A nuclease specific to lepidopteran insects suppresses RNAi

Abstract

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer , we identified a gene, termed, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both and Of note, we found that this up-regulation of expression is faster than that of the gene encoding the key RNAi-associated nuclease knockdown in significantly enhanced RNAi efficiency. Moreover, overexpression in suppressed RNAi efficiency. Finally, knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.

Molecular characterization of DNA methyltransferase 1 and its role in temperature change of armyworm Mythimna separata Walker

“…However, our previous work demonstrated that exogenous dsRNA was sufficient to induce an RNAi effect in M. separata (Wang, Chen, et al, 2018; Wang et al, 2019). Therefore, we attributed this phenomenon to one of the following two reasons: Elder instar larvae were refractory to RNAi due to the activity of dsRNase (Guan et al, 2018); or MsDnmt1 is not sensitive to RNAi, because genes in the central nervous systems are not easily knocked down (Terenius et al, 2011). Obviously, more work needs to be done to illustrate the function of MsDnm1 in M. separata by other up‐to‐date technologies including CRISPR–Cas9 (Jin et al, 2018).…”

Prospects, challenges and current status of RNAi through insect feeding

“…This nuclease had the capability of digesting single‐stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo . The nuclease was renamed by the authors as protein RNAi efficiency–related nuclease ( REase ) . Two nuclease genes exclusively expressed in the gut of Colorado potato beetle were also recently identified and characterized : Ld_dsRNase1 and Ld_dsRNase2 .…”

“…In addition, exposure through diets generally requires large amounts of dsRNA to trigger RNAi effects. Sometimes those effects are highly variable even among the same species . In addition, some level of degradation can also occur before ingestion, since dsRNases can also be present in the food.…”

Gene silencing by RNAi via oral delivery of dsRNA by bacteria in the South American tomato pinworm, Tuta absoluta

“…Species of Lepidoptera are known to be highly recalcitrant to gene silencing by dsRNA . In Lepidoptera, the alkaline pH of the larvae gut and the action of RNaseIII have been indicated as possible causes for a lack of sensitivity to dsRNA treatment in this order, due to the degradation of supplied dsRNA . It has also been hypothesized that the presence of virus in insect hemolymph might be associated with the recalcitrance of Lepidoptera to RNAi, possibly by saturating the RNAi core machinery .…”

“…It has also been hypothesized that the presence of virus in insect hemolymph might be associated with the recalcitrance of Lepidoptera to RNAi, possibly by saturating the RNAi core machinery . A recently described novel exonuclease, denominated ‘RNAi‐efficiency related nuclease’ (REase), has been proposed as one of the mechanisms responsible for the recalcitrance of Lepidoptera to RNAi . The REase gene is up‐regulated by exposure to dsRNA and the enzyme is able to digest single and double stranded RNA in Ostrinia furnacalis , with homologous sequences found in seven other lepidopteran species.…”