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Drosophila endogenous small RNAs bind to Argonaute2 in somatic cells

ABSTRACT: 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

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE10277 | GEO | 2008/05/08

SECONDARY ACCESSION(S): PRJNA108475

REPOSITORIES: GEO

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Project description:Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing mechanisms that repress TEs expression: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2 that primarily helps to direct siRNAs for loading into Ago2. We provide deep sequencing evidence consistent with the idea that R2D2 and Loqs-PD can function in part redundantly. Certain transposons display a preference for either dsRBD-protein for production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in the germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway. small RNA sampling experiment; small RNAs were prepared from head & thorax as well as dissected ovaries of Adult female Drosophila melanogaster. We used homozygous mutants of the dsRBD proteins Loqs and r2d2 to determine their contribution to the biogenesis of transposon-derived small RNAs. Heterozygous mutant animals served as control. For each RNA sample, we performed one deep-sequencing run without any treatment, and in parallel one sequencing run after periodate oxidation and beta-elimination. After this treatment, only Ago2, Piwi, Aub and Ago3-loaded small RNAs remain as they carry a 2'-O-methyl modification at their 3'-end. This helps to determine the loading status of the small RNAs detected. In total 8 different RNA samples were prepared and 16 libraries were sequenced.

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Drosophila endogenous small RNAs bind to Argonaute2 in somatic cells

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