RNA polymerase II (Pol II) is classically described as a DNA-dependent RNA polymerase (DdRP) that transcribes all mRNAs within the eukaryotic cell. Curiously, Pol II also possesses a secondary function as an RNA-dependent RNA polymerase (RdRP), using the mouse B2 non-coding RNA as both a template and a substrate for the addition of a distinct 18-nt extension to the B2 RNA 3' end. This extension results in altered stability and function of B2 RNA, suggesting Pol II RdRP activity may modulate RNA metabolism at the post- transcriptional level. While the mechanism and regulation of Pol II DdRP transcription has been extensively studied, little is known about the underlying mechanistic steps of Pol II RdRP 3' extension, the magnitude of RdRP activity within the cell, or the effects of RdRP activity on the stability of RdRP-derived RNAs. The studies proposed here will identify B2 RNA secondary structures that are required for the Pol II RdRP reaction in vitro and within cells. Furthermore, a novel RdRP-sequencing (RdRP-seq) approach will be developed that will allow detection of all cellular RdRP-derived RNAs. After uncovering members of the mammalian RdRPome, the Pol II-specific RdRP-generated RNAs will be identified, and the effects of RdRP activity on RNA half-life will be investigated. These studies will provide valuable insight into the mechanism and biological significance of this unique Pol II RdRP activity.