contrast, neither of the Rpb4/5/6/7/9 subunits showed positive interaction with TFIIIA-7ZF. Interesting, Rpb3 and Rpb12 cannot interact with TFIIIA-7ZF in non-infected samples (Fig 1B), indicating that PSTVd RNA is essential to bring TFIIIA-7ZF and the remodeled Pol II into one complex.

Based on RIP (between PSTVd RNA and Pol II subunits) and coIP (between TFIIIA-7ZF and Pol II subunits), Rpb4/5/6/7/9 are not part of the remodeled Pol II on PSTVd RNA templates, which is consistent with our previous analysis using an in vitro transcription system [14].

Structure overview of the TFIIIA-7ZF/Pol II complex on the (+) PSTVd RNA templates

Previous structural studies have resolved multiple high-quality Pol II complexes with general transcription factors at different transcription stages [22,36–42]. The structure of a canonical TFIIIA protein with nine zinc finger domains (ZFs) is also available (PDB 1tf6) [43]. However, the structure illustrating the complex of Pol II and RNA-specific transcription factors (i.e., HDAg-S and TFIIIA-7ZF) is lacking. Nevertheless, these resolved structures provided the basis for predicting a Pol II complex with other factors. Taking the advantage of the emergence of AlphaFold3 [44], we predicted the structure of the TFIIIA-7ZF/Pol II complex on the (+) PSTVd RNA template (359 nt)(S1 Video). For comparison, we also predicted the Pol II complex (with 12 subunits) on a DNA template of 180 bp, using the sequence from an expression vector pRTL2 with the 35S promoter. As shown in S3 Fig, the predicted models have very high confidence for the Pol II and remodeled Pol II complexes, which reflects the presence of high-quality Pol II data. The structures for the nucleic acid portions generally have low confidence, which is expected since AlphaFold3 is generally thought to have less accuracy in predicting nucleic acid structures.

The remodeled Pol II lacks the “Stalk” that consists of Rpb4/7 and its adjacent Rpb6 (Fig 1C). This absence is unlikely caused directly by TFIIIA-7ZF occupancy because TFIIIA-7ZF does not share any common binding sites with these three subunits (Fig 1C). The absence of Rpb5 and Rpb9 may be caused by TFIIIA-7ZF occupancy since their binding sites crossover with the TFIIIA-7ZF binding site on the surface of Rpb1.

Testing the geometry of TFIIIA-7ZF in the Pol II/TFIIIA-7ZF/PSTVd complex

We noticed that TFIIIA-7ZF wraps the RNA template while attaches to the remodeled Pol II complex (Fig 1C). The N-terminal region of TFIIIA-7ZF is involved in binding with the RNA template, and the TFIIIA-7ZF C-terminus appears to be critical for binding with the Pol II core, particularly Rpb2. As shown in Fig 2A, residues R243, R245, and K257 of TFIIIA-7ZF are predicted to be involved in binding with Rpb2 Q51 in the Protrusion domain and E549/E550 in the External 2 domain. Residues R243, R245, and K257 are outside of the seven zinc finger domains towards the C-terminus of the TFIIIA-7ZF. We also noticed that the C-terminus end of TFIIIA-7ZF is relatively close to the N-terminus of Rpb12 but away from the N-terminus of Rpb3 (Fig 2B).

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Fig 2. Relative position of TFIIIA-7ZF and Pol II subunits.

(A) Predicted interactions between Rpb2 and TFIIIA-7ZF. Rpb2 and TFIIIA-7ZF are depicted in magenta and blue, respectively. (B) Predicted structure shows the relative position between the C-terminus of TFIIIA-7ZF (labeled by an arrow head) and the N-terminus of Rpb3 and Rpb12 (labeled by arrow heads). (C) Bi-fluorescence complementation (BiFC) confirms that the C-terminus of TFIIIA-7ZF is close to the N-terminus of Rpb12 but not the N-terminus of Rpb3. Mu, TFIIIA-7ZF^C-del. Scale Bar, 10 μm. (D) RNA gel blots show that transiently over-expressed WT TFIIIA-7ZF, but not TFIIIA-7ZF^C-del, enhanced PSTVd accumulation in planta. TFIIIA-7ZF proteins were detected by α-HA antibody. Ponceau S staining serves as loading control for immunoblots. PSTVd was detected by RNA gel blots, and ethidium bromide staining of rRNAs serves as the loading control.

