A novel paperclip double-stranded RNA structure demonstrates clathrin-independent uptake in the mosquito Aedes aegypti.

Roohollah Abbasi, Daniel Heschuk, Brandon Kim, Steve Whyard

Insect biochemistry and molecular biology 2020 Dec

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RNA interference (RNAi) has become a widely used technique of knocking down a gene's expression in insects, but its efficacy in some species is limited by a reduced ability of the cells to take in and disperse the double-stranded RNA (dsRNA) throughout the cytoplasm. While RNA transport proteins such as SID-1 and its orthologues can facilitate dsRNA uptake in some invertebrate species, dsRNA uptake in many insects examined to date appears to be facilitated by clathrin-mediated endocytosis (CME). In this study, we used pharmacological inhibitors and RNAi-mediated knockdown of endocytic genes to provide evidence that CME is the primary means of dsRNA uptake in the mosquito Aedes aegypti. Inhibition of clathrin-mediated endocytosis was sufficient to supress uptake of short (21 nt) interfering RNAs (siRNAs), short (23 nt) hairpin RNAs (shRNAs), and long (>200 nt) dsRNA molecules in Aedes aegypti cultured cells and larvae. In contrast, we observed that short (23 nt) "paperclip" RNAs (pcRNAs), with partially closed ends, efficiently enter cells via a clathrin-independent pathway and effectively facilitate transcript knockdown. This alternative dsRNA structure may prove useful in insects generally considered recalcitrant to RNAi and in insect populations where resistance to RNAi-insecticides may arise through changes in dsRNA uptake mechanisms. Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

Citation

Roohollah Abbasi, Daniel Heschuk, Brandon Kim, Steve Whyard. A novel paperclip double-stranded RNA structure demonstrates clathrin-independent uptake in the mosquito Aedes aegypti. Insect biochemistry and molecular biology. 2020 Dec;127:103492