Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila

Abstract

Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.

Figure 1.

Effect of piwi, zucchini, and armitage knockdown on transposon silencing in OSS cells. OSS cells were treated with dsRNA against piwi, zuc, or armi for 6 d. qPCRs were normalized to internal controls rp49 (A) or bantam (B). Fold changes relative to cells treated with gfp-dsRNA are shown on a linear scale. Error bars represent one standard deviation over three biological replicates. Transcripts (A) and two abundant piRNAs (B) corresponding to gypsy and idefix retroelements were detected by qPCR. (C) Small RNAs coimmunoprecipitating with Piwi in untreated cells and cells treated with dsRNA against gfp, armi, zuci, or piwi were labeled with ³²P at their 5′ termini.

Figure 2.

Piwi protein localization and levels upon knockdown of armitage or zucchini. (A) Piwi subcellular localization was determined by immunostaining in OSS cells treated with zuc-dsRNA, armi-dsRNA, or piwi-dsRNA. Gfp-dsRNA-treated cells were used as control. (B) Piwi protein levels in total cell extracts were determined by Western blotting. Tubulin was used as a loading control. (D) Piwi qPCRs were normalized to rp49. Fold changes relative to cell treated with gfp-dsRNA are shown on a linear scale. Error bars represent one standard deviation over three biological replicates.

Figure 3.

MudPIT analysis of Piwi, Zucchini, and Squash complexes. (A) A proposed model for the function of piRNA pathway components is shown. (B) Protein associations identified by MudPIT are depicted. Arrows point from the immunoprecipitated protein to the coimmunoprecipitated protein. Numbers of identified peptides and corresponding unique sequences (shown in parentheses) of two biological replicates are indicated. Only peptides above the significance threshold were considered (see the Materials and Methods). (C) Zucchini, Squash, and Piwi were immunoprecipitated (IP) from OSS cells. The presence of Piwi was assessed by Western blotting.

Figure 4.

piRNA populations from zucchini and squash mutant ovaries. Heterozygous siblings serve as control. (Left panel) Size profiles of small RNAs mapping to repeats normalized to total read counts are shown. (Right panel) Densities of uniquely mapping piRNAs are plotted over the flamenco locus in reads per million (only small RNAs matching the plus strand are depicted). (A) Small RNAs cloned from total RNA. (B) Small RNAs from Piwi immunoprecipitates (IP). Regions of flamenco measured in Figure 5D are indicated by asterisks.

Figure 5.

Transposons, piRNAs, and piRNA precursors in zucchini and squash mutant ovaries. (A) Transcripts of gypsy, idefix, and ZAM transposons were detected by qPCR. (B) Individual piRNAs targeting gypsy and idefix were detected by qPCR. (C) Piwi protein levels in mutant and heterozygous ovary extracts were measured by Western blotting. Tubulin serves as loading control. Celera sequencing strain (S-strain) is shown in addition. (D) Three ∼100-nt regions of flamenco that are normally highly processed into piRNAs were detected by qPCR. The positions of these segments are indicated in Figure 4. qPCR data were normalized to internal controls rp49 (A,C) or bantam (B). Fold changes relative to heterozygous siblings are shown on a linear scale. Error bars represent one standard deviation over three technical replicates.