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

Area of Science:

• Molecular Biology
• Plant Pathology
• Virology

Background:

• Viroids are subviral agents consisting of single-stranded circular noncoding RNAs.
• Nuclear-replicating viroids use DNA-dependent RNA polymerase II (Pol II) for transcription from their RNA genome.
• The mechanism of Pol II utilizing RNA templates is not well understood.

Purpose of the Study:

• To investigate the mechanism of viroid RNA-templated transcription by Pol II.
• To characterize the Pol II complex involved in viroid RNA replication.
• To elucidate the role of transcription factors in viroid transcription.

Main Methods:

• Reconstitution of an in vitro transcription system.
• Purification of the Pol II complex using nano-liquid chromatography-tandem mass spectrometry.
• Analysis of TFIIIA-7ZF zinc finger domains.

Main Results:

• Pol II can accept minus-strand viroid RNA as a template to generate plus-strand RNAs.
• A remodeled Pol II complex, lacking subunits Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, was identified.
• This remodeled Pol II requires TFIIIA-7ZF but not canonical general transcription factors or elongation factors.
• The absence of Rpb9 correlates with the higher mutation rate of viroids.

Conclusions:

• A distinct Pol II organization facilitates viroid RNA-templated transcription.
• The findings offer new insights into viroid replication and transcription machinery evolution.
• The study reveals the critical role of the first three zinc finger domains of TFIIIA-7ZF in RNA template binding.