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To empirically test the predicted structural geometry of the TFIIIA-7ZF/Pol II complex, we used bimolecular fluorescence complementation assay (BiFC) to assess the relative distance between TFIIIA-7ZF (with a C-terminal half of YFP fused to the end of the TFIIIA-7ZF C-terminus) and Rpb12 or Rpb3 (with a N-terminal half of YFP fused to the subunit’s N-terminus). As shown in Fig 2C, only the Rpb12/TFIIIA-7ZF pair, but not the Rpb3/TFIIIA-7ZF pair, could complement the YFP fluorescence. We then designed a C-terminus-lacking variant of TFIIIA-7ZF (TFIIIA-7ZF^C-del; aa 1–228) that still keeps all seven zinc finger domains but lacks the critical residues for Rpb2 binding. This TFIIIA-7ZF^C-del could not complement YFP fluorescence with Rpb12 (Fig 2C). Furthermore, TFIIIA-7ZF^C-del could not enhance PSTVd accumulation as compared with WT TFIIIA-7ZF (Fig 2D). These results confirm that the C-terminus of TFIIIA-7ZF is functionally important for binding with the remodeled Pol II complex. Importantly, the BiFC data corroborate the predicted structure of the complex in terms of the relative distance between TFIIIA-7ZF and Pol II subunits (i.e., Rpb3 and Rpb12).

Structure of TFIIIA-7ZF/viroid complex

In our Pol II-based in vitro transcription assay, incubating RNA template with TFIIIA-7ZF prior to the addition of Pol II is critical for optimized transcription efficiency [24], which implies that TFIIIA-7ZF and PSTVd RNA-protein complex is formed first before recruiting Pol II. However, it is unclear how TFIIIA-7ZF coordinates its seven ZFs for RNA template binding. Therefore, we were interested in understanding the structure of TFIIIA-7ZF and the PSTVd (+) genomic RNA by using AlphaFold3. Previously, it was speculated that the left terminal region of PSTVd (+) RNA genome forms either a rod-shape structure or a bifurcated structure during transcription [27]. However, the model predicted by AlphaFold3 provided an unexpected conformation (Fig 3A). While PSTVd RNA bases still pair similarly as in the rod-shape structure, the TFIIIA-7ZF protein bends the overall rod-shape structure that leads to the formation of an enlarged left terminal region. We noticed that base A51, which resides in loop 8, is right at the bending site. Using an established mutant that changes loop 8 to a double-stranded conformation [45], we demonstrated that the rigid double-stranded conformation impairs PSTVd RNA binding with TFIIIA-7ZF (Fig 3C), which is in support of bending at loop 8 in the TFIIIA-7ZF/PSTVd complex. It also aligns with the previous observation that this loop 8 mutant exhibited reduced replication efficiency [45]. Together, the data support the importance of RNA bending for interacting with TFIIIA-7ZF protein.

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Fig 3. Structure of TFIIIA-7ZF and (+) PSTVd RNA.

(A) Predicted structure of TFIIIA-7ZF and PSTVd RNA. Different zinc finger domains are color highlighted. Regions between U356 and U324 are predicted to be covered by TFIIIA-7ZF. (B) A zoom-in view of the critical zinc fingers for RNA binding. (C) EMSA demonstrating that loop 8, which is predicted as the bending site, is critical for TFIIIA-7ZF binding. (D) RNA-immunoprecipitation demonstrating the function of the zinc fingers. HA-tagged proteins were expressed in PSTVd-infected plants. α-HA and α-MYC were used for immunoprecipitation (IP) and control treatments, respectively. Semi quantitative RT-PCR were used for examining PSTVd enrichment in IP fractions, with U6 snRNA served as negative control. All experiments were repeated at least twice.

