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

Authors

Bradley P Klemm, Andrew P Sikkema, Emily R Schugardt, Traci M Tanaka Hall

Published in

Nucleic acids research. Volume 54. Issue 14. Jul 17, 2026.

Abstract

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.

PMID:
42475678
Bibliographic data and abstract were imported from PubMed on 21 Jul 2026.

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