Transposon Defense by Endo-siRNAs, piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

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 activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during 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 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

Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster

Small interfering RNAs (siRNAs) defend the organism against harmful transcripts from exogenous (e.g. viral) or endogenous (e.g. transposons) sources. Recent publications describe the production of siRNAs induced by DNA double-strand breaks (DSB) in Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster and human cells, which suggests a conserved function. A current hypothesis is that break-induced small RNAs ensure efficient homologous recombination (HR). However, biogenesis of siRNAs is often intertwined with other small RNA species, such as microRNAs (miRNAs), which complicates interpretation of experimental results. In Drosophila, siRNAs are produced by Dcr-2 while miRNAs are processed by Dcr-1. Thus, it is possible to probe siRNA function without miRNA deregulation. We therefore examined DNA double-strand break repair after perturbation of siRNA biogenesis in cultured Drosophila cells as well as mutant flies. Our assays comprised reporters for the single-strand annealing pathway, homologous recombination and sensitivity to the DSB-inducing drug camptothecin. We could not detect any repair defects caused by the lack of siRNAs derived from the broken DNA locus. Since production of these siRNAs depends on local transcription, they may thus participate in RNA metabolism-an established function of siRNAs-rather than DNA repair

Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster

Small interfering RNAs (siRNAs) defend the organism against harmful transcripts from exogenous (e.g. viral) or endogenous (e.g. transposons) sources. Recent publications describe the production of siRNAs induced by DNA double-strand breaks (DSB) in Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster and human cells, which suggests a conserved function. A current hypothesis is that break-induced small RNAs ensure efficient homologous recombination (HR). However, biogenesis of siRNAs is often intertwined with other small RNA species, such as microRNAs (miRNAs), which complicates interpretation of experimental results. In Drosophila, siRNAs are produced by Dcr-2 while miRNAs are processed by Dcr-1. Thus, it is possible to probe siRNA function without miRNA deregulation. We therefore examined DNA double-strand break repair after perturbation of siRNA biogenesis in cultured Drosophila cells as well as mutant flies. Our assays comprised reporters for the single-strand annealing pathway, homologous recombination and sensitivity to the DSB-inducing drug camptothecin. We could not detect any repair defects caused by the lack of siRNAs derived from the broken DNA locus. Since production of these siRNAs depends on local transcription, they may thus participate in RNA metabolism-an established function of siRNAs-rather than DNA repair

Length distribution of transposon-matching small RNAs identified in this study.

<p>A) Reads of each library originating from soma and germline were mapped to the reference containing a transposon sequence collection. Transposon matching small RNAs were analyzed for their size distribution and normalized to total genome matching reads. The normalized counts were expressed as reads per thousand (RPT). B) The steady state transcript levels of <i>297, TNFB, roo</i> and <i>blood</i> transposable elements were examined by qRT-PCR. RNA was isolated from three biological replicates of heterozygous <i>loqs-D</i> and <i>r2d2</i> mutants separated in somatic and germline tissue, respectively. The <i>doc, 412</i> and <i>copia</i> transposons were included for comparison. Ct-values for each transposon were normalized to the <i>rp49</i> control (delta Ct). Values are mean ± SD (n = 3). C) The length distribution of transposon matching small RNAs in <i>r2d2</i> and <i>loqs-D</i> mutants after exclusion of <i>roo</i>, <i>297</i>, <i>TNFB</i> and <i>blood</i> transposons.</p

The counts of <i>297, TNFB, roo</i> and <i>blood</i> matching small RNAs.

<p>The counts of <i>297, TNFB, roo</i> and <i>blood</i> matching small RNAs.</p

Orientation bias for pilRNAs in soma and piRNAs in germline.

<p>Small RNA libraries generated with β-eliminated RNA samples were mapped to the transposon sequence collection. The RPM for sense (+) and antisense (–) transposon matching small RNAs for 23 nt to 29 nt were depicted for soma (A) and germline (B) to demonstrate the orientation bias. Note that the apparent increase of somatic pilRNAs is due to the removal of certain miRNAs and endo-siRNAs in homozygous mutants, which are either less efficiently produced and/or mis-directed into Ago1. Upon β-elimination, these RNAs no longer contribute to the sequenced pool, hence other RNA classes appear to be more abundant. We did not observe this increase if untreated libraries were analyzed.</p

Analysis of steady state level of transposons by qRT-PCR.

<p>RNA was isolated from heterozygous and homozygous <i>r2d2</i> and <i>loqs-D</i> mutants. DNA was digested with DNase I, the RNA was reverse transcribed and used for transposon profiling by qRT-PCR. Each transposon was normalized to the average of <i>rp49</i> and <i>gapdh</i> controls and depicted as the fold change of homozygous to heterozygous mutant in soma and germline, respectively (p<0.05(*), p<0.009(**) student’s T-test, n = 3).</p

Changes in processing and loading of small RNAs matching to individual transposons in <i>r2d2</i> and <i>loqs-D</i> mutants.

<p>Transposon mapping endo-siRNA were normalized to total genome matching reads and expressed as reads per million (RPM). Each dot in the plot represents an individual transposon consensus sequence. The upper two panels compare of heterozygous <i>r2d2</i> and <i>loqs-D</i> mutants during processing (left) and loading (right) for soma and germline. The lower panels compare homozygous <i>r2d2</i> with homozygous <i>loqs-D</i> mutants. For example, a higher amount of endo-siRNAs in <i>r2d2</i> homozygous mutant than in <i>loqs-D</i> homozygous mutants means that these endo-siRNAs are <i>r2d2</i> independent but <i>loqs-D</i> dependent. They are thus situated below the diagonal, whereas transposons that require <i>loqs-D</i> but not <i>r2d2</i> will fall above the diagonal.</p

Analysis of strand asymmetry and 5’-nucleotide preference in deep sequencing data.

<p>A) The thermodynamic asymmetry was calculated for transposon mapping endo-siRNAs of the indicated genotypes. We calculated the difference in free energy of duplex formation at either end of the presumed siRNA precursor for each sequence read using the nearest neighbor method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084994#pone.0084994-Xia1" target="_blank">[51]</a>, then calculated the average difference (ΔΔG0'). A positive value indicates that on average the 5’ ends of the reads are less stably base paired than the opposite ends. B) The relative frequency for each nucleotide at the 5’-end is depicted as a function of genotype and RNA treatment.</p