Locus structure of Su(Ste), whose transcripts silence Stellate function

Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome (Balakireva, 1992).

The organization and transcription is reported of diverged tandemly repeated Y-linked Su(Ste) genes that are considered as suppressors of testis-expressed X-linked-repeated Stellate genes that encode a protein sharing extensive homology with beta-subunit of casein kinase 2. Clustering of restriction variants is confirmed. Size variants of Su(Ste) repeats appear to be nonhomogeneously distributed among the P1 phage clones. Different ways of Su(Ste) RNA processing because of the appearance of new splice sites and polyadenylation signals were detected. The high extent of homology between Stellate and Su(Ste) repeats suggests the possibility of Stellate suppression by antisense transcription of Su(Ste) elements. The detection of only 'sense' Su(Ste) cDNAs in testis cDNA library allows this proposal to be rejected. The genomic and cDNA clones are shown to be equally diverged. This indicates widespread rather than restricted transcription capacity of these repeats (Kalmykova, 1998).

Involvement of Homeless in regulating Stellate

The homeless gene of Drosophila encodes a member of the DE-H family of ATPase and RNA helicase proteins. Loss-of-function homeless mutations were previously found to cause female sterility with numerous defects in oogenesis, including improper formation of both the anterior-posterior and dorsal-ventral axes and failure to transport and localize key RNAs required for axis formation. One homeless mutation was also found to affect male meiosis, causing elevated X-Y nondisjunction. The role of homeless in male meiosis has been further analyzed. homeless mutations cause a variety of defects in male meiosis including nondisjunction of the X-Y and 2-2 pair, Y chromosome marker loss, meiotic drive, chromosome fragmentation, chromatin bridges at anaphase, and tripolar meiosis. In addition, homeless mutations interact with an X chromosomal factor to cause complete male sterility. These phenotypes are similar to those caused by deletion of the Suppressor of Stellate locus. Like Su(Ste) deficiencies, homeless mutants also exhibit crystals in primary spermatocytes and derepression of the X-linked Stellate locus. To determine whether the regulatory role of hls is specific for Stellate or includes other repeated sequences as well, testis RNA levels were compared for nine transposable elements; all but one, copia, are expressed at the same levels in hls mutants and wild type. Copia, however, is strongly derepressed in hls mutant males. It is concluded that hls functions along with Su(Ste) and other recently described genes to repress the Stellate locus in spermatocytes, and that it may also play a role in repressing certain other repeated sequences (Stapleton, 2001).

RISC assembly defects in the Drosophila RNAi mutant armitage: armitage is required for the silencing of Stellate

The RNA helicase Armitage is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. armi has been shown to be required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).

Silencing of the X-linked Ste gene by the highly homologous Y-linked Su(Ste) locus is an example of endogenous RNAi. In Drosophila testes, symmetrical transcription of Su(Ste) produces dsRNA, which is processed into siRNAs (Gvozdev, 2003). Su(Ste) siRNAs direct the degradation of Ste mRNA. Inappropriate expression of Ste protein in testes is diagnostic of disruption of the RNAi pathway. Both the Argonaute protein, aub, and the putative DEAD-box helicase, spn-E, have been shown to be required for RNAi in Drosophila oocytes. Both mutants fail to silence Ste, as evidenced by the accumulation of Ste protein crystals in the testes of aub and spn-E mutants. No Ste protein is detected in wild-type testes. Strikingly, Ste protein accumulates in testes of two different armi alleles, armi1 and armi72.1. Neither allele is expected to be a true null because armi1 is caused by a P element insertion 5' to the open reading frame, whereas armi72.1, which was created by an imprecise excision of the armi1 P element, corresponds to a deletion of sequences in the 5' untranslated region. Ste silencing is re-established in males homozygous for the revertant chromosome, armirev 39.2 (henceforth, armirev; Cook, 2004), which was generated by excision of the armi1 P element. These data suggest a role for Armi in Drosophila RNAi (Tomari, 2004).

Immunofluorescent detection of Ste protein in testes implicates both armi alleles in endogenous RNAi, but provides only a qualitative measure of allele strength. Since Ste protein in males reduces their fertility , the percent of embryos that hatch when mutant males are mated to wild-type (Oregon R) females provides a more quantitative measure of Ste dysregulation. Hatch rates were measured for the offspring of wild-type, armi1, armi72.1, and spn-E1 homozygous males mated to Oregon R females. For spn-E1 males, 82% of the progeny hatched. Seventy-five percent of the progeny of armi1 males hatched, but only 45% for armi72.1. In contrast, 97% of the offspring of wild-type males hatched. Thus, armi72.1 is a stronger allele than armi1, at least with respect to the requirement for armi in testes (Tomari, 2004).

In contrast to wild-type, lysates prepared from armi72.1 ovaries do not support siRNA-directed target cleavage in vitro: no cleavage product was observed in the armi72.1 lysate after 2 hr. This result was observed for more than ten independently prepared lysates. To determine if the RNAi defect was allele specific, ovaries from armi1 were tested. Phenotypically, this allele is weaker than armi72.1 in its effects on both male fertility and oogenesis. For armi72.1 females, 92% of the eggs lacked dorsal appendages, compared to 67% for armi1 eggs, and some armi1 eggs had wild-type or partially fused dorsal appendages. Consistent with its weaker phenotype, the armi1 allele showed a small amount of RNAi activity in vitro. The two alleles were analyzed together at least four times using independently prepared lysates. In all assays, total protein concentration was adjusted to be equal. Lysate from the revertant allele, armirev, which has wild-type dorsal appendages, showed robust RNAi, demonstrating that the RNAi defect in the mutants is caused by mutation of armi, not an unlinked gene (Tomari, 2004).

The rate of target cleavage was much slower for armi1 than for wild-type . Since the rate of target cleavage in this assay usually reflects the concentration of RISC, it was hypothesized that armi mutants are defective in RISC assembly. To test this hypothesis, a method to measure RISC was developed that requires less lysate than previously described techniques. Double-stranded siRNA was incubated with ovary lysate in a standard RNAi reaction. To detect RISC, a 5' 32P-radiolabled, 2'-O-methyl oligonucleotide complementary to the antisense strand of the siRNA was added. Like target RNAs, 2'-O-methyl oligonucleotides can bind to RISC containing a complementary siRNA, but unlike RNA targets, they cannot be cleaved and binding is essentially irreversible (Hutvagner, 2004). RISC/2'-O-methyl oligonucleotide complexes were then resolved by electrophoresis through an agarose gel (Tomari, 2004).

To validate the method, RISC formation was examined in embryo lysate. Four distinct complexes (C1, C2, C3, C4) were formed when siRNA was added to the reaction. Formation of these complexes required ATP and was disrupted by pre-treatment of the lysate with the alkylating agent N-ethylmaleimide (NEM), but it was refractory to NEM treatment after RISC assembly; these are all properties of RNAi itself. No complex was observed with an siRNA unrelated to the 2'-O-methyl oligonucleotide. The amount of complex formed by different siRNA sequences correlated well with their capacity to mediate cleavage. The four complexes were also detected in wild-type ovary lysate, suggesting that the same RNAi machinery is used during oogenesis and early embryogenesis. The lower amount of RISC formed in ovary compared to embryo lysates can be explained by the lower overall protein concentration of ovary lysates (Tomari, 2004).

The 2'-O-methyl oligonucleotide/native gel assay was used to analyze RISC assembly in armi mutant ovary lysates. armi mutants are deficient in RISC assembly. The extent of the deficiency correlated with allele strength: less C3/C4 complex formed in lysate from the strong armi72.1 allele than from armi1. Compared to the phenotypically wild-type armirev, >10-fold less RISC was produced in armi72.1 (Tomari, 2004).

The defect in RISC assembly in armi mutants is similar to that observed in lysates from aubHN2 ovaries. aub mutants do not support RNAi following egg activation and fail to silence the Ste locus in testes, and lysates from aubHN2 ovaries do not support RNAi in vitro. Aub is one of five Drosophila Argonaute proteins, core constituents of RISC. It is therefore not surprising that Aub is required for RISC assembly. Since RISC assembly in vitro was not detectable in aubHN2 lysates, the data suggest that Aub is the primary Argonaute protein recruited to exogenous siRNA in Drosophila ovaries. In contrast, ovaries from nanosBN, a maternal effect mutant not implicated in RNAi, are fully competent for both RISC assembly and siRNA-directed target RNA cleavage (Tomari, 2004).

Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line

To date, few natural cases of RNA-silencing-mediated regulation have been described. Repression has been analyzed of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, temporal control was found of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line (Aravin, 2004).

Stellate gene transcription yields only sense transcripts, while Su(Ste) repeats yield both sense and antisense transcripts. Expression of Stellate and sense Su(Ste) transcripts is repressed in wild-type males but antisense Su(Ste) transcripts escape silencing despite their complementarity to short RNAs. Antisense RNAs accumulate in the nucleoplasm and are not transported into the cytoplasm. This result supports the proposal that nonpolyadenylated antisense RNAs escape the cytoplasmic degradation machinery because they are sequestered in the nucleus (Aravin, 2004).

Sense transcripts are localized in nuclei of mature wild-type primary spermatocytes. In cry1Y males, in which the Stellate genes are derepressed, these transcripts are found only in the cytoplasm. These results correspond to the accumulation of the Stellate-coded protein as crystalline aggregates in the cytoplasm of mature primary spermatocytes of cry1Y males. The total amount of Stellate and Su(Ste) sense transcripts is greatly increased in cry1Y males. The absence of a nuclear signal in cry1Y males, contrasts with the presence of sense transcripts in wild-type nuclei, therefore suggests that these transcripts are never released from the wild-type nucleus. Nuclear retention of sense transcripts in the wild type might be explained by the interaction between sense and antisense transcripts. Nuclear localization of sense and antisense transcripts has also been observed for bidirectionally transcribed white transgenes, which induce RNAi of the endogenous white gene (Aravin, 2004).

The distinct sharp dots observed in the nuclei for both sense and antisense RNAs in mature primary spermatocytes may correspond to the accumulation of the native transcript at the sites of transcription. The signals are often located at the border between the chromatin (DAPI stained) and the nucleoplasmic areas of the nucleus, where actively transcribed loci are thought to be located. Restricted nuclear signals corresponding to the sites of transcription have been observed for a number of genes, whereas transcripts in the process of export from the nucleus are usually below the detection sensitivity of the standard in situ hybridization technique. The colocalization of the sense and antisense transcripts suggests the formation of dsRNA in the nucleus, thus placing the initiation of Stellate silencing in the nucleus. It is proposed that these nuclear dsRNA species may involve hybrids between sense and antisense Su(Ste) transcripts, as well as between sense Stellate and antisense Su(Ste) transcripts, and that these hybrids are essential for Stellate silencing by Su(Ste) (Aravin, 2004).

A strong correlation is observed between Stellate silencing and the presence in testes of sense and antisense 25- to 27-nt RNAs homologous to Stellate and Su(Ste) sequences. The short RNAs are absent when Stellate genes are derepressed as a consequence of either a Su(Ste) locus deletion or mutations in the aub and spn-E genes. The cloning of short RNA from D. melanogaster testes also demonstrates the presence of short RNAs that are derived from Su(Ste) and are highly homologous to Stellate. A rigid size restriction of 21 to 23 nt has, however, been observed for siRNA in various in vitro studies of D. melanogaster RNAi. Examination of Dicer activity with different dsRNAs suggests a strong specificity of processing to 21- to 23-nt fragments in both Drosophila embryo extracts and cell culture. Furthermore, investigation of the functional anatomy of chemically synthesized siRNAs in embryo extracts defined the optimal length of siRNAs as 21 to 23 nt, while RNAs longer than 24 nt have practically no cognate-mRNA cleavage activity. It has been proposed that only RNAs that meet this size requirement can be loaded into the RISC. However, examples of the existence of two size classes of short RNAs (21 or 22 nt and 24 to 26 nt) involved in silencing have also been reported. Two different size variants of short RNAs were observed during artificial silencing in plants, with the short variant responsible for posttranscriptional gene silencing and the long one most likely participating in DNA methylation and spreading of the silencing signal. Furthermore, only RNAs from the long class have been detected that correspond to endogenous plant transposable elements. Two size classes of short RNAs are produced from dsRNA in plant extracts, and the activity of different Dicer proteins was shown to be responsible for producing each class. Cloning of endogenous short RNAs from D. melanogaster has also identified two size classes of short RNAs, with the short class (21 to 23 nt) including microRNAs and the long class (24 to 26 nt) comprising sequences derived from transcripts of transposable elements and other repetitive heterochromatic sequences (Aravin, 2004).

The larger size of the short Su(Ste) RNA may be explained by specific sequences affecting dsRNA processing by Dicer or by the presence in testes of specific factors that change the cleavage interval of dsRNA. However, exogenous Su(Ste) dsRNA is cleaved into 21- to 23-nt siRNA in testis extracts, most likely reflecting the activity of the same Dicer protein that acts in somatic tissues. The hypothesis is favored that the 25- to 27-nt Su(Ste) RNAs detected in vivo are produced by a mechanism at least partially different from conventional siRNA production. A clue to the origin of the short Su(Ste) RNAs comes from the finding that Su(Ste) dsRNA formation occurs in the nucleus, unlike that of artificial RNAi, in which dsRNA is believed to be processed in the cytoplasm. Both conventional-size siRNA and a longer short RNA have been observed during viroid replication in the plant nucleus. Two size classes of short RNAs may be produced in D. melanogaster by different Dicer proteins, as has been demonstrated in plants. Alternatively, specific nuclear factors may affect how a single Dicer protein processes dsRNA in the nucleus (Aravin, 2004).

