Drosophila endogenous small RNAs bind to Argonaute2 in somatic cells

RNA silencing is a conserved mechanism in which small RNAs trigger various forms of sequence specific gene silencing by guiding Argonaute complexes to target RNAs via base-pairing. RNA silencing is thought to have evolved as a form of nucleic-acid-based immunity to inactivate viruses and transposable elements (TEs). Although the activity of TEs in animals has been thought to be largely restricted to the germline, recent studies have shown that they may also actively transpose in somatic cells, creating somatic mosaicism in animals3. In the Drosophila germline, piRNAs arise from repetitive intergenic elements including retrotransposons by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. Here we show that in Drosophila cultured S2 cells, Argonaute2 (AGO2), an AGO subfamily member of Argonautes, associates with endogenous small RNAs of 20-22 nucleotides in length, which we have collectively named esiRNAs (endogenous siRNAs). esiRNAs can be divided into two groups: one that mainly corresponds to a subset of retrotransposons, and the other that arises from stem-loop structures. esiRNAs are produced in a Dicer-2-dependent manner from distinctive genomic loci, are modified at their 3’ ends and can direct AGO2 to cleave target RNAs. Depletion of Dicer2 or AGO2 in S2 cells coincided with higher levels of retrotransposon transcripts. Together, our findings indicate that in Drosophila, different types of small RNAs and Argonautes are utilized to repress retrotransposons in germline and somatic cells. Keywords: AGO2, small RNA 454 sequencing - high throughput Cloning of small RNAs associated with AGO2 in S2 cells and deep sequence analysis. Presented here is a list of non-redundant AGO2-associated small RNAs.

RNA沉默（RNA silencing）是一种保守的生物学机制，小型RNA通过碱基配对引导Argonaute复合物（Argonaute complexes）靶向靶RNA，从而介导多种序列特异性基因沉默过程。学界普遍认为，RNA沉默演化形成了基于核酸的免疫形式，用于灭活病毒与转座因子（transposable elements, TEs）。尽管此前的研究认为动物体内的转座因子活性主要局限于生殖系，但近期研究表明，这类因子亦可在体细胞中活跃转座，进而在动物体内引发体细胞嵌合现象³。

在果蝇生殖系中，Piwi互作RNA（Piwi-interacting RNA，简称piRNAs）由包括逆转座子在内的重复基因间元件通过不依赖Dicer的通路生成，并通过Argonaute蛋白的PIWI亚家族发挥功能，以确保逆转座子的沉默。本研究在果蝇培养的S2细胞中发现，Argonaute蛋白亚家族成员Argonaute2（AGO2）可与长度为20~22个核苷酸的内源性小型RNA结合，我们将这类RNA统称为内源性小干扰RNA（endogenous siRNAs，简称esiRNAs）。esiRNAs可分为两类：一类主要对应部分逆转座子序列，另一类则源自茎环结构。esiRNAs由特定基因组位点以依赖Dicer-2的方式生成，其3'端存在修饰，且能够引导AGO2切割靶RNA。在S2细胞中敲低Dicer-2或AGO2的表达，会伴随逆转座子转录本水平的显著升高。

综上，本研究结果表明，在果蝇中，不同类型的小型RNA与Argonaute蛋白分别在生殖系与体细胞中发挥抑制逆转座子的作用。

关键词：AGO2、小型RNA、454高通量测序（454 sequencing）——本研究通过高通量克隆S2细胞中与AGO2结合的小型RNA并开展深度测序分析，本文呈现了一组非冗余的AGO2结合小型RNA列表。