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A close look at the predicted structure showed that ZF2 and ZF3 clamp the major groove of the RNA template (Fig 3B). ZF6 is also critical as it is central to coordinate TFIIIA-7ZF in bending the PSTVd RNA (Fig 3B). The C-terminus of TFIIIA-7ZF binds to the RNA template when Pol II is not present, which is in contrast to binding with Rpb2 in the Pol II/TFIIIA-7ZF/PSTVd complex shown in Fig 2A. The TFIIIA-7ZF binding sites are estimated from G328 to G354 (Fig 3A), consistent with our foot-printing assay that mapped the binding region from U331 to G346 [13]. This region is also proximal to the transcription initiation site between U359 and C1 [46]. The significant overlapping between the experimental data and the prediction model supports that the predicted model indeed reflects the structure of the (+) PSTVd/TFIIIA-7ZF complex. To further map the ZFs that are critical for RNA templates binding, we introduced a point mutation changing the first histidine to asparagine to disrupt each C2H2 ZF as previously reported [47,48] and performed RIP using these variants with an HA tag. We could not detect the expression of the zf5 variant in plants, potentially attributable to poor stability of the protein. Nevertheless, the rest six variants were successfully expressed and detected in the eluted RIP fractions. Since our primer sets can detect both (+) and (-) PSTVd strands simultaneously in one-step RT-qPCR reactions, we only used semi-quantitative RT-PCR to test for the enrichment of the (+) PSTVd RNA in the immunoprecipitated fractions. As shown in Fig 3D, the zf2, zf3, and zf6 variants consistently failed to precipitate PSTVd RNA, indicating that these domains are critical for RNA binding, which provides a strong support for the predicted model of the RNP.

To further test the critical zinc fingers involved in (+) PSTVd RNA binding, we performed electrophoretic mobility shift assays (EMSAs) using each zf variant. Actin protein was used as a negative control (S4 Fig). As shown in Fig 4, disruption of either ZF3 or ZF6 significantly impacts RNA binding, as these variants lost the ability to achieve a complete RNA shifting in comparison to wildtype and other zf variants. Interestingly, the rest of zf variants demonstrated similar or even higher affinity to the PSTVd RNA as indicated by the concentration producing half occupation (K)(Fig 4). Notably, the zf2 variant could bind with (+) PSTVd RNA in EMSA tests, which is different from the RIP outcomes presented in Fig 3D. This discrepancy implies that the ZF2 domain may be involved in other interactions in the cellular environment. Alternatively, ZF1 might contribute to stabilizing RNA binding in vitro through working together with ZF3 for RNA binding when ZF2 is impaired, which may be implied by the close distance between ZF1 and ZF3 on PSTVd RNA in the predicted model (Fig 3B). In spite of this discrepancy of ZF2, RIP, EMSA, and the structural model all support the critical roles of ZF3 and ZF6 in (+) PSTVd binding.

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Fig 4. Interaction of TFIIIA-7ZF variants with PSTVd in EMSAs.

(A) Representative EMSA result for the binding between TFIIIA-7ZF zinc finger variants and PSTVd. Protein concentrations are listed for each lane. Solid arrows depict the position of free RNA substrate. Hollow arrows depict the position of protein-RNA complexes. (B) Representative binding curves for each TFIIIA-7ZF variants. Each data point was calculated based on three replicates. Protein concentrations are listed on X axes.

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Discussion

The results presented in this study demonstrate a distinct organization of Pol II transcribing viroid RNA template in planta, a new layer of transcriptional regulation in a natural biological process. By using RNA-immunoprecipitation and co-immunoprecipitation, we showed that a subset of Pol II subunits is not associated with PSTVd RNA and TFIIIA-7ZF, in line with of our previous results from an in vitro setting [14]. Interestingly, we could not detect the interactions between TFIIIA-7ZF and Rpb3/Rpb12 in non-infected plants, suggesting that the abundance of the suitable RNA templates is a critical factor for TFIIIA-7ZF and Pol II interaction. Rpb1/2/3/8/10/11/12 constitute the remodeled Pol II for RNA-templated transcription (Fig 5). During the assembly of Pol II complex, there are several intermediate complexes/subassemblies, including the Rpb1 subassembly containing Rpb1/5/6/8, the Rpb2 subassembly containing Rpb2/9, the Rpb3 subassembly containing Rpb3/10/11/12 and the Rpb4/7 subassembly (Stalk) [49]. Interestingly, the remodeled Pol II contains the intact Rpb3 subassembly, a modified Rpb1 subassembly lacking Rpb5 and Rpb6, and a modified Rpb2 subassembly lacking Rpb9 [50,51]. The stalk portion of the Pol II is completely absent in the remodeled Pol II. It is known that Rpb4, Rpb5, Rpb7, and Rpb9 tend to dissociate from the Pol II complex upon alpha-Amanitin treatment [50], suggesting that these subunits are prone to leave the Pol II complex under stress. Our findings also align with a recent finding of possible heterogeneous organization of Pol II in mammalian cells [31], suggesting that the transcriptional regulation at the polymerase organization level is more complex than previously recognized.

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Fig 5. A model of distinct Pol II complexes for DNA and RNA templates.

Numbers in the Pol II complexes represent the corresponding subunits. Question marks represent factors yet-to-be-identified.

