Background

RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation.

Methods

Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced.

Results

Depleting 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 obtained siRNAs mapped 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 preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals.

Conclusions/general significance

The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding 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.

中文翻译：

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缺失Argonaute 2的德国小att中的siRNA富集

背景

RNA干扰（RNAi）是一种用于抵抗各种威胁的细胞机制，包括转座子，异常RNA和某些类型的病毒。这种机制依赖于dsRNA分子的检测，该分子通过涉及Dicer-2（Dcr-2）和Argonaute 2（AGO2）的途径产生与结合RNA结合的小干扰RNA（siRNA），触发它们的降解。

方法

使用蟑螂德国小Bl作为模型，我们通过使用RNAi消耗其mRNA并分析产生的表型来检查AGO2的活性。

结果

耗尽AGO2表达对若虫发育或繁殖没有显着影响。dsRNA处理独立于Dcr-2的作用触发了AGO2表达的立即和短暂增加。另外，我们分析了在对照和AGO2缺乏动物中注射异源dsRNA后产生的siRNA 。结果表明，获得的siRNA沿dsRNA序列分布不均匀。在消耗AGO2的动物中，22个核苷酸读数的比例更高，并且读数的积累出现在对照中代表性较差的区域。我们还检测到对胞嘧啶的偏爱，因为它是对照组中的第一个核苷酸，在缺失AGO2的个体中显着减弱。

结论/一般意义

从dsRNA产生的siRNA沿着dsRNA的长度异质定位，这种排列取决于dsRNA序列。AGO2在siRNA双链体上发挥其核酸酶作用，独立于其对相应mRNA的作用。这项研究除了设计出更有效，更具体，更环保的害虫控制策略外，还为实验室反向遗传学提供了极为有用的流程。