Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

Abstract

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2(1) mutants have striking developmental defects. r2d2(1) mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2(1) mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.

The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2

“…Notably, expression of either UAS- PACT or UAS- r2d2 in r2d2 heterozygous flies did not influence fertility or hatch rates. Consistent with a previous report 27 , flies homozygous mutant for r2d2 had a strong reduction in both the total number of eggs laid and the hatch rate compared to wild-type ( Figure 3). We observed a rescue of both total number of eggs (p=0.0512) and hatch rate (p=0.0671) in r2d2 mutant flies by ubiquitous ( tub -GAL4) transgenic expression of UAS- r2d2 that is approaching statistical significance and consistent with previous reports 27 .…”

“…Flies mutant for r2d2 have reduced fertility 27 . Therefore, we sought to determine the extent to which PACT can suppress r2d2 mutant fertility defects.…”

Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis

“…Currently, the known small RNA-mediated silencing pathways in Drosophila that impinge upon gamete generation function at the earliest stages of germ line development. Genes encoding components of the miRNA machinery such as Loqs and Ago1 act at the level of germ line stem cell self-renewal in females (21,47,48), and a Dicer1-dependent role for R2D2 has been suggested in somatic follicle cells (49). A role for the miRNA pathway in sperm development has not yet been fully investigated, though loqs hypomorphic males are incompletely fertile and show a derepression of stellate (21).…”

R2D2 Organizes Small Regulatory RNA Pathways in Drosophila∇†

“…S2A in the supplemental material), indicating its functionality. Unexpectedly, although R2D2 was previously described to be important for fertility (24), r2d2 [1] flies homozygous for FLAG-AGO2 (i.e., bearing 4 copies of AGO2) could be maintained as a stable homozygous stock. Extra copies of AGO2 may partially compensate for the absence of R2D2; alternatively, meiotic recombination might have removed background mutations from the parental r2d2 [1] chromosome.…”

“…Although there is competition between AGO1 and AGO2 loading machineries (13,20,55), a UV cross-linking assay showed that AGO1 can largely reject synthetic siRNAs even in the absence of AGO2 loading machinery (55). On the other hand, genetics suggested possible interactions between these pathways, since double mutants of miRNA and siRNA pathway genes exhibited synthetic phenotypes (24,40). Moreover, the sorting of miR-277, whose mature strand resides in both AGO1 and AGO2 complexes, is altered by the availability of the two Argonautes (13,20).…”