RISC-bound small RNA sequencing provides insights into guide-strand selection and siRNA trimming and tailing following insecticidal dsRNA delivery

Abstract

RNA interference (RNAi) offers a sequence-specific approach to pest control. In insects, Dicer-2 processes double-stranded RNA (dsRNA) into small interfering RNA (siRNA) duplexes, from which the RNA-induced silencing complex (RISC) retains a guide strand. However, how the sequence of delivered dsRNA shapes the RISC-bound siRNA pool in insect pests remains poorly understood, limiting opportunities for sequence optimization. Here, we profiled RISC-bound siRNAs following injection of 34 insecticidal dsRNAs targeting 11 essential genes in Tribolium castaneum larvae. We computationally reconstructed 7,879 siRNA duplexes and examined associations between sequence features and strand representation. Differences in GC identity at terminal paired positions 1–5, used as a proxy for local pairing asymmetry, correlated with strand bias, with the strongest correlations at the first two paired positions. ORF targeting and reduced predicted antisense self-folding were also associated with higher antisense fractions. Analysis of non-templated terminal additions revealed predominantly 3′ uridylation. Among ORF-associated siRNA pairs, sense strands showed higher ratios of U-tailed and putatively trimmed/U-tailed reads to perfect 21-nt reads than did antisense strands. Antisense strands with the least predicted self-folding also showed low modification ratios. These observations are consistent with contributions from guide-strand selection and differential post-loading modification but do not distinguish initial loading from subsequent turnover or directly demonstrate siRNA degradation. The identified associations provide a basis for testing improved predictions of dsRNA-derived RISC-bound siRNA pools and their relevance to pest-control efficacy and off-target activity. Graphical abstract Highlights RISC-bound siRNAs were profiled after delivery of 34 dsRNAs in T. castaneum. Terminal paired positions 1–5 correlate with siRNA strand representation. ORF targeting and reduced self-folding correlate with higher antisense fractions. ORF-associated sense siRNAs show higher uridylation/trimming ratios.