Abstract
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 (-)-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 (-)-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.
Highlights
Besides their canonical role in DNA-templated RNA transcription, some DNA-dependent RNA polymerases (DdRPs) have an RNAdependent RNA polymerase (RdRP) activity
We further show using various biochemical, molecular, and subcellular localization studies that the shorter variant TRANSCRIPTION FACTOR IIIA (TFIIIA)-7ZF acts as a genuine transcription factor that directly guides Pol II-catalyzed transcription using Potato spindle tuber viroid (PSTVd) RNA genome as a template in plants
Several lines of evidence support the role of Pol II in PSTVd replication (Flores et al, 2005; Ding, 2009), the most direct physical evidence showing the binding of Pol II to the (+)- and (2)-strands of PSTVd in vivo has been elusive
Summary
Besides their canonical role in DNA-templated RNA transcription, some DNA-dependent RNA polymerases (DdRPs) have an RNAdependent RNA polymerase (RdRP) activity. High-resolution crystallographic structures revealed that yeast Pol II uses the same site for binding to DNA and RNA templates, leading to the hypothesis that the RdRP activity of a DdRP may represent a missing link in molecular evolution; a DdRP may have evolved first to use an RNA before switching to a DNA template (Lehmann et al, 2007) This relic RdRP activity of a DdRP has persisted in modern life forms in catalyzing the transcription of some noncoding RNA templates to regulate gene expression and is part of the reprogramming by noncoding (viroids) and limited-coding (HDV) RNA pathogens vital for their replication.
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