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Confirming the remodeled Pol II organization in vivo paves the way for further investigations on the different regulations of DNA-dependent versus RNA-templated transcription. For instance, the Pol II stalk is involved in binding with capping enzymes for adding G-cap to nascent mRNAs [52,53]. This explains the lack of G-cap in viroid RNAs [54]. Whether HDV RNA-templated transcription also uses a remodeled Pol II is not immediately clear. A small fraction of HDV-derived mRNAs contains a G-cap, but Pol II-transcribed genomic or antigenomic RNAs do not [34,55].

Although the detailed structural basis has largely been determined for almost every step in RNA polymerases transcribing DNA templates, the molecular basis underlying the widely existing RNA-templated transcription remains elusive. Previous work using analytical ultracentrifugation and electron microscopy provided limited visual insights into the Pol II-PSTVd complex [56]. However, TFIIIA-7ZF was not identified at the time, and thus was not included in the complex in the study. Therefore, it is unknown whether a remodeled Pol II, the active catalytic enzyme on PSTVd RNA template, was observed under the electron microscope. To date, the only high-resolution structure, which contained 12-subunit Pol II, TFIIS, and a synthetic mosaic RNA template with partial HDV sequence, was determined by using a crystallographic analysis [57]. This complex does not include the RNA-specific transcription factor (HDAg-S) for HDV RNA templates. In addition, this complex does not possess strong processivity for yielding long products (longer than 100 nt) that occur in nature. Moreover, the TFIIS-based cleavage allows Pol II to catalyze primed transcription on some RNA templates, but it is not required for transcribing PSTVd as Pol II catalyzes de novo transcription using PSTVd RNA template [24]. Therefore, alternative structure model is needed for understanding RNA-templated transcription.

Here, we obtained predicted structures of the transcription complex and experimentally tested the structural model. In the predicted TFIIIA-7ZF/PSTVd structural model, notably, TFIIIA-7ZF binds around the left terminal part of PSTVd (+) RNA template and possibly bends the RNA to form an enlarged tertiary structure (Fig 3). The loop 8 mutant that makes the bending region more rigid impairs TFIIIA-7ZF and PSTVd interaction as well as PSTVd replication, supporting the functional relevance of this possible RNA bending. This enlarged overall structure possibly contributes to the recruiting of Pol II. The predicted model also suggests that the viroid RNA promoters may not entirely rely on primary sequences. Instead, an overall bended tertiary structure is likely critical for transcription. This organization is different from previously reported hairpin-like RNA promoter used for testing a chimeric HDV RNA template [57]. This model may help to explain that over 30 nuclear-replicating viroids with distinct sequences all use Pol II for replication [32].

When Pol II is recruited to the PSTVd/TFIIIA-7ZF complex, the C-terminus of TFIIIA-7ZF somehow moves from RNA binding to the association with Rpb2. A C-terminus deletion impairs TFIIIA-7ZF binding with the remodeled Pol II and abolishes its function in aiding PSTVd accumulation. Therefore, these data suggest a possible structural transition of the TFIIIA-7ZF C-terminus from bending the RNA template to connecting the RNA template with the polymerase. The details of the structural transition, from the association with the bended RNA template to the Rpb2 in the remodeled Pol II, deserve future investigations.

Materials and methods

Plant materials

Nicotiana benthamiana plants were grown in a growth room with 28°C and a 14-h/10-h light/dark cycle. Arabidopsis thaliana plants were grown in a growth room with 22°C and a 10-h/14-h light/dark cycle. During agroinfiltration period, plants were moved into a growth room with 22°C and a 14-h/10-h light/dark cycle. PSTVd-infection and validation steps were the same as described previously [58,59]. Protoplasts generation and transfection were reported in detail previously [60].

Molecular cloning

Following clones were ordered from Arabidopsis Biological Resource Center (ABRC) at the Ohio State University: Rpb4 (DKLAT5G09920) and Rpb7 (DKLAT5G59180). ORFs of Rpb5 (At3g22320), Rpb6 (At2g04630), Rpb9 (At4g16265), Rpb12 (At5g41010), TFIIF1 (At1g75510), and TFIIS (AT2G38560) were cloned via conventional RT-PCR and inserted into the pENTR-D-TOPO vector (ThermoFisherSci, Waltham, MA) and recombined to destination vectors. All clones were verified by Sanger Sequencing. Primers were listed in the S1 Table. Loop 8 mutant was reported previously [45]. TFIIIA-7ZF WT and point mutation variants were reported previously [14]. The C-terminal deleted TFIIIA-7ZF mutant (aa 1–228) was cloned into the pENTR-D-TOPO vector (ThermoFisherSci) using WT clone as template, followed by LR recombination to festination vectors. 35S:plasmid was reported previously [61]. The cDNA of Arabidopsis actin (AT3G46520) was cloned via RT-PCR and inserted into the pENTR-D-TOPO vector. The actin entry clone was then recombinated to pDEST17 (ThermoFisherSci) to generate actin plasmid for bacterial expression.

