InteractiveFly: GeneBrief

zucchini: Biological Overview | References

RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. This study reports 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. These defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs (Pane, 2007).

In eukaryotic organisms, RNAi, or “RNA interference,” controls a wide variety of biological processes, including development, genome organization, and virus and transposable elements defense. RNAi is triggered by small RNA molecules, which can be grouped in three classes: siRNAs, micro-RNAs (miRNAs), and repeat-associated small interfering RNAs (rasiRNAs). In Drosophila, Dcr2 is responsible for the maturation of the siRNAs from long dsRNA, while the Dcr1/Loquacious complex produces miRNAs from hairpin structures. siRNAs and miRNAs are then incorporated into specific RNP complexes, which are named, respectively, RISC (RNA-induced silencing complex) and miRNP. Core components of the RISC and miRNP complexes are members of the Argonaute (Ago) family, like Ago1 and Ago2. While RISC has been shown to target the transcripts for destruction, the miRNP complex is implicated in the control of mRNA translation. The third class of small RNAs, the so-called rasiRNAs, shares sequence complementarity with mobile elements, satellite and microsatellite DNA, and tandem repeats (Aravin, 2003). It has recently been reported that the biogenesis of the rasiRNAs does not proceed through Dcr1 and Dcr2, thus pointing to a novel mechanism for the maturation of these molecules (Vagin, 2006). rasiRNAs are thought to assemble into RNP complexes containing members of the PIWI family, such as Piwi and Aubergine (Aub), which are involved in chromatin organization as well as in triggering target mRNA destruction to protect the fly genome from selfish genetic elements (Saito, 2006; Pane, 2007 and references therein).

RNAi has been shown to be involved in axial polarization in the Drosophila germline (Cook, 2004; Tomari, 2004). In this species, establishment of dorsal-ventral (DV) and anterior-posterior (AP) axes is achieved through the localized translation of specific mRNAs. The protein products of gurken (grk) and oskar (osk) genes are essential for this process. Early during oogenesis, grk RNA encoding a TGFα-like molecule is localized to the posterior of the oocyte, where it signals the posterior fate to the adjacent follicle cells. Following the reorganization of the microtubule cytoskeleton at stage 8, the oocyte nucleus and grk RNA are relocalized to the dorsal-anterior corner of the oocyte. Grk protein now induces dorsal cell fates in the surrounding epithelial cells. In contrast to Grk, which is expressed throughout oogenesis, osk mRNA is kept silenced early during oocyte development. At later stages, Osk protein is found at the posterior of the oocytes, where it directs the organization of the germ plasm as well as abdomen formation of the future embryo. The silencing of oskar translation from stage 1 to 6 is controlled by a set of genes, including armitage (armi), maelstrom (mael), spindle-E (spn-E), and aubergine (aub), which have been shown to be required for RNAi phenomena (Cook, 2004). Mutations in these genes induce ectopic expression of Osk at early stages of oocyte development. This observation revealed a connection between the RNAi machinery and the establishment of the AP axis during Drosophila oogenesis. armi encodes the homolog of Arabidopsis SDE-3 helicase (Cook, 2004), which plays a role in post-transcriptional gene silencing (PTGS), a mechanism closely related to RNAi. mael encodes an evolutionarily conserved protein that is required for the proper localization of Ago2 and Dicer, two components of the RNAi machinery. aub and spn-E encode a member of the PIWI class of Argonaute proteins and a DExH RNA helicase, respectively. Aub and spn-E are involved in the silencing of some classes of transposable elements and tandem repeats in the germline, in heterochromatin formation, in double-stranded RNA (dsRNA)-mediated RNAi in embryos, and in the defense against viruses. Interestingly, spn-E and aub are also involved in telomere regulation (Savitsky, 2006). In most eukaryotes, the telomeres are maintained through the action of telomerase, the enzyme that ensures the addition of six- to eight-nucleotide arrays to the chromosome ends. However, in Drosophila, telomere elongation occurs after the transposition of non-long-terminal repeat (non-LTR) HeT-A, TAHRE, and TART retrotransposons. Mutations in spn-E and aub cause the upregulation of Het-A and TART expression in the germline, which, in turn, increases the frequency of telomeric element attachments to chromosome ends (Pane, 2007).

