Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in

In many eukaryotes, RNA-dependent RNA poly-merases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recogni-tion and processing of aberrant transcripts trigger-ing the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mecha-nisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, trun-cated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs sugges-tive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth