Endogenous RNA interference provides a somatic defense against Drosophila transposons

Abstract

Background: Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are approximately 24-30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity.

Results: Here, we describe a second system that represses Drosophila TEs by using endogenous small interfering RNAs (siRNAs), which are 21 nucleotide, 3'-end-modified RNAs that are dependent on Dicer-2 and Argonaute-2. In contrast to piRNAs, we find that the TE-siRNA system is active in somatic tissues, and particularly so in various immortalized cell lines. Analysis of the patterns and properties of TE-derived small RNAs reveals further distinctions between TE regions and genomic loci that are converted into piRNAs and siRNAs, respectively. Finally, functional tests show that many transposon transcripts accumulate to higher levels in cells and animal tissues that are deficient for Dicer-2 or Argonaute-2.

Conclusions: Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs).

Figure 1. Drosophila Transposable Elements Generate Two Classes of Small RNAs with Distinct Size and Expression Characteristics

The inset tabulates the number of total reads, miRNA reads, and TE reads in each set of libraries; only the TE reads were considered in the main graph. Analysis of all transposon-derived small-RNA reads from all libraries (light blue dataset) reveals a 21 peak that is distinct from the 24–27 nt peak. The latter corresponds to piRNAs, which are especially abundant in the female body and early embryos (green dataset). However, these samples contain a distinct peak of 21 nt reads corresponding to siRNAs. Adult heads (purple dataset) and S2 + Kc cells (red dataset) generate TE-small RNAs that are nearly exclusively 21 nt in size.

Figure 2. 5′ and 3′ Characteristics of TE-siRNAs

(A–D) 5′ nucleotide bias of TE-siRNAs; i.e., 21 nt reads matching the sense or antisense strand of TEs. (A) TE-siRNAs in our S2 and Kc cell libraries. (B) TE-siRNAs in our male and female head libraries. (C) TE-siRNAs in the S2 data of Seitz and Zamore [26]; combined reads from non-β-eliminated and β-eliminated RNA. (D) TE-siRNAs in the head data of Seitz and Zamore [26]; combined reads from non-β-eliminated and β-eliminated RNA. In all datasets, there is preference for 5′ U and mild bias against 5′ G. (E and F) TE-siRNAs from S2 RNA and β-eliminated S2 RNA (E), and head RNA and β-eliminated head RNA (F) in the data of Seitz and Zamore [26]. Counts in each dataset were normalized per 100,000 total reads. In both S2 and head data, there is strong enrichment for TE-siRNAs after β-elimination.

Figure 3. Genomic Origins of TE-siRNA and piRNAs

(A) Overall transposon density across the ~21 megabases of chromosome 2R. (B–D) Density of 21 nt TE reads from S2 and Kc cells; graphs depict all such reads mapped to all locations (B), reads normalized for multiple mapping (C), or just the uniquely mapped reads (D). (E–G) Similar analyses were performed for the ≥ 24 ntTE reads from female body and 0–1 hr embryos. siRNAs and piRNAs generally map all over the chromosome (B and E). Normalization for multiple mappings does not significantly change the TE-siRNA landscape (other than causing a ~30-fold reduction in overall numbers (C), whereas this treatment severely dampens the TE-piRNA landscape, leaving only the predominant 42AB cluster (F). Analysis of uniquely mapped siRNAs results in another ~30-fold reduction in overall numbers, leaving behind a few modest clusters in addition to 42AB (D). In contrast, the uniquely mapped piRNAs collapse to only the 42AB cluster and other regions of the pericentric heterochromatin but maintain > 50% of the density values of the normalized, total piRNA population (G).

Figure 4. Chemical Properties and Biogenesis Requirements of TE-siRNAs

(A) TE probes that detect mdg1- and 297-derived piRNAs in early embryos (E) and female bodies (F) instead detect siRNAs in S2 cells (S2); 2S indicates ethidium staining of 2S rRNA. (B) S2 cells were treated with the designated dsRNAs and tested for the accumulation of 21 nt TE-siRNAs. The accumulation of both TE-siRNAs is highly dependent on Dcr-2 and AG02, and 297.1 is mildly dependent upon Loqs. Because individual siRNAs are present at low levels, the exposure of the main portion of the blot was adjusted separately from the RNA ladders. Longer gels were used in (B) compared to (A) and resolved a ~19 nt band that hybridized to the 297.1 probe. Although its identity is unclear, it exhibited similar sensitivity to the 21 nt band. (C) TE-siRNAs are resistant to β-elimination (β) and show the same mobility as untreated RNA (null sign). In contrast, miRNAs such as miR-8 exhibit increased mobility after β-elimination.

Figure 5. Dcr-2- and AG02-Deficient Cells Exhibit Increased Levels of TE Transcripts

Relative RNA levels (mean ± SD) are shown. (A) TE transcripts were measured by quantitative RT-PCR of S2 cells treated with dsRNA against Dcr-2 or AGO2, normalized to GFP dsRNA-treated cells. (B) The heads of dcr-2 or ago2 homozygous flies similarly exhibit increased levels of several TE transcripts relative to Canton S heads.