The in vivo dsRNA Cleavage Has Sequence Preference in Insects

Abstract

Exogenous dsRNA enters the insect body and can induce the RNAi effect only when it is cleaved into siRNA. However, what kinds of base composition are easier to cut and what kinds of siRNA will be produced in vivo is largely unknown. In this study, we found that dsRNA processing into siRNA has sequence preference and regularity in insects. We injected 0.04 mg/g dsRNA into Asian corn borers or cotton bollworms according to their body weight, and then the siRNAs produced in vivo were analyzed by RNA-Seq. We discovered that a large number of siRNAs were produced with GGU nucleotide residues at the 5′- and 3′-ends and produced a siRNA peak on the sequence. Once the GGU site is mutated, the number of siRNAs will decrease significantly and the siRNA peak will also lost. However, in the red flour beetle, a member of Coleoptera, dsRNA was cut at more diverse sites, such as AAG, GUG, and GUU; more importantly, these enzyme restriction sites have a high conservation base of A/U. Our discovery regarding dsRNA in vivo cleavage preference and regularity will help us understand the RNAi mechanism and its application.

Strategies for enhancing the efficiency of RNA interference in insects

“…The mechanisms that cause the variability in the sensitivity of different portions of a target gene to RNAi are not entirely clear. However, in some insects, researchers have shown that cleavage of dsRNAs can be affected by their sequence 25 . In Asian corn borer ( Ostrinia furnacalis ) and cotton bollworm ( Helicoverpa armigera ), injected dsRNAs tended to produce siRNAs specifically cut at GGU nucleotide sequences, and dicing was reduced if these consensus sites were removed 25 .…”

Prospects, challenges and current status of RNAi through insect feeding

“…This knowledge might be useful to predict functional siRNA fragments that result from a specific dsRNA sequence. Thus, we can achieve higher RNAi effects by incorporating these sites into dsRNA …”

“…Thus, we can achieve higher RNAi effects by incorporating these sites into dsRNA. 123,124 Length of dsRNA: Natural uptake of dsRNAs is length dependent and researchers have tested dsRNA molecules ranging from 50 bp to 2000 bp, which generates around 10-25 siRNA fragments after Dicer processing. However, 200-550 bp are more effective in triggering RNAi effects.…”

Knockout of the HaREase Gene Improves the Stability of dsRNA and Increases the Sensitivity of Helicoverpa armigera to Bacillus thuringiensis Toxin

“…However, RNAi efficiency is relatively low in lepidopteran insects and this is a core factor that frustrates the application of this technology (Terenius et al, 2011; Cooper et al, 2019). Many factors affect RNAi efficiency in insects, including the absence of RNA-dependent RNA polymerase (RdRP)-mediated synthesis of secondary small interfering RNAs (siRNAs) (Sijen et al, 2001), the rate of dsRNA processing into siRNAs (Guan et al, 2018a), the dsRNA uptake and transport mechanism (Luo et al, 2012), and the degradation rate of dsRNA (Christiaens et al, 2014; Spit et al, 2017; Song et al, 2019).…”