Initiation by a eukaryotic RNA-dependent RNA polymerase requires looping of the template end and is influenced by the template-tailing activity of an associated uridyltransferase.

A conserved family of eukaryotic RNA-dependent RNA polymerases (RDRs) initiates or amplifies the production of small RNAs to provide sequence specificity for gene regulation by Argonaute/Piwi proteins. RDR-dependent silencing processes affect the genotype-phenotype relationship in many eukaryotes, but the principles that underlie the specificity of RDR template selection and product synthesis are largely unknown. Here, we characterize the initiation specificity of the Tetrahymena RDR, Rdr1, as a heterologously expressed single subunit and in the context of its biologically assembled multisubunit complexes (RDRCs). Truncation analysis of recombinant Rdr1 revealed domain requirements different from those of the only other similarly characterized RDR, suggesting that there are subfamilies of the RDR enzyme with distinct structural requirements for activity. We demonstrate an apparently obligate Rdr1 mechanism of initiation in which the template end is looped to provide the hydroxyl group priming the synthesis of dsRNA. RDRC subunits with poly(U) polymerase activity can act on the template end prior to looping to increase the duplex length of product, thus impacting the small RNA sequences generated by the RDRC-coupled Dicer. Overall, our findings give new perspective on mechanisms of RDR initiation and demonstrate that non-RDR subunits of an RDRC can affect the specificity of product synthesis.