RNA-Immunoprecipitation (RIP) and co-immunoprecipitation (coIP)

RIP and coIP were performed following our established protocol [13]. For RIP, PSTVd-infected N. benthamiana plants were agro-infiltrated with destination vectors to express GFP-tagged Pol II subunits and HA-tagged TFIIIA-7ZF variants. Three days post infiltration, protein expressions were verified by microscopy observation of green fluorescence before RIP procedures. Collected leaf samples were lysed in RIP buffer (25 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.2% NP-40, protease inhibitor cocktail following manual) as described before [13]. The lysates were incubated with GFP-trap beads (ProteinTech, Rosemont, IL), anti-HA magnetic beads (ThermoFisherSci) or anti-Myc magnetic beads (ThermoFisherSci) for 2 hr at 4°C with rotation. The beads were washed twice in IP buffer and once in RNase-Free water, before elution using acidic Glycine buffer following manual instructions (ThermoFisherSci). The protein fractions were analyzed via immunoblotting as described before in details [13]. The precipitated RNAs were analyzed by semi-quantitative RT-PCR or RT-qPCR (please see S1 Table for primer sequences). For RT-qPCR, 5.8S rRNA was used as negative control for normalizing PSTVd enrichment folds. Data from three biological replicates were used for T-test analysis.

For coIP, the same procedures were used to express GFP-tagged Pol II subunits in PSTVd- or mock-infected N. benthamiana plants. Collected samples were lysed overnight with RIPA buffer (with SDS concentration reduced to 0.05%), supplemented with 2.5 mM MgCl₂, 0.5 mM CaCl₂, 4 U Turbo DNase (ThermoFisherSci), 60 U murine RNase inhibitor (New England Biolabs, Ipswich, MA). The lysate was collected and incubated with GFP-trap beads as described above. After incubation, beads were washed three times with 1X TBST (20 mM Tris pH7.4, 150 mM NaCl, 0.1% w/v Tween-20). For elution, 2X SDS loading buffer (120 mM Tris pH 6.8, 20% v/v glycerol, 4% w/v SDS, 0.04% w/v bromophenol blue, 10% v/v β-mercaptoethanol) was used according to the GFP-trap manual. For FLAG-tagged Rpb2 [7], we used L5 monoclonal anti-FLAG antibody (ThermoFisherSci) for immunoprecipitation using magnetic protein A/G beads (ThermoFisherSci) and anti-FLAG M2 antibody (MilliporeSigma, St. Louis, MO) for immune-detection. Immunoblotting was performed following our established protocol as reported in details previously [13]. Anti-TFIIIA antibody has been reported previously [13].

Bimolecular fluorescence complementation (BiFC) and Microscopy

For BiFC, PSTVd-infected N. benthamiana seedlings were used for agroinfiltration of various combinations of constructs, all including 35S:RFP-Histone 2B [58] as the nucleus marker. The N split (aa 1–174) YFP was fused in front of the N-terminus of RPB3 or RPB12. The C split YFP (aa 175-end) was fused after the C-terminus of TFIIIA-7ZF and its variant. We analyzed 10 randomly chosen regions of infiltrated leaves from at least three plants for each treatment. ECHO Revolve microscope (Discover Echo Inc., San Diego, CA) was used for observing the fluorescence signals.

Electrophoretic mobility shift assays (EMSA)

For EMSA, TFIIIA-7ZF and its variants were expressed using IMPACT system (New England Biolabs, Ipswich, MA) in bacteria and purified using Chitin beads as previously described [13]. The HIS₆-tagged actin protein was purified using a protocol that was described previously [62]. Protein-RNA binding reactions (20 mM Tris-HCl pH 7.5, 35 mM KCl, 5 µM ZnCl₂, 3.5 mM MgCl₂, 10 nM yeast tRNA and 10% (v/v) glycerol) were performed at 25°C using ³²P-labeled RNAs (20,000 dpm) with increasing concentrations of recombinant proteins (described in Fig 4). Electrophoresis was carried out on ice using 6% (w/v) polyacrylamide (29:1) gels for 2 h at 120 V with Tris borate buffer (65 mM Tris, 22.5 mM boric acid, pH 8). PhosphorImager cassette was used to visualize the signal in a Personal Molecular Imager (Bio-Rad Laboratories, Hercules, CA). The intensity of each band on the gel was quantified by using Quantity One software (Bio-Rad Laboratories). The binding curves were obtained by plotting the fraction of RNA bound with proteins as previously described [13].

