shutdown is a component of the Drosophila piRNA biogenesis machinery

Abstract

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.

FIGURE 1.

Shutdown is the only FKBP-family protein required for transposon silencing. (A) Above are shown the critical residues for the FKBP family peptidyl-prolyl cis-trans isomerase active site and the HSP90-interacting region in the TPR, as indicated, comparing the site in Drosophila Shutdown with those present in other family members. Residues in green indicate highly conserved residues with a known impact on PPIase activity, while those in yellow indicate a more poorly conserved region that has also been implicated. Below are evolutionary trees comparing each domain with family members present in other species. (B) The domain structures of the eight Drosophila FKBP family members are shown schematically. (C) Relative expression levels of Drosophila FKBP family members are shown for ovary and OSS RNAseq data sets. Relative enrichment in ovary versus other tissues is also shown. (D) Shown are relative HetA expression levels detected in ovaries from Drosophila engineered to express dsRNAs corresponding to each family member in the germline lineage. To the right is indicated whether dsRNA-expressing females are fertile (+) or sterile (−).

FIGURE 2.

Phenotypes of Drosophila with germline-specific shu knockdown. (A) Depletion of shu in the germline results in derepression of multiple, unrelated transposons from the LINE and LTR families. Derepression, relative to white RNAi, is displayed as log2 fold change in heat map form. Analysis of flies with germline knockdown of Armi and Piwi, two known piRNA components, is displayed for comparison. (B) Germline-knockdown of shu causes patterning defects as indicated by the presence of fused dorsal appendages. (C) Depletion of shu causes female sterility. shu RNAi females lay fewer eggs compared with controls or animals depleted of other piRNA pathway factors. Hatching rates for all knockdown animals are zero, indicating complete sterility. (D) Depletion of shu in the germline using nos-GAL4 results in Piwi delocalization from nuclei and in Aub and Ago3 delocalization from nuage. Vasa localization is not changed. Depletion of white is shown as control. (E) Tj-GAL4–driven knockdown of shu in somatic follicle cells also causes Piwi delocalization. RNAi against white is shown as control.

FIGURE 3.

Knockdown of shu causes loss of cluster-derived piRNAs in both somatic and germline tissues. (A) At the top is shown a histogram of small RNAs mapping to the germline-specific 42AB cluster in flies expressing the indicated dsRNAs specifically in germ cells. In the middle, the size distribution of RNAs derived from each strand of the 42AB and flamenco clusters is shown as a histogram. At the bottom are histograms reflecting the relative enrichment of RNAs overlapping by the indicated number of nucleotides, plotted by Z-score, for the 42AB and flamenco clusters in the indicated knockdown animals. The peak at position 9 (arrow) is indicative of a ping-pong interaction. (B) A histogram shows relative piRNA levels for a series of germline and somatic clusters. Total reads were normalized across libraries to piRNAs mapping to flamenco, which is unaffected in germline-specific knockdowns. For each cluster, changes in mapping piRNAs are shown with reference to the white control, which is set to 100%. C and D are similar to A and B except that dsRNA expression is driven by a follicle cell–specific tj-GAL4 driver. In C, at the top, reads are shown mapping to the soma-specific flamenco cluster. In D, reads are normalized across libraries to those derived from 42AB, whose activity is not affected in the soma-specific knockdown.

FIGURE 4.

Loss of transposon control in shu knockdowns is a consequence of piRNA loss. (A) The heat map displays changes in piRNA abundance for each germline knockdown (as indicated) for the 75 elements most heavily targeted in our strain. Sense and antisense, with respect to the transposon coding strand, are quantified separately (gray heat maps), and their ratio is also indicated (red-blue heat map). (B) For three transposons, piRNAs are plotted along the length of the consensus sequence (upper) and a histogram of overlap between sense and antisense species (lower) is presented to indicate the degree of ping-pong (arrow highlights peak at position 9). Data are presented for shu and piwi knockdown and for a control (white). Two transposons with strong expression in the germline, Rt1b and roo (top and middle), are shown in comparison to a somatically biased element, ZAM. Since knockdown is germline specific, ZAM piRNAs are unaffected.