Intrinsic catalytic parameters drive specificity of non-templated poly(UG) tail synthesis.

RNA interference (RNAi) in nematodes is amplified through the generation of secondary small interferring RNA (siRNA) from products of primary siRNA cleavage. This process requires RDE-3, a unique ribonucleotidyltransferase that adds a poly(UG) tail of alternating U and G nucleotides without a template. Here we demonstrated using in vitro enzymatic assays that RDE-3 is intrinsically specific for substrate combinations that correctly extend the pUG tail and optimized for effective pUGylation in vivo. Specificity for cognate substrate pairs (3'-G RNA with UTP or 3'-U RNA with GTP) was driven primarily by a faster turnover rate, whereas non-cognate GG or UU extensions were dramatically slower. RDE-3 could also extend 3'-A or 3'-C RNA substrates with GTP or UTP, allowing it to initiate pUGylation of primary RNAi products, but at slower rates and with little GTP/UTP preference. We established an assay where products of both 3'-G and 3'-U RNA substrates in a reaction with GTP and UTP were followed simultaneously. We found that pUG extension was optimal and most accurate under conditions where GTP/UTP concentrations corresponded to their relative KMNTP values and typical cellular conditions.