Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells

Abstract

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.

Fig. 1

High-throughput pyrosequencing revealed 3′-terminally modified 21-nt RNAs in the fly soma. (A) Length and sequence composition of the small RNA sequences from a library of total small RNA from the heads of flies expressing an inverted repeat (IR) silencing the white gene and for a parallel library enriched for RNAs modified at their 3′ ends. (B) Similar analysis for small RNA sequences from Drosophila S2 cells. For data labeled “without miRNAs,” pre-miRNA–matching sequences were removed computationally.

Fig. 2

Endo-siRNAs correspond to transposons. (A) Distribution of annotations for the genomic matches of endo-siRNA sequences. Bars total more than 100% because some siRNAs match both LTR and non-LTR retrotransposons or match both mRNA and transposons. (B) Transposon-derived siRNAs with more than 50 21-nt reads mapped about equally to sense and antisense orientations. (C) Alignment of endo-siRNA sequences to Drosophila transposons. The abundance of each sequence is shown as a percentage of all transposon-matching siRNA sequences. LTR, long terminal repeat; TIR, terminal inverted repeat. Here and in subsequent figures, data from high-throughput pyrosequencing and sequencing-by-synthesis were pooled for wild-type heads.

Fig. 3

Transposon silencing requires Dcr-2 and Ago2, but not Dcr-1. (A and B) The change in mRNA expression (mean ± SD, N = 3) for each transposon between dcr-2^L811fsX (A) or ago2⁴¹⁴ (B) heterozygous and homozygous heads was measured by quantitative reverse transcription polymerase chain reaction. The data were corrected for differences in transposon copy number between the paired genotypes. (C) The change in transposon expression (mean ± SD, N = 3) in S2 cells was measured for the indicated RNAi depletion relative to a control dsRNA.

Fig. 4

The composition of somatic small RNAs is altered in the absence of Ago2. (A and B) Size distribution (A) and sequence composition (B) of sequences from a library of total 18- to 29-nt RNA from the heads of ago2 null mutant flies or a library enriched for 3′-terminally modified RNAs. Reads matching pre-miRNA sequences were removed. (C) Distribution of annotations for the genomic matches of small RNA sequences from the two ago2 libraries.