This study shows that the genes zucchini (zuc) and squash (squ) are required early during oogenesis for the translational silencing of osk mRNA and at later stages for proper expression of the Grk protein. It is proposed that insufficient levels of Grk protein in zuc and squ mutants are at least partially due to activity of a checkpoint that affects Grk translation, similar to the effects of DNA repair mutants in meiotic oocytes. zuc encodes a member of the phospholipase-D/nuclease family (Koonin, 1996; Ponting, 1996), while squ encodes a protein with limited similarity to RNAase HII. Like Aub, Mael, and Armi proteins, Zuc and Squ localize to nuage, an electron-dense structure surrounding the nurse cell nuclei implicated in RNAi and RNA processing and transport. Zuc and Squ physically interact with Aub, thus pointing to a direct role for these proteins in the RNAi mechanisms. In further support of this conclusion, it has been demonstrated that zuc and squ are required for the biogenesis of rasiRNAs in ovaries and testes. Accordingly, mutations in these genes abolish the production of this class of siRNAs and lead to the deregulation of transposable elements and tandem repeats in the Drosophila germline (Pane, 2007).

zucchini and squash cause dorso-ventral patterning defects and egg chamber abnormalities during oogenesis: zuc and squ were identified in a screen for female sterile mutations on chromosome II of Drosophila (Schüpbach, 1991). zuc and squ mutant females are viable, but produce eggs with a range of DV patterning defects. Flies with the most severe allele of zuc, zuc^HM27, lay few eggs, all of which are completely ventralized and often collapsed, whereas those with the weaker alleles, zuc^SG63 and zuc^RS49, produce some eggs with a more normal eggshell phenotype in addition to the ventralized eggs). In addition, a P element insertion in the coding region of the gene also acts as a strong loss-of-function allele with ventralized eggshell phenotypes. Three independent alleles of squ were recovered from the screen, namely squ^HE47, squ^PP32, and squ^HK3, and these alleles also generate a range of ventralized eggshell phenotypes (Pane, 2007).

Similar eggshell phenotypes have been described for mutations in other spindle class genes, which include both DNA repair enzymes such as spindle-B (spn-B) or okra (okr), as well as the RNAi components spn-E, aub, and mael. Similar to the spindle class mutants, several additional developmental defects can be observed in the zuc and squ mutants during oogenesis. In the wild-type oocyte, the nucleus condenses in a compact sphere, known as the karyosome. In contrast, the DNA in the nuclei of zuc and squ oocytes appears dispersed or in separate structures. Since compaction of chromatin in the karyosome occurs at stage 3, the defects observed in zuc and squ egg chambers indicate a function for the genes in the early development of the oocyte. Similar to spnE mutants, in a small number of zuc and squ egg chambers the oocyte is not positioned at the posterior as in wild-type, but is found in the middle of the egg chamber. Finally, fusion of egg chambers can also be observed in zuc mutants, resulting in egg chambers with 30 nurse cells and two oocytes. Many egg chambers in the zuc mutant undergo degeneration at different stages (Pane, 2007).