Mutations in the aub and spn-E genes lead to elimination of short Su(Ste) RNA in testes. However, neither mutation affects processing of exogenously provided dsRNA to 21- to 23-nt siRNA in testis extracts. It has been observed that both aub and spn-E mutations block RNAi in oocytes produced by injected dsRNA. It has been proposed that both proteins affect RNAi because of their involvement in translational control, but the results suggest that Aub and Spn-E may be involved in the production and/or stabilization of siRNA. Similarly, the rde-1 and mut-7 genes of Caenorhabditis elegans are required for the production of siRNA in vivo but are dispensable for dsRNA processing in vitro. The corresponding proteins are required for long-term stabilization of siRNA rather than for dsRNA processing (Aravin, 2004).

The aub and spn-E mutations eliminate the short Su(Ste) RNA without affecting the abundance of two different microRNAs in testes. It is proposed that distinct protein complexes mediate production and/or stabilization of short Su(Ste) RNA and microRNAs in testes. Similarly, different members of the Argonaute family participate in artificial RNAi and in microRNA processing in C. elegans and plants, despite the central role of Dicer in both processes (Aravin, 2004).

Homologous silencing mediated by short RNA may occur by posttranscriptional degradation of mRNA and by DNA and chromatin modification leading to transcriptional repression. The nuclear antisense RNA accumulation and dsRNA formation that was found in this study raises the question of whether posttranscriptional or transcriptional mechanisms of silencing operate in Stellate repression. For animals, it is generally believed that artificial RNAi caused by dsRNA leads to posttranscriptional degradation of mRNA. However, it has been shown that dsRNA or short RNA can affect transcription and chromatin structure of homologous sequences in plants and Saccharomyces cerevisiae. In plants, for example, transcriptional silencing of reporter constructs can be caused if the dsRNA produced by hairpin constructs or virus infection is homologous to the untranscribed promoter region of the target gene, while posttranscriptional degradation of the corresponding mRNA occurs if there is homology between the dsRNA and the transcribed sequence (Aravin, 2004).

Constructs containing the Stellate coding sequence driven by a heterologous promoter are regulated by Su(Ste) repeats in the same manner as native Stellate genes or reporter constructs with Stellate sequence fused to lacZ. In contrast, expression of the endogenous ßNac-like genes, having a putative promoter region with high levels of sequence similarity (95%) to Stellate but an unrelated transcribed sequence, shows no response to the deletion of Su(Ste). In the present study, it was also found that nucleotide substitutions in the transcribed region of a Stellate fragment homologous to the Su(Ste) sequence lead to a release of silencing. Thus, homology to Su(Ste) in the untranscribed region is dispensable for repression, while local disturbance of complementarity in the transcribed sequence impairs silencing. The possibility that regulatory sequences important for transcriptional silencing may be present in the transcribed region cannot be ruled out, but the results are more simply explained by a posttranscriptional Stellate silencing mechanism (Aravin, 2004).

The two blocks of tandemly repeated Stellate genes are located in intercalary and constitutive heterochromatin of the X chromosome, and Su(Ste) repeats are located in the heterochromatic Y chromosome. siRNA-mediated transcriptional repression of centromeric heterochromatin repeats has been recently demonstrated in S. cerevisiae. The results do not exclude participation of transcriptional repression of genomic Stellate repeats acting in concert with a posttranscriptional mechanism. Similarly, both transcriptional and posttranscriptional mechanisms have been shown to operate in the repression of multicopy transgenes associated with the presence of homologous short RNA in D. melanogaster. Thus, both transcriptional and posttranscriptional mechanisms might act in Stellate silencing, and further studies will be directed to understanding the contribution of each of them (Aravin, 2004).

Loquacious and processing of Stellate

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqsf00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).

loqsf00791 males are incompletely fertile. When Oregon R females were mated to loqsf00791 homozygous mutant males, only 17% of embryos hatched; for loqsf00791 heterozygous males, 47% of embryos hatched. Ninety percent of embryos hatched for wild-type Oregon R males. Genes required for RNA silencing often reduce male fertility, because the X-linked gene Ste is epigenetically silenced in testes by dsRNA derived from the bi-directionally transcribed Suppressor of Stellate (Su(Ste)) locus. Ste silencing is genetically similar, but not identical, to RNAi, in that like RNAi it requires the function of the gene armitage (armi), but unlike RNAi does not require r2d2. In the absence of Ste silencing, Stellate protein accumulates as protein crystals in the testes. loqsf00791 mutants contain Stellate crystals in their testes, much like armi72.1 mutants, identifying a second role for loqs in silencing by endogenous RNA triggers, distinct from its function in miRNA biogenesis (Forstemann, 2005).

A distinct small RNA pathway silences selfish genetic elements in the germline

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. These data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways (Vagin, 2006).

In plants and animals, RNA silencing pathways defend against viruses, regulate endogenous gene expression, and protect the genome against selfish genetic elements such as retrotransposons and repetitive sequences. Common to all RNA silencing pathways are RNAs 19 to 30 nucleotides (nt) long that specