An endogenous small interfering RNA pathway in Drosophila

Abstract

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.

Figure 1. AGO2 binds endogenous small RNAs

a, RNA was isolated from AGO1 and AGO2 immunoprecipitates (IP) from embryos and S2 cells. b, Length profiles for small RNAs isolated from S2 cells and ovaries are shown. Species are split into miRNAs and remainder, and are compared to those obtained from individual Argonaute complexes (as indicated). c, Annotation of AGO1- and AGO2-associated small RNAs from ovaries and S2 cells. rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA.

Figure 2. A subset of endo-siRNAs originates from transposons

a, Indicated are the cloning frequencies of AGO2-bound siRNAs in ovaries and S2 cells that match individual transposons. b, RNA levels of twelve transposons and two control genes in ovaries mutant for AGO2 as compared to AGO2 heterozygotes (four biological replicates; error bars indicate technical variation). c, Distributions of AGO2-bound siRNAs (black) and Piwi-bound piRNAs (orange) from ovaries on the piRNA cluster at cytological position 42AB (ref. ; relative abundances of both populations can be estimated from Supplementary Fig. 2d).

Figure 3. Two types of genic endo-siRNA loci in Drosophila

a, FlyBase gene structure at the esi-2 locus indicating the twenty ~260-bp repeats of esi-2 (black bars). Below, three repeats are magnified and the small RNA density is depicted. Most siRNAs match up to 20 times and cannot be unambiguously assigned to a chromosomal location. Each repeat is a palindromic sequence, offering a multitude of possible structures. One structure is shown with the 5′ ends of cloned siRNAs as black bars (the height correlates with cloning frequency; phasing is indicated by brackets). b, An abundant siRNA from esi-2 (indicated by an arrow in a) is highly complementary to the mus308 mRNA. Shown is the mus308 target site and cloned siRNAs (solid dots indicate canonical base pairs; open dots indicate GU base pairs). The only detected cleavage site within this duplex is indicated above. c, Shown are mus308 transcript levels from AGO2 and Dcr-2 mutant (mut.) flies compared to their respective heterozygotes (error bars indicate standard deviation). To the right, average reporter levels (error bars indicate standard deviation) of a construct containing two mus308 target sites in S2 cells depleted of the indicated genes by RNAi are shown. d, An example of siRNAs arising from convergent transcription units. A 30-kb region containing multiple instances of convergently transcribed genes is displayed, with the density of AGO2-associated RNAs in ovaries shown above.

Figure 4. Genetic requirements for siRNA biogenesis

a, Length distributions of small RNAs from total RNA libraries obtained from wild-type (black), Dcr-2 mutant (red) and loqs mutant (yellow) ovaries. Excluding miRNAs and piRNAs (23–29 nucleotides), the population of 21-nucleotide siRNAs mapping to structured loci, genes and repeats is lost from Dcr-2 mutants and those mapping to structured loci are strongly reduced in loqs mutants (all libraries are adjusted to the same total small RNA count). b, Northern blots showing levels of three siRNAs encoded from structured loci esi-1 and esi-2 (esi-2.1, esi-1.1 and esi-1.2) in S2 cells treated with dsRNA against the genes indicated. As controls, northern blots of bantam (pre-miRNA indicated by the asterisk) and 2S rRNA are shown below. c, Renilla luciferase reporter assays are shown for the siRNAs examined in b and an additional esi-1-derived species (esi-1.3) in S2 cells treated with dsRNAs against the indicated genes (error bars indicate standard deviation; n = 3).