siRNA enrichment in Argonaute 2-depleted Blattella germanica

Abstract

Background RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNA, and some types of viruses. It relies on dsRNA detection and, through a mechanism involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), together with a small RNA fragment (siRNA) as a complementary guide, binds to an RNA and cleaves it. Methods Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA levels using RNAi and analyzing the phenotypes produced. Results Silencing AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that siRNAs were distributed non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a slight preference for cytosine as the first nucleotide in controls to be lost in AGO2-depleted individuals. Conclusions/General significance The siRNAs produced from a dsRNA are heterogeneously distributed along the length of the dsRNA and this arrangement depends on the sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the homologous mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies. Highlights - RNA interference is used to fight cellular threats including transposons and viruses - Silencing AGO2 in Blattella germanica does not affect development or reproduction - siRNA distribution along the dsRNA sequence is non-uniform and sequence dependent - AGO2 depletion increased the proportion of 22 nt reads in certain areas