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. 2007 Jun;12(6):851-62.
doi: 10.1016/j.devcel.2007.03.022.

zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline

Attilio Pane  1 , Kristina Wehr, Trudi Schüpbach
Affiliations

zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline

Attilio Pane et al. Dev Cell. 2007 Jun.

Abstract

RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. Here we report the identification of Drosophila zucchini (zuc) and squash (squ), which function in germline RNAi processes. Zuc and Squ contain domains with homologies to nucleases. Mutant females are sterile and show dorsoventral patterning defects during oogenesis. In addition, Oskar protein is ectopically expressed in early oocytes, where it is normally silenced by RNAi mechanisms. Zuc and Squ localize to the perinuclear nuage and interact with Aubergine, a PIWI class protein. Mutations in zuc and squ induce the upregulation of Het-A and Tart, two telomere-specific transposable elements, and the expression of Stellate protein in the Drosophila germline. We show that these defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs.

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Figures

Fig 1
Fig 1. Grk expression pattern and karyosome defects in squ and zuc egg chambers
In the wild type oocyte (A) Grk protein is found in a cap above the nucleus by stage 9 of oogenesis, where it signals the dorsal fate to the surrounding epithelial cells. In zuc (B) and squ (C) oocytes, the levels of Grk protein are severely reduced and often the protein is completely absent. However, in situ hybridization with a grk probe reveals that similar to the wildtype oocyte (D), the grk transcript is for the most part correctly localized in zuc (E) and squ (F) oocytes. By stage 3 of oogenesis the DNA of the oocyte nucleus forms a compact sphere called karyosome in the wild type (G). In zuc (H) and squ (I) oocytes, the DNA is more dispersed or fragmented, thus indicating a function in early oogenesis for these genes.
Fig 2
Fig 2. Structure of Zuc and Squ proteins and alignment of Zuc and Squ with homologous proteins
(A) Alleles of zuc and squ. zucHM27 contains a stop codon at residue 5, zucRS49 a substitution of the Alanine 47 with an Aspartic Acid residue, zucSG63 a substitution of the Hystidine 169 in the conserved HKD domain with a Tyrosine. squHE47 and squPP32 are generated by insertion of stop codons at residues 100 and 111 respectively. (B) Alignment of Zuc with the putative human homologue LOC201164 (GenBank) and the bacterial nuclease Nuc. The HKD domain (black box) is conserved in all the proteins. (C) Alignment of Drosophila Squ protein with Agrobacterium tumefaciens RNAse HII (Agrt RNAse HII). These proteins share significant identities in their N-terminal regions. Asterisks mark the conserved residues. Dashes mark similar aminoacids.
Fig 3
Fig 3. Zuc and Squ proteins localize to the nuage and physically interact with Aub
The nuage is a perinuclear fibrous structure, which has been implicated in RNAi. (A) Expression of a EGFP-Zuc fusion protein in nurse cells under the control of a Nos-Gal4-VP16 driver. Live imaging on transgenic egg chambers shows a perinuclear localization of EGFP-Zuc. EGFP-Zuc protein is also detectable in cytoplasmic particles in nurse cells. (B) Expression of HA-Squ protein under the control of the Nos-Gal4-VP16 driver visualized with anti HA antibody. Squ also localizes to the nuage and to cytoplasmic particles. (C) Physical interaction of Zuc and Squ with Aub. Nos-Gal4-VP16 UAS-aub-gfp lines were crossed to UAS-HA-zuc transgenic strains and IP was performed on doubly transgenic ovaries using an anti GFP antibody. A strong band corresponding to the HA-Zuc protein can be detected in the ovarian extracts of doubly transgenic flies, while no signal above the background is visible in the IP lane of the control HA-Zuc lines. Similarly, nos-Gal4-VP16 UAS-aub-gfp lines were crossed to UAS-HA-squ transgenic strains and IP was performed on doubly transgenic flies. A band corresponding to HA-Squ can be observed in the lane of doubly transgenic ovaries, which is not present in the control lane. Additional bands are generated by an unspecific cross-reaction of the antibody. SN represents the unbound fraction of the IP.
Fig 4
Fig 4. Osk expression pattern in early oocytes
In wild type egg chambers (A), Osk translation is inhibited by RNAi mechanisms from stage 1 to 6. Consistent with a role in RNAi processes, zuc (B) and squ (C) mutations activate Osk expression in early oocytes. Osk protein forms clumps in the oocyte of the mutants and is found in punctae surrounding the nurse cells nuclei in the egg chambers.
Fig 5
Fig 5. Het-A and Tart retro-transposable elements and Ste tandem repeats are upregulated in ovaries of zuc and squ mutants
A) Mutations in aub and spnE impair the RNAi processes thus leading to higher expression levels of some classes of transposable elements, including the telomere-specific Het-A and Tart retro-transposons (Aravin et al. 2001). Using qRT-PCR we detected approximately a 10 fold increase of Het-A levels in the germline of aub, spnE and squ mutants compared to heterozygous control flies, while the upregulation is much higher in zuc ovaries, where it reaches nearly 1500 fold increase. The Tart element seems to be less sensitive to mutations in RNAi-related genes. A 10 to 15 fold increase in the levels of Tart expression are detected in spnE and zuc, while no significant increase is observed in the germline of aub and squ. It is possible that the heterozygous control would already show a light upregulation of the transposable elements over wildtype. The levels of upregulation as calculated here are therefore a conservative estimate. B-D) The Stellate protein is down-regulated in the testis of wild type males through RNAi mechanisms involving the Su(Ste) locus and the RNAi-related proteins SpnE, Armi and Aub. Mutations in these genes lead to inhibition of the RNAi machinery and ectopic expression of the Stellate proteins, which in turn form needle-shaped aggregates. Such crystals are absent from testis of wild type males (5B), while they can be easily detected in testis of squ (5C) and zuc (5D) mutant males.
Fig 6
Fig 6. Analyses of rasiRNAs production in ovaries and testes of squ and zuc mutants
A) Northern blot analysis on total RNA extracted from fly ovaries. Membranes were probed with an antisense oligonucleotide to the abundant roo rasi (Brennecke et al., 2007) to monitor the expression of rasiRNAs in the various mutant backgrounds. Similar to aub and spnE, mutations in the zuc gene impair the production of these siRNAs. In contrast, mutations in the squ gene do not completely abolish the production of rasiRNAs in ovaries. The same membranes were stripped and reprobed with an oligonucleotide antisense to miR310 (Saito et al., 2006). None of the mutants analysed affects the biogenesis of microRNAs. As a loading control the membranes were finally hybridized with a probe antisense to the 2S rRNA. B) Northern blot analysis of total RNA extracted from fly testes. In this experiment the membranes were probed with an antisense oligonucleotide to the abundant Su(Ste) rasi (Brennecke et al., 2007). In accordance with the results described above, mutations in zuc, aub and spnE abolish the production of rasiRNAs also in testes. Mutations in squ also strongly affect the biogenesis of the rasiRNAs in this tissue. The membranes were probed with an antisense oligonucleotide to the 2S rRNA as a loading control.
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