Reorganizing the RNA polymerase II complex for replication of an infectious noncoding RNA in vivo

DNA-dependent RNA polymerases (DdRPs) recognize not only DNA but also RNA templates. This RNA-dependent RNA polymerase (RdRP) activity is exploited by viroids in plants and human hepatitis delta virus in animals. A major knowledge gap exists regarding the molecular basis conferring this RdRP activity. Here, we provide evidence supporting the reorganization of the 12-subunit Pol II to 7-subunit in vivo for PSTVd transcription. Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9 are not involved in PSTVd transcription in planta. A splicing variant of transcription factor IIIA with seven zinc finger domains (TFIIIA-7ZF) aids the remodeled Pol II in transcribing PSTVd. Using AlphaFold3, the structure of the remodeled Pol II with PSTVd RNA and TFIIIA-7ZF was generated. The predicted structure and experimental data both show that the N-terminus of TFIIIA-7ZF binds to the left terminal domain of PSTVd, while the C-terminus interacts with Rpb2. Interestingly, AlphaFold3 also predicts the bending at PSTVd loop 8 in the TFIIIA-7ZF/PSTVd complex. Replacing this loop 8 with a rigid double-stranded conformation impairs the TFIIIA-7ZF/PSTVd interaction. Altogether, our demonstrate an active form of heterogenous organization of the essential Pol II enzyme in vivo and provide structural insights into the organization of Pol II transcription complex on RNA template.