Argonaute-2 role in dsRNA processing in cockroaches

Abstract

Interference of RNA (RNAi) is a cellular process that allows the cell to protect itself against harmful agents, especially alien RNA. Through the treatment with double-stranded RNA (dsRNA), this mechanism is being used to perform specific knockdown of the expression of a given gene, in order to do laboratory functional genetic studies or even to control insect pests. The aim of the present work is to study an aspect of the functioning of RNAi in insects, in particular, to determine the functions the protein Argonaute-2 (Ago2). Using the cockroach Blattella germanica as a model, specimens treated with dsRNA against Ago2 (dsAgo2), and control, were subsequently treated with a heterologous dsRNA (dsPolyh). Four days after the second treatment, libraries of small RNAs were obtained from control and dsAgo2 depleted specimens. The small interfering RNAs (siRNAs) corresponding to dsPolyh were analyzed. siRNAs found in the libraries were not equally distributed along the whole length of both RNA strands from the dsRNA injected, but enriched in certain areas. This unequal distribution was similar both in Control- and dsAgo2-treated specimens. Nevertheless, dsAgo2 samples contained 4 times more 22 nt siRNAs (the most usual size in the libraries) than Control samples. The unequal distribution of siRNAs along a heterologous dsRNA sequence indicates that there is a selection of siRNAs independent of their use in the process of degradation of the target RNA. The similar distribution of siRNAs in control and dsAgo2 samples indicates that Ago2 is not the protein responsible for doing siRNA selection. Nevertheless, the higher amount of 22 nt siRNAs in dsAgo2-treated specimens shows Ago2 nuclease activity on ¿mature¿ siRNAs.