RNA polymerase II acts as an RNA-dependent RNA polymerase to extend and destabilize a non-coding RNA

Wagner, Stacey D.; Yakovchuk, Petro; Gilman, Benjamin; Ponicsan, Steven L.; Drullinger, Linda F.; Kugel, Jennifer F.; Goodrich, James A.

doi:10.1038/emboj.2013.18

Cited by 52 publications

(54 citation statements)

References 36 publications

(71 reference statements)

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“…Mouse B2 and human Alu ncRNAs both repress mRNA transcription by binding to RNAP II and blocking contacts with promoter DNA (Yakovchuk et al, 2009). Remarkably, RNAP II can utilize the B2 RNA as both a transcription template and substrate, using an RNA-dependent RNAP activity to extend the 3′-end of the B2 RNA in an internally templated reaction (Wagner et al, 2013). Extension of the B2 RNA in this way destabilizes the RNA and relieves the RNAP II inhibition.…”

Section: Discussionmentioning

confidence: 99%

“…Our results help explain the specificity of Eco 6S RNA for Eσ 70 , and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP. Functional parallels between bacterial and eukaryotic ncRNA RNAP inhibitors (Wagner et al, 2013; Yakovchuk et al, 2009) suggest that the structural principles for 6S RNA binding and inhibition of Eco RNAP delineated here are widely applicable.…”

Section: Introductionmentioning

confidence: 99%

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6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase

et al. 2017

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Summary Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ70-holoenzyme (Eσ70) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ70 determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3′-endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ70, and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase

et al. 2017

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“…These results strongly suggest that the TERT RdRP produces RNAs in the IP-RdRP assay. RNA polymerase II (Pol II) is reportedly responsible for RdRP activity in Saccharomyces cerevisiae (13) and mice (14); therefore, we excluded the possibility that Pol II is responsible for RNA production in this assay. For this purpose, we performed the IP-RdRP assay with ␣-amanitin, a well-characterized Pol II inhibitor.…”

Section: Resultsmentioning

confidence: 99%

De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

Maida¹,

Yasukawa²,

Masutomi³

2016

Molecular and Cellular Biology

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RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such as Caenorhabditis elegans and Neurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAs de novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However, de novo synthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5=-triphosphorylated ends, which indicates de novo synthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.T elomerase reverse transcriptase (TERT) is known as the catalytic subunit of telomerase and is expressed at high levels in cancer cells but only at low levels in normal human somatic cells. TERT elongates telomeres by its RNA-dependent DNA polymerase (RdDP) activity, using the telomerase RNA component (TERC) as the template. TERT and TERC assemble and form telomerase; however, there is a population of TERT proteins that are not assembled into the telomerase complex (1). Several lines of evidence indicate that TERT plays roles independent of telomere maintenance; therefore, nonassembled TERT may be involved in complexes other than telomerase.RNA silencing is a sequence-specific gene regulatory mechanism mediated by double-stranded RNAs (dsRNAs). RNAdependent RNA polymerase (RdRP) is a key player in RNA silencing, and the polymerase is found in a variety of organisms, including fungi, plants, and worms (2). Although insects and mammals lack sequence homologues of cell-encoded RdRPs, phylogenetic and structural analyses of TERT revealed that TERT is closely related to RdRPs of RNA viruses as well as to retroviral RdDPs (3). In fact, we found that TERT generates dsRNA in a primer-dependent manner and works as an RdRP by a mechanism similar to that for cell-encoded RdRPs (4, 5). Both viral RdRPs and cell-encoded RdRPs transcribe singlestranded RNA (ssRNA) from template RNA, not only in a primer-dependent manner but also in a primer-independent manner. However, primer-independent initiation of RNA synthesis by TERT, a human RdRP, remains to be elucidated.To analyze the characteristics of the RdRP activity of human TERT, we established an in vitro RdRP assay in which we analyzed the RdRP activity of TERT immune complexes immunoprecipitated from cell lysates by use of an anti-human TERT monoclonal antibody (MAb) (IP-RdRP assay) (5). Here we investigated the detailed characteristics of RNAs processed through the IP-RdRP assay. The results indicate that TERT RdRP produces short RNAs in a primer-independent manner. The relationship between TERT protein levels and the RdRP acti...

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“…Mammalian Pol II can bind to a hairpin formed by the noncoding B2 RNA and use the longer strand of the hairpin sequence as a template to extend the short strand by transcription. This extension by Pol II in turn destabilizes the B2 RNA and represents a novel posttranscriptional mechanism for altering the stability of B2 RNA (Wagner et al, 2013). Thus, in addition to its function in pathogen replication, the RdRP activity of DdRPs is emerging as an essential mechanism for regulating gene expression across kingdoms.…”

Section: Introductionmentioning

confidence: 99%

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

Wang

et al. 2015

The Plant Cell

106

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Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (2)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (2)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes.

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RNA polymerase II acts as an RNA-dependent RNA polymerase to extend and destabilize a non-coding RNA

Cited by 52 publications

References 36 publications

6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase

6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase

De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

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