Molecular basis of viroid RNA-templated transcription

Transcription is a fundamental process catalyzed by DNA-dependent RNA polymerases (DdRPs). Interestingly, some DdRPs can use both DNA and RNA as templates for transcription. This RNA-dependent RNA polymerase (RdRP) activity of DdRPs is used by RNA-based pathogens such as viroids and hepatitis delta virus for replication. In addition, RdRP activity of DdRPs widely occurs in various organisms to regulate gene transcription. Despite the importance of this intrinsic RdRP activity of DdRPs, associated factors and mechanisms are in their infancy stage. We employed potato spindle tuber viroid (PSTVd) as a model to study RNA-templated transcription. Here, we present evidence showing that circular PSTVd templates are critical for the synthesis of longer-than-unit-length (-) strand products. Further, we show transcription factor IIS is dispensable for PSTVd replication supporting de novo transcription on PSTVd RNA templates. The absence of canonical general transcription factor, TFIIS from PSTVd-templated transcription complex led to the hypothesis that RNA-templated transcription has a distinct organization on the RNA template. To test this hypothesis, we used our well-established in vitro transcription (IVT) system and demonstrated that RNA polymerase II (Pol II) accepts minus-strand for transcription. In addition, transcription factor TFIIIA-7ZF is needed to aid Pol II transcription activity. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed that the first three zinc finger domains are pivotal for template binding. Notably, we identified a remodeled Pol II complex for viroid transcription that is missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9. General transcription factors for DNA-templated transcription are also absent in the transcription complex on the RNA template. This remodeled Pol II complex still possesses the transcription activity on PSTVd RNA template. Collectively, our data illustrate 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

Evidence Supporting That RNA Polymerase II Catalyzes De Novo Transcription Using Potato Spindle Tuber Viroid Circular RNA Templates

Transcription is a fundamental process that mediates the interplay between genetic information and phenotype. Emerging evidence indicates that RNA polymerase II (Pol II) can catalyze transcription using both DNA and RNA templates. It is well established that Pol II initiates de novo transcription on DNA templates. However, it is unclear whether Pol II performs de novo transcription or relies on primers for initiation (primed transcription) on RNA templates. Using potato spindle tuber viroid (PSTVd) as a model, we presented evidence showing that circular PSTVd templates are critical for the synthesis of longer-than-unit-length (−)-strand products, which supports the de novo transcription based on the asymmetric rolling circle model of PSTVd replication. We further showed that the crucial factor for primed transcription, transcription factor IIS (TFIIS), is dispensable for PSTVd replication in cells. Together, our data support the de novo transcription on PSTVd RNA templates catalyzed by Pol II. This result has significant implications in understanding the mechanism and machinery underlying Pol II-catalyzed transcription using other RNA templates

Potato Spindle Tuber Viroid RNA-Templated Transcription: Factors and Regulation

Viroids are circular noncoding RNAs that infect plants. Without encoding any protein, these noncoding RNAs contain the necessary genetic information for propagation in hosts. Nuclear-replicating viroids employ DNA-dependent RNA polymerase II (Pol II) for replication, a process that makes a DNA-dependent enzyme recognize RNA templates. Recently, a splicing variant of transcription factor IIIA (TFIIIA-7ZF) was identified as essential for Pol II to replicate potato spindle tuber viroid (PSTVd). The expression of TFIIIA-7ZF, particularly the splicing event, is regulated by a ribosomal protein (RPL5). PSTVd modulates its expression through a direct interaction with RPL5 resulting in optimized expression of TFIIIA-7ZF. This review summarizes the recent discoveries of host factors and regulatory mechanisms underlying PSTVd-templated transcription processes and raises new questions that may help future exploration in this direction. In addition, it briefly compares the machinery and the regulatory mechanism for PSTVd with the replication/transcription system of human hepatitis delta virus

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

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

The Intriguing Conundrum of a Nonconserved Multifunctional Protein of Citrus Tristeza Virus That Interacts with a Viral Long Non-Coding RNA

Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed

Potato Spindle Tuber Viroid Modulates Its Replication through a Direct Interaction with a Splicing Regulator

Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic makeup and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5 [RPL5]) in vitro and in vivo. PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can regulate their own replication and modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions. IMPORTANCE Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants, often resulting in disease symptoms. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control