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A Somatic piRNA Pathway in the Drosophila Fat Body Ensures Metabolic Homeostasis and Normal Lifespan

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A Somatic piRNA Pathway in the Drosophila Fat Body Ensures Metabolic Homeostasis and Normal Lifespan

Brian C Jones et al. Nat Commun.

Abstract

In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. Here we report a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health.

Figures

Figure 1
Figure 1. Canonical signatures of a piRNA pathway in the adult fly fat body.
(a) Expression of primary and secondary piRNA pathway genes generated from total RNA-seq libraries of head, thorax, eviscerated abdomen and ovary. piRNA pathway genes are more highly expressed in the eviscerated abdomen than in the head or thorax. Data values for ovary libraries that exceed the range of the plot are shown above each relevant bar. RPKM, reads per kilobase per million. Error bars represent s.e.m.; n=3 replicate libraries. In comparing the eviscerated abdomen with head and thorax controls, 10 of 11 genes (excluding tj) are statistically significant (P<0.0001). See Supplementary Dataset 1 for statistics. (b) Piwi protein localizes to the nuclei of abdominal fat body cells. DAPI labels fat body nuclei. Staining in the membrane is autofluorescence typical of fat body cells. Scale bars represent 20 μm. (c) Piwi protein is present in the fat body. All piRNA argonautes are present in the ovary samples. Actin serves as a loading control. (d) Fat body smRNA size profile from oxidized smRNA-seq libraries. Oxidation allows for enrichment of 2′-O-methylated smRNAs. Peak at 21 nt likely represents short interfering RNA (siRNA) population. Broader peak from 23 to 29 nt represents putative fat body piRNAs. Reads aligning to rRNA and miRNA were excluded from analysis. (e) Fat body piRNAs (23–29 nt) aligned to the fly genome map primarily to TEs. (f) Sequence composition of TE-mapping fat body piRNAs (23–29 nt) displays a first position nucleotide bias for uracil.
Figure 2
Figure 2. The piRNA pathway is active in the fat body.
(a) Heat map of log2 fold change of TE transcript levels (total RNA-seq) >1.2 fold change and corresponding piRNAs (smRNA-seq) in piwi mutant (piwi−/−) compared with heterozygous control (piwi−/+) fat bodies; n=3 replicate libraries. *False discovery rate (FDR) <0.05. (b) Fat body smRNA size profile from oxidized smRNA-seq libraries of piwi mutant fat bodies (red) and heterozygous controls (black). Shown are total smRNAs (top), TE-mapping smRNAs (middle) and 3′UTR-mapping smRNAs (bottom). Panels at right show levels for 23–29 nt smRNAs for each genotype. (c) A fat body-specific transposition reporter line, gypsy-TRAP/r4-GAL4::UAS-GFP, in a piwi mutant background (see Methods). GFP-positive cells are cells in which a transposition event has activated reporter function. The 10-day-old piwi mutants show elevated levels of GFP-positive cells compared with heterozygous controls. Images are of representative flies exhibiting high, medium and low levels of GFP-positive cells (left panels). Scale bars represent 150 μm. Panel on right shows the distribution of flies for each group by genotype; numbers within each bar are corresponding percentages for each group. Fisher's exact test compared the combined ‘High'+‘Medium' groups and the ‘Low' group of each genotype. piwi−/+: n=140 flies; piwi−/−: n=142 flies; P<0.0001. (d) Fold ratio of uniquely mapping cluster piRNAs in piwi mutants compared with heterozygous controls. The flamenco cluster shows the greatest response to loss of piwi. (e) Unique piRNA reads (23–29 nt) map to the flamenco locus in wild-type (wt) and piwi heterozygotes and are lost in piwi mutants. (f,g) Unique piRNA reads (23–29 nt) map to the tj gene body in wt and piwi heterozygotes and are lost in piwi mutants. Thick lines in gene model (g) represent coding sequence, and thin lines represent 5′ and 3′UTRs. See Supplementary Dataset 2 for raw data.
Figure 3
Figure 3. Loss of the piRNA pathway disrupts normal metabolic functions of the fat body.
(a) Representative images of γ-H2A.v staining in piwi mutants and heterozygous controls. piwi mutants exhibit higher levels of γ-H2A.v staining compared with heterozygous controls. Scale bars represent 20 μm. (b) Quantification of γ-H2A.v staining in (a). piwi−/+ and piwi−/−: error bars are s.e.m. Student's two-tailed t-test compared with heterozygous control; n=104 nuclei per genotype. *P<0.0001. (c) Representative images of Nile red staining of fat body lipid droplets in piwi mutants and heterozygous controls. piwi mutants exhibit smaller lipid droplets relative to heterozygous controls. Scale bars represent 20 μm. (d) Quantification of lipid droplet staining in (c). piwi−/+: n=6 flies, piwi−/−: n=5 flies. See Supplementary Dataset 3 for raw data. (e) Box plot showing distribution of lipid droplets >200 μm2 from (d) comparing piwi mutants to heterozygous controls. (fi) Measurements of whole-body adult fly TAGs (f,g) and glycogen (h,i) of piwi (f,h) or flamenco (g,i) mutants compared with heterozygous controls. Data were normalized to total protein concentration of each sample and represented as a percent of the heterozygous control. Error bars are s.e.m. For each assay, n=5 biological replicates per genotype. Student's two-tailed t-test compared with heterozygous control. *P<0.01; **P<0.001. All assays were performed using 10-day-old flies.
Figure 4
Figure 4. piRNA pathway mutants are stress sensitive and short-lived.
(a) Survivorship curves for starvation of piwi mutants and heterozygous controls. piwi mutants are more sensitive to starvation than heterozygous controls. Log rank test compared with heterozygous controls; n≈50; P<0.0005. (b) Survivorship curves for immune challenge of piwi mutants and heterozygous controls. piwi mutants are more sensitive to infection than heterozygous controls. Flies were either infected with a mock EtOH control (−) or a culture of E. carotovora (+). Log rank test compared with heterozygous or mock EtOH control (−); n≈50; P<0.0005. (c) Survivorship curves of piwi mutants and heterozygous control. piwi mutants are shorter lived compared with heterozygous controls. Wilcoxon rank sum test compared with heterozygous control; n≈250; P<0.0005. (d) Survivorship curves of flam mutants and heterozygous control. flam mutants are shorter lived compared with heterozygous controls. Wilcoxon rank sum test compared with heterozygous control; n≈250; P<0.0005. (e) Survivorship curves of flam mutants fed 10 mM 3TC. flam mutant flies fed 3TC live longer than untreated controls. Wilcoxon rank sum test compared with control; n≈250; P<0.0005. See Supplementary Table 1 for assay parameters and statistics. All assays were repeated twice with similar results.

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