An evolutionarily conserved N-terminal domain of RRF-3 governs GTSF-1 binding in nematodes.

GTSF1 is an essential activating cofactor for PIWI proteins in many metazoans. In the nematode Caenorhabditis elegans, however, GTSF-1 does not bind PIWI, but is associated with the RNA-dependent RNA polymerase RRF-3, supporting endo-siRNA (26G-RNA) biogenesis. Here, we demonstrate that this rewiring is deeply conserved across nematodes. For C. briggsae and Pristionchus pacificus, we show that GTSF-1 interacts with RRF-3 and is essential for 26G-RNA production and fertility. We map this interaction to an N-terminal domain of RRF-3, termed the GTSF-1 interacting domain (GID), and show that the GTSF-1 zinc finger region alone is sufficient for binding. Mutagenesis identifies critical residues mediating this interaction and reveals that GTSF-1 stability depends on RRF-3. Other RdRPs possess GID-like domains, which we propose to bind GTSF-1-related proteins. Phylogenomic and structural analyses support GTSF-1-RRF-3 interactions across all major nematode lineages and map the shift in GTSF-1 activity to the last common nematode ancestor. We propose that binding of GTSF-1 induces conformational changes in RRF-3 that facilitate RdRP complex assembly and activate its function, paralleling its role as a PIWI activator.