Supporting information

S1 Table. Primer sequences.

https://doi.org/10.1371/journal.ppat.1014200.s001

(XLSX)

S1 Fig. The G435E mutation in Rpb2 affects DNA-dependent transcription but not RNA-templated transcription.

PSTVd RNA and 35S:GFP plasmid were co-transfected in protoplasts yielded from wild type or the nrpb2–3 hypermorphic mutant. GFP express from plasmid was repressed in the nrpb2–3 mutant, but PSTVd RNA replication was not affected. Scale bar, 50 μm.

https://doi.org/10.1371/journal.ppat.1014200.s002

(PDF)

S2 Fig. Co-immunoprecipitation of Rpb1/TFIIIA-7ZF and Rpb2/TFIIIA-7ZF.

https://doi.org/10.1371/journal.ppat.1014200.s003

(PDF)

S3 Fig. AlphaFold 3 confidence scores.

https://doi.org/10.1371/journal.ppat.1014200.s004

(PDF)

S4 Fig. EMSA with actin protein as a negative control.

https://doi.org/10.1371/journal.ppat.1014200.s005

(PDF)

S1 Video. The predicted transcription complex on PSTVd RNA template.

https://doi.org/10.1371/journal.ppat.1014200.s006

(MP4)

S1 Dataset. AlphaFold3 predicted transcription complex containing Pol II and a DNA fragment.

https://doi.org/10.1371/journal.ppat.1014200.s007

(ZIP)

S2 Dataset. AlphaFold3 predicted transcription complex containing remodeled Pol II, TFIIIA-7ZF, and (+) PSTVd RNA.

https://doi.org/10.1371/journal.ppat.1014200.s008

(ZIP)

S3 Dataset. AlphaFold3 predicted complex containing TFIIIA-7ZF and (+) PSTVd RNA.

https://doi.org/10.1371/journal.ppat.1014200.s009

(ZIP)

Acknowledgments

We are grateful for the technical support of Shachinthaka D. Dissanayaka Mudiyanselage. FLAG-tagged Rpb2 genomic clone was a gift from Dr. Craig S. Pikaard (Indiana University).

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Reorganizing the RNA polymerase II complex for replication of an infectious noncoding RNA in vivo

Reorganizing the RNA polymerase II complex for replication of an infectious noncoding RNA in vivo

This is an uncorrected proof.

Figures

Abstract

Author summary

Introduction

Results

The composition of in vivo Pol II complex on RNA templates

Structure overview of the TFIIIA-7ZF/Pol II complex on the (+) PSTVd RNA templates

Testing the geometry of TFIIIA-7ZF in the Pol II/TFIIIA-7ZF/PSTVd complex

Structure of TFIIIA-7ZF/viroid complex

Discussion

Materials and methods

Plant materials

Molecular cloning

RNA-Immunoprecipitation (RIP) and co-immunoprecipitation (coIP)

Bimolecular fluorescence complementation (BiFC) and Microscopy

Electrophoretic mobility shift assays (EMSA)

Supporting information

S1 Table. Primer sequences.

S1 Fig. The G435E mutation in Rpb2 affects DNA-dependent transcription but not RNA-templated transcription.

S2 Fig. Co-immunoprecipitation of Rpb1/TFIIIA-7ZF and Rpb2/TFIIIA-7ZF.

S3 Fig. AlphaFold 3 confidence scores.

S4 Fig. EMSA with actin protein as a negative control.

S1 Video. The predicted transcription complex on PSTVd RNA template.

S1 Dataset. AlphaFold3 predicted transcription complex containing Pol II and a DNA fragment.

S2 Dataset. AlphaFold3 predicted transcription complex containing remodeled Pol II, TFIIIA-7ZF, and (+) PSTVd RNA.

S3 Dataset. AlphaFold3 predicted complex containing TFIIIA-7ZF and (+) PSTVd RNA.

Acknowledgments

References