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bioRxiv · 10.1101/2022.05.31.494195

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

Dissanayaka Mudiyanselage, S. D.·Ma, J.·Pechan, T.·Pechanove, O.·Liu, B.·Wang, Y.
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Abstract

Viroids, a group of plant pathogens, are mysterious subviral agents composed of single-stranded circular noncoding RNAs. Nuclear-replicating viroids exploit host 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 for transcription remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts the minus-strand viroid RNA template to generate plus-strand RNAs. Based on this reconstituted system, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis. We identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, which contrasts to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. This remodeled Pol II is active in transcription with the aid of TFIIIA-7ZF. Interestingly, this remodeled Pol II appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), indicating a different mechanism/machinery regulating RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the transcription complex on RNA templates. We further analyzed the critical zinc finger domains in TFIIIA-7ZF for aiding Pol II activity on RNA template and revealed the first three zinc finger domains pivotal for template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into the evolution of transcription machinery, the mechanism of RNA-templated transcription, as well as viroid replication.

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Dissanayaka Mudiyanselage, S. D., Ma, J., Pechan, T., Pechanove, O., Liu, B., Wang, Y.. 2022-05-31. A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription. https://doi.org/10.1101/2022.05.31.494195

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