Grk expression is affected in zuc and squ mutants: The DV patterning defects suggested that the Gurken protein is not properly expressed in the mutant egg chambers. In earlier stages of oogenesis, Grk protein is detected in the oocyte similar to the wild-type egg chambers. At stage 9 in wild-type oocytes, Grk is localized in a cap above the oocyte nucleus, where it specifies the dorsal fate of the adjacent follicle cells. In zuc mutants, the amount of Grk protein found in the dorsal-anterior corner of the oocyte is strongly reduced or absent, suggesting that zuc controls the expression of Grk during mid-oogenesis. To further address this question, the distribution pattern of the grk transcript was analyzed in wild-type and zuc mutant egg chambers. In wild-type, grk mRNA localization mirrors the distribution of the protein and is found in the dorsal-anterior corner of the oocyte. Similarly, in zuc mutant egg chambers, grk mRNA is properly localized during mid-oogenesis. zuc therefore affects accumulation of the Grk protein in mid-oogenesis, most likely affecting the translation of the transcripts. This phenotype is also characteristic of the spindle class mutants in general (Pane, 2007).

In squ mutants, Grk protein also fails to accumulate properly in the oocyte at stage 9. Similar to zuc, analysis of grk transcripts in these mutants revealed that the grk mRNA is correctly localized in the majority of the squ egg chambers in mid-oogenesis. This result suggests that squ is also required for Grk translation (Pane, 2007).

zuc and squ do not belong to the spindle class of dna repair genes: The analysis of the zuc and squ egg chambers revealed defects, which place them into the spindle class genes. The spindle genes can be grouped into different categories: the DNA repair genes, the RNAi genes, and a class of translational regulators. The DNA repair genes are implicated in the repair of DNA double-strand breaks which are induced during meiotic recombination by the topoisomerase Mei-W68, a homolog of yeast Spo11. Mutations in these DNA repair genes result in the activation of a meiotic checkpoint mediated by mei-41, the Drosophila ATR homolog. Mei-41 activates the kinase Chk2 also called Mnk in Drosophila, and the activity of Chk2 results in a downregulation of Gurken translation. The resulting reduction in Gurken protein accumulation leads to the ventralized eggshell phenotype. As predicted for a mediator between DNA damage and grk translation, mutations in mei-41 and chk2 are able to suppress the phenotypes caused by mutations in the DNA repair genes. Accordingly, wild-type morphology is restored, for instance, in the eggs of flies doubly mutant for spn-B and mei-41. To assess whether zuc and squ belong to the DNA repair genes, zuc; mei-41 and squ; mei-41 double mutant flies were generated, and the eggs laid by these females were checked for the presence of DV patterning defects. In both cases, the persistence of dorso-ventral patterning defects was observed, indicating that zuc and squ do not likely belong to the class of DNA repair enzymes. Flies doubly mutant for zuc and chk2 and squ and chk2 were generated. Interestingly, it was found that while patterning defects persist in the eggs of zuc chk2 flies, wild-type morphology is restored in the eggs laid by squ chk2 homozygous females. Suppression of the eggshell ventralization phenotypes was also observed in chk2 aub mutants, but not in chk2; spn-E or chk2 piwi double mutants. This demonstrates that a checkpoint mediated by Chk2 is largely responsible for the low levels of Grk protein in aub and squ mutants. The fact that zuc, spnE, and piwi phenotypes are not suppressed by chk2 mutations suggests that they may have multiple effects on oogenesis, some of which may act independent of checkpoint activity (Pane, 2007).

Molecular analysis of the zuc and squ genes: A set of deficiencies was used to map the zuc mutation to region 33B5 of chromosome II. Transformation rescue experiments narrowed the region to a candidate region of 5 kb, containing two transcripts: CG12314 and CG16969. Sequence analysis revealed that all the zuc mutations reside in CG12314. zuc encodes a member of the phospholipase-D/nuclease family and is characterized by one copy of a conserved H(X)K(X₄)D (HKD) motif (Koonin, 1996; Ponting, 1996). Notably, members of the family having two HKD domains are classified as phospholipase-D proteins, while members with one HKD domain have been shown to catalyze the hydrolysis of double-stranded RNA and DNA molecules in vitro. Hence, Zuc is likely to be a nuclease. The Histidine (H) residue of the HKD domain is essential for the function of the phospholipase-D/nuclease proteins, since substitution of the H residue results in a strong reduction of the catalytic activity in vitro (Sung, 1997). Interestingly, the substitution of the H of the catalytic domain with a Tyrosine in the zuc^SG63 allele generates a strong loss-of-function allele. zuc^HM27 is generated by the introduction of a stop codon at residue 5, resulting in a putative protein null allele. Finally, the zuc^RS49 allele contains a substitution of the Serine47 with an aspartic acid residue. Transformation rescue experiments confirmed that CG12314 corresponds to zuc (Pane, 2007).

Recombination mapping placed squ on the left arm of the second chromosome at map position 2-53. Deficiency mapping and P-element-mediated male recombination placed squ into a region containing six candidate genes including her and grp. Complementation tests and sequence analysis argued against the six genes as candidates to be squ. Upon closer inspection of the grp locus a gene, CG4711, was seen to be nested in the first intron, which had previously been predicted to encode an alternate splice exon of grp. CG4711 as sequenced in squ^HE47, squ^PP32 and squ^HK35 and squ^HE47 and squ^PP32 were found to both contain single nucleotide changes resulting in nonsense codons in CG4711 at residues 100 and 111, respectively. No mutations were identified in the predicted CG4711 coding region in squ^HK35. Transformation rescue experiments confirmed that CG4711 corresponds to squ. This gene encodes a protein with similarity to RNAase, which is known (Itaya, 1990) to catalyze the degradation of RNA moieties in DNA-RNA hybrids (Pane, 2007).

Zuc and Squ localize to the nuage and physically interact with Aub: The “nuage” is a cytoplasmic organelle that is widely conserved in evolution. Homologous structures exist in all eukaryotic organisms and are thought to play a fundamental role in germline functions. In Drosophila, the nuage appears as an electron-dense, punctate fibrous structure that surrounds the nuclei of the nurse cells in the egg chambers. This organelle is thought to be a staging site where ribonucleoprotein complexes originating in the nuclei are remodeled, before they are transported to specific localizations in the cells. Recent studies have also shown that the nuage is implicated in RNAi. For instance, in human cell lines Ago1 and Ago2 proteins localize to cytoplasmic bodies, called P bodies, which are thought to be homologous to the Drosophila nuage. Similar to the P bodies, Drosophila nuage hosts molecules required in RNAi phenomena like Aub, Armi, and Mael. In addition, mutations in mael, another component of the RNAi machinery, disturb the nuage granules, resulting in a displacement of the RISC components Ago2 and Dcr1. To analyze the expression pattern of Zuc during oogenesis, transgenic lines were produced that express Zuc fused to EGFP. Live imaging on ovaries dissected from these lines show a strong accumulation of Zuc in the nuage. Zuc is also found in cytoplasmic particles. Immunostaining on lines expressing Zuc fused to triple HA tag confirmed these observations. Similar to Zuc, Squ protein localizes to the nuage and in cytoplasmic particles as demonstrated by the immuno localization analysis of triple-HA-Squ transgenic lines (Pane, 2007).

These results show that Zuc and Squ localize to the nuage similar to Aub. aub encodes a member of the Piwi class of Ago proteins and has been shown to be implicated in different RNAi processes in Drosophila germline. Furthermore, the inability of aub mutants to assemble RNAi complexes in the germline led to the hypothesis that Aub might be a core component for RNAi-induced complexes in this tissue. Remarkably, both Zuc and Squ were found interact with Aub in vivo, consistent with the cellular localization of these proteins. AubGFP lines were crossed to triple-HA-Zuc and triple-HA-Squ strains, respectively. CoIP was performed with GFP- and HA-specific antibodies on ovaries of doubly transgenic flies. Bands corresponding to HA-Zuc and HA-Squ are detected in the IP lanes, while no signal above background is present in the control lanes (Pane, 2007).

Mutations in zuc and squ Activate the Expression of Osk in early oocytes: A hallmark of the spindle class genes that are involved in RNAi is the control of Osk translation at early stages of development. In wild-type oocytes, osk mRNA is silenced from stage 1 to 6 through RNAi dependent mechanisms. The translational repression of osk mRNAs at these stages is thought to involve the miRNA miR280. In contrast, ectopic translation of Osk is observed in early stages of armi, aub, spnE, and mael mutant egg chambers. To assess whether zuc and squ are involved in RNAi, the expression pattern of Osk was analyzed in zuc and squ mutant egg chambers. It was found that Osk is properly translated and localized at late stages of oogenesis, where it is found at the posterior pole of the oocyte. However, in early egg chambers Osk expression is ectopically activated, and clumps of Osk protein can be observed in the developing oocyte in zuc and squ egg chambers. Osk protein is also found in punctae surrounding the nurse cell nuclei. These results suggest that zuc and squ are involved in the RNAi silencing of osk mRNAs in the nurse cells and the oocyte (Pane, 2007).

Het-A and Tart expression is regulated by zuc and squ: To further test the involvement of zuc and squ in RNAi, the expression levels of Het-A and Tart, two telomere-specific retrotransposons, were analyzed in the ovaries of zuc and squ mutants. In Drosophila, telomere maintenance is achieved through the transposition of retrotransposons to the chromosome ends. The telomere elements in Drosophila are non-LTR-containing retrotransposons, which transpose to the chromosome ends via a poly(A)⁺ RNA intermediate. The mechanism of transposition is well characterized, and recent work has shown that the RNAi machinery is involved in the maintenance of the telomeres. Aub and spnE have been shown to regulate the expression of a number of transposable elements in the germline of Drosophila. In particular, mutations in aub and spnE were discovered to trigger the upregulation of the Het-A and Tart elements, two telomere-specific retrotransposons. This process occurs in the germline of Drosophila, but not in the soma, and results in the addition of extra elements to the telomere array. Since Zuc and Squ are found in a complex with Aub, whether they also share a similar function in this process was tested. To this aim, quantitative RT-PCR was performed on total RNA extracted from heterozygous zuc^Hm27/+ and transheterozygous zuc^Hm27/Df(2L)PRL ovaries. Df(2L)PRL is a deletion that uncovers the genomic region containing the zuc gene. Comparison of the two samples reveals more than 1000-fold upregulation of the Het-A element in the germline of zuc^Hm27/Df(2L)PRL flies. A significant increase in the expression levels of Tart can be observed in zuc mutants, where this element is upregulated by 15-fold. Elevated levels of Het-A, but not Tart, can be observed in the ovaries dissected from squ^HE47/squ^PP32 mutant females as compared to the control squ^HE47/+ flies. It is possible that the levels in the heterozygous control flies are already somewhat elevated over wild-type, but since different wild-type backgrounds may vary, heterozygous flies were used as control. These results show clearly that, similar to aub and spnE, zuc and squ are required for the silencing of retrotransposons in the Drosophila germline (Pane, 2007).

Stellate silencing is impaired in testes of zuc and squ mutants: The Stellate (Ste) locus in Drosophila resides on the X chromosome and encodes a protein with homology to the β-subunit of protein kinase CK2. While the protein is normally expressed in wild-type females, it is downregulated in wild-type males through the activity of RNAi-based mechanisms. The Y chromosome of Drosophila contains the crystal locus, also called Suppressor of Stellate [Su(Ste)], which shares 90% degree of identity with Ste. The insertion of a Hoppel transposon in the region 3′ to Su(Ste) causes the transcription of antisense transcripts in addition to the sense mRNAs. Sense and antisense RNAs are thought to drive the dsRNA-mediated degradation of Ste target mRNAs. This mechanism is required in males to silence the approximately 200 repeats of the Ste locus located on the X chromosome. In males carrying a deletion of the bulk cry locus, or mutations in RNAi genes like spnE, aub, and armi, expression of Ste is relieved, which in turn leads to the accumulation of needle-shaped crystals in testes and meiotic abnormalities. To test whether zuc and squ are required for the RNAi silencing of Ste tandem repeats, testes of mutant males were stained with a Ste-specific antibody. While no signal can be detected in wild-type males, Ste crystals can be easily observed in zuc and squ mutant testes. These results demonstrate that zuc and squ are required for the silencing of tandem repeats in the Drosophila germline (Pane, 2007).

rasiRNAs biogenesis is impaired in zuc and squ mutants: The upregulation of transposable elements and tandem repeats in the germline of zuc and squ mutants pointed to a role for the Zuc and Squ proteins in the rasiRNA pathway. Hence, attempts were made determine whether these proteins are involved in the biogenesis of the rasiRNAs or rather in the mechanism which causes the silencing of selfish genetic elements. To this aim, northern blot analysis was performed on total RNA extracted from fly ovaries and testes and probed for abundant rasiRNAs. In particular, the level of expression of two recently cloned rasiRNAs, namely the roo rasi and the Su(Ste) rasi, was measured. To minimize the background effects, the production was compared of rasiRNAs in homozygous or transheterozygous mutants versus heterozygous flies. Hybridization with an antisense oligonucleotide to roo rasi reveals that rasiRNAs are not produced in the ovaries of flies mutant for zuc, aub, and spnE. A reduction of rasiRNA levels can also be observed in the ovaries of squ mutant flies, though the production of these small RNAs is not completely abolished like in zuc, aub, and spnE mutants. Hybridization of the same membranes with an antisense oligonucleotide to miR310 shows that miRNA levels are not affected in the mutants analyzed. As a loading control a final hybridization was performed with a 2S rRNA antisense probe (Pane, 2007).

Northern blots on total RNA extracted from testes were probed with an antisense oligonucleotide to Su(Ste) rasi. This experiment revealed that, similar to aub and spnE, rasiRNAs are not produced in testes of flies mutant for zuc and squ. Also in this case, hybridization with a probe corresponding to 2S rRNA was used as a loading control (Pane, 2007).

These results demonstrate a role for zuc and squ in the biogenesis of rasiRNA in the Drosophila germline (Pane, 2007).

These studies have shown that Drosophila zuc and squ control the expression of Grk and Osk, thus affecting the axial patterning of the oocyte and future embryo. The silencing of Osk at early stages is known to be controlled by RNAi-dependent mechanisms (Cook, 2004), suggesting that Zuc and Squ are involved in RNAi processes. In support, it was found that Zuc and Squ localize to the nuage and interact with Aub, a PIWI/PAZ protein that is required for the assembly of RISC complexes in the Drosophila germline. In this tissue, RNAi ensures genomic stability by silencing selfish genetic elements (Vagin, 2006). Consistent with a role in a silencing RNAi process, the upregulation of some classes of transposable elements was observed in ovaries and expression of tandem repeats in testes of zuc and squ mutants (Pane, 2007).

Osk translation is silenced at early stages of oocyte development by the activity of RNAi-related proteins, namely Armi, Mael, Aub, and spn-E (Cook, 2004). Similar to armi, mael, aub, and spn-E, mutations in zuc and squ lead to early expression of Osk protein in stage 1–6 oocyte. miRNAs have been shown to mediate translational repression of target mRNAs by base-pairing with their 3′UTR. A computational approach revealed that osk 3′UTR contains a sequence complementary to miR-280, which is also found in a number of putative target genes, including kinesin heavy chain mRNA (Cook, 2004). However, the results reported here together with previous data (Vagin, 2006) show that miRNA biogenesis is not affected by mutations in squ, zuc, aub, armi, and spnE. Therefore, it is proposed that Zuc and Squ, together with Aub, Armi, Mael, and spn-E, might act in concert to allow the assembly of a miR-280 miRNP complex and the silencing of osk and other target genes (Pane, 2007).

Previous studies demonstrated that Aub and spn-E are implicated in the suppression of transposable element mobilization in the Drosophila germline (Aravin, 2001). This process is based on RNAi mechanisms and requires a class of siRNAs called rasiRNAs. rasiRNAs are particularly abundant in the Drosophila germline and are complementary to tandem repeats, transposable elements, and satellite DNA (Aravin, 2003). It was recently reported that rasiRNAs corresponding to retro-elements, like SINE, LINE and LTR retrotransposons, are also present in mouse oocytes, thus suggesting that a conserved RNAi machinery exists in eukaryotes that ensures genome stability by silencing selfish genetic elements. This study shows that, like aub and spn-E, zuc and squ regulate the expression of some classes of transposable elements and tandem repeats in the Drosophila germline. The expression of the Het-A and Tart retrotransposable elements was analyzed and it was found that they are upregulated in zuc and squ mutant egg chambers. In addition, expression of Ste protein, which is downregulated by dsRNA-mediated degradation of Ste mRNA in wild-type males, is activated in squ and zuc mutant males. Consistent with a role in RNAi, Zuc and Squ were shown to localize to the nuage together with Aub, and physically interact with Aub, a member of the PIWI class of Argonaute proteins. Interestingly Het-A and Tart are two non-LTR retrotransposable elements, which are implicated in the maintenance of telomere length in Drosophila. Upregulation of these transposons in the egg chambers of aub and spn-E mutant flies leads to a higher rate of transposition to the chromosome ends, resulting in telomere elongation and chromosomal abnormalities. This study shows that zuc and squ regulate the expression of Het-A and Tart, strongly suggesting that they might be involved in telomere regulation in the Drosophila germline (Pane, 2007).

In wild-type egg chambers, Grk localizes in a cap above the oocyte nucleus where it signals the dorsal identity to the surrounding follicle cells. In zuc and squ mutant egg chambers, Grk protein fails to accumulate properly in the dorsal-anterior corner of the oocyte, which results in the production of eggs with various degree of ventralization. A similar phenotype was reported for spn-B, spn-D,spn-A, and okra mutants, in which the DNA double-strand breaks induced during the meiotic recombination are not efficiently repaired. These mutations activate a meiotic checkpoint that involves the Drosophila ATR homolog Mei-41 and Chk-2/mnk. The latter is likely to promote the posttranslational modification of Vasa, a helicase with homology to eIF4A. This modification event is thought to cause the inhibition of Vasa activity and, consequently, the downregulation of grk translation. However, mutations in zuc and squ are not suppressed by mutations in mei-41, supporting the conclusion that these genes do not belong to the DNA repair class. Surprisingly, mutations in chk2/mnk are able to suppress the effects of mutation in squ and aub (Chen, 2007), but not zuc, spn-E, or piwi. This result indicates that squ and aub mutations activate a checkpoint mechanism that involves Chk2, but is not absolutely dependent on Mei-41. Similar to the DNA repair mutants, the checkpoint activity of Chk2 acts to cause the ventralized eggshell phenotype in these mutants. In contrast, zuc and spn-E mutants are not suppressed in combination with the chk2 mutant, even though it was found that Vas is posttranslationally modified in the zuc background, as has been reported for spnE mutations. This suggests that zuc and spnE may also activate the chk2-dependent checkpoint in oogenesis that modifies Vasa, a translational regulator of Grk, as seen in the DNA repair mutants. But Zuc and SpnE appear to affect oogenesis through additional mechanisms, acting not only through Chk-2. Similarly, mutations in armi were also observed to affect oogenesis at multiple levels (Cook, 2004). It is therefore plausible that Zuc, Squ, SpnE, Armi, and Aub all participate in the downregulation of selfish genetic elements, and that the retrotransposons and tandem repeats activity results in activation of Chk-2. Yet Zuc and Spn-E might have additional effects in oogenesis, similar to Armi, and those effects may be more direct and not mediated by a checkpoint mechanism (Pane, 2007).

Zuc is conserved in evolution and belongs to the phospholipase-D/nuclease superfamily, which contains several proteins with diverse functions. All the members share a conserved HKD domain that is fundamental for the catalytic activity. However, two different groups of proteins can be identified within this family. A group of proteins with two HKD domains includes human and plant PLD enzymes, cardiolipin synthase, phosphatidylserine synthase, and the murine toxin from Yersinia pestis. Members of the superfamily with one HKD domain include several bacterial endonucleases, like Nuc, and a helicase-like protein from E. coli. Zuc contains only one HKD domain and thus belongs to the subgroup of the nucleases. These enzymes have been shown to hydrolyze double-stranded RNA and DNA molecules in vitro, but little is known about their function in vivo. The results of this study demonstrate that zuc is involved in RNAi. Interestingly, it was shown that the biogenesis of the rasiRNAs does not require Dcr1 and Dcr2 and that this class of small RNAs has a different size and structure when compared to other siRNAs (Vagin, 2006). Mutations in the zuc gene impair the production of rasiRNAs, both in ovaries and testes. Therefore, Zuc is involved in the maturation of rasiRNAs and may replace Dcr1 and Dcr2 in the germline rasiRNAs mechanisms. It was recently proposed that Aub is required for the production of the rasiRNAs 5′ ends, while the nuclease implicated in the cleavage of the 3′ termini remains elusive. Given the strong interaction between Zuc and Aub and the absence of rasiRNAs in the zuc mutants, it is tempting to speculate that Zuc might be the nuclease responsible for the production of rasiRNAs 3′ ends in Drosophila. squ encodes a protein with similarity to RNase HII, which is known to degrade the RNA moiety in RNA-DNA hybrids (Itaya, 1990). Mutations in squ do not completely abolish the production of rasiRNAs in ovaries, thus suggesting that this protein might act in the actual silencing mechanism of target genes rather than in the biogenesis of the rasiRNAs. However, the analysis of Su(Ste) rasiRNAs in testes of squ mutants reveals that the Squ protein is essential for the production of rasiRNAs in this tissue. A possible explanation for these data is that Squ exerts a key function in testes together with Zuc, Aub, spnE, and Armi to ensure the proper processing of rasiRNAs. Differently, in ovaries Squ might be partially redundant since a squ paralog exists in Drosophila and might replace in part the function of Squ during oogenesis. Neither Zuc nor Squ are required for biosynthesis of microRNAs, suggesting that they are specific for the production of rasiRNAs (Pane, 2007).

In summary, this study identified the phospholipase-D/nuclease Zucchini and the RNase HII-related protein Squash as members of RNAi processes that function in the germline of Drosophila. Similar requirements for RNAi processes have also been reported for the normal development of the mammalian germline and the germline of C. elegans (Sijen, 2003), and it will be interesting to determine in the future whether Zuc and Squ homologs also participate in germline RNAi in other organisms (Pane, 2007).

Search PubMed for articles about Drosophila Zucchini

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Chen, Y., Pane, A. and Schüpbach, T. (2007). Cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila, Curr. Biol. 17: 637-642. PubMed citation: 17363252

Cook, H. H., Koppetsch, B. S., Wu, J. and Theurkauf, W. E. (2004). The Drosophila SDE3 homolog armitage is required for oskar mRNA silencing and embryonic axis specification, Cell 116: 817-829. PubMed citation: 15035984

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Koonin, E. V. (1996). A duplicated catalytic motif in a new superfamily of phosphohydrolases and phospholipid synthases that includes poxvirus envelope proteins, Trends Biochem. Sci. 21: 242-243. PubMed citation: 8755242

Pane, A., Wehr, K., Sch¸pbach, T. (2007). zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. Dev. Cell 12(6): 851-62. PubMed citation: 17543859

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date revised: 10 May 2008

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