Key factors determining variations in RNA interference efficacy mediated by different double‐stranded RNA lengths in Tribolium castaneum

Abstract

Double‐stranded RNA (dsRNA) length may affect RNA interference (RNAi) efficacy. Herein, variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum. Through in vitro experiments with T. castaneum midgut, dsRNA accumulation in the midgut, degradation by midgut homogenates and persistence in haemolymph after injection were tested to determine the causes of RNAi efficacy variation. The comparative efficacies of dsRNAs were 480 bp ≈ 240 bp > 120 bp > 60 bp >> 21 bp. The combined midgut dsRNA accumulation and midgut homogenate‐induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning, but was likely not the main determinant of the variation in longer dsRNAs’ (≥60 bp) bioactivity. In vitro RNAi experiment with T. castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes, suggesting little variation in intracellular RNAi machinery’s affinity for different dsRNA lengths. In vivo haemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. In addition, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of haemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length.

Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

“…A similar relationship between the length of the dsRNA and RNAi efficacy was observed in other organisms 35,36,53,54 , however, the length of dsRNA required for efficient RNAi differs. For example, unlike in T. urticae, dsRNAs of 100-200 bp in length induce highly efficient RNAi in Tribolium 36,55 , western corn rootworm 53,54 , Drosophila S2 cells 47 and the nematode C. elegans 35 . However, while in T. urticae the in vivo effects of dsRNAs of different length on RNAi efficacy tightly correlated with the processivity of dsRNA by Dicer in vitro assay, Fig.…”

Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

“…A similar relationship between the length of the dsRNA and RNAi efficacy was observed in other organisms 35,36,53,54 , however, the length of dsRNA required for efficient RNAi differs. For example, unlike in T. urticae, dsRNAs of 100-200 bp in length induce highly efficient RNAi in Tribolium 36,55 , western corn rootworm 53,54 , Drosophila S2 cells 47 and the nematode C. elegans 35 . However, while in T. urticae the in vivo effects of dsRNAs of different length on RNAi efficacy tightly correlated with the processivity of dsRNA by Dicer in vitro assay, Figure 2D and E, in Drosophila S2 cells, the length of dsRNA affected its cellular uptake and not the processivity 47 , indicating differences in the RNAi machinery.…”

Double-Stranded RNA-Degrading Enzymes Reduce the Efficiency of RNA Interference in Plutella xylostella

“…The dsRNA degradation [2][3][4] or the inability of dsRNA to enter the cytoplasm [3,5,6] are the main factors affecting RNAi efficiency. Studies have also shown that dsRNA needs to last long enough in the midgut or hemolymph to be absorbed into cells to produce an effective RNAi response [7]. DsRNases are the key factors affecting the stability of dsRNA in insects.…”