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

• Published in: PLoS pathogens Vol. 18; no. 9; p. e1010850
• Main Authors: Dissanayaka Mudiyanselage, Shachinthaka D, Ma, Junfei, Pechan, Tibor, Pechanova, Olga, Liu, Bin, Wang, Ying
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.09.2022; Public Library of Science (PLoS)
• Subjects: Analysis; Bacteria, Phytopathogenic; Biology and Life Sciences; Circular RNA; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA-directed RNA polymerase; Elongation; Genetic aspects; Genetic transcription; Genomes; Identification and classification; Intermediates; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Mutation; Mutation rates; Physical Sciences; Proteins; Ribonucleic acid; RNA; RNA - metabolism; RNA polymerase; RNA polymerase II; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA polymerases; RNA, Circular - genetics; RNA-Dependent RNA Polymerase - genetics; RNA-directed RNA polymerase; Structure; TFIID protein; Transcription elongation; Transcription Factor TFIIA - genetics; Transcription Factor TFIIA - metabolism; Transcription Factor TFIIB - genetics; Transcription Factor TFIID - genetics; Transcription Factor TFIID - metabolism; Transcription Factor TFIIIA - metabolism; Transcription factors; Transcription Factors, General - genetics; Transcription Factors, General - metabolism; Transcription initiation factor TFIIA; Transcription initiation factor TFIIB; Transcription, Genetic; Viroids; Viroids - genetics; Viroids - metabolism; Zinc; Zinc finger proteins; United States
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1010850

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.