Transcription is a major step governing the genetic flow from genome to phenotype. It has been known for a long time that some DNA-dependent RNA polymerases can catalyze transcription using both DNA and RNA templates, but the underlying mechanism is unclear. Nuclear-replicating viroids employ DNA-dependent RNA polymerase II (Pol II) for replication, a process that makes a DNA-dependent enzyme to recognize RNA templates. Using potato spindle tuber viroid (PSTVd)-replication as a model, we identified a splicing variant of transcription factor IIIA (TFIIIA-7ZF) that is critical for Pol II to transcribe PSTVd RNA templates through a combination of multiple molecular and biochemical assays. Mutational and biochemical analyses revealed essential zinc finger domains as the functional sites. In addition, PSTVd directly interacts with a splicing regulator (RPL5) of TFIIIA transcripts to favor the expression of TFIIIA-7ZF, thereby enhancing PSTVd replication. These studies provide new insights into RNA-templated transcription by Pol II and have significant implications for exploring functional mechanisms of RNA-templated transcription in the future. (Dedicated to the memory of the late Professor Biao Ding at the Ohio State University)