The cleavage of dsRNA by StaufenC into siRNA affects the RNAi efficiency in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata

The efficacy of RNA interference (RNAi) in insects hinges critically on the precise conversion of double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs), which are essential for initiating effective gene silencing. While our recent work identified HvStaufenC as a key regulator of RNAi efficiency in the coleopteran pest Henosepilachna vigintioctopunctata, the underlying mechanism remained unknown. To define HvStaufenC's role, we employed complementary in vitro and in vivo strategies. In vitro, recombinant HvStaufenC protein efficiently processed dsRNA into siRNAs primarily under 35 nucleotides (nt) in length. Crucially, this catalytic activity was completely abolished in a mutant protein harboring disruptions in its essential LNN motif (HvStaufenC-Mutant). In vivo, small RNA sequencing of larvae injected with dsRNA targeting HvTH gene revealed striking differences: wild-type produced siRNAs overwhelmingly enriched for 21-nt species (49.08 % of 19–25-nt siRNAs), characterized by 5’-GAU/UUG/GAA and 3’-UUU/GUU/UUG cleavage motifs. In contrast, HvStaufenC knockout mutants (HvStaufenCKO) showed significantly impaired 21-nt siRNA generation (22.73 %) and exhibited altered cleavage preferences (5’-UUC/GAU/AUU; 3′-GAG/UAC/GGA). Functional validation confirmed the biological significance of these processing differences. Chimeric dsRNA constructs incorporating most abundant wild-type-derived 21-nt siRNA sequences triggered significantly stronger RNAi effects compared to constructs based on sequences derived from HvStaufenCKO mutants. Moreover, no phenotypic changes were observed in HvStaufenCKO 3rd instar larvae following the injection of chimeric dsRNAs. Collectively, these findings demonstrate that HvStaufenC is indispensable for efficient RNAi in H. vigintioctopunctata. It specifically mediates dsRNA cleavage to generate functional 21-nt siRNAs with distinct sequence motifs, providing novel mechanistic insights into RNAi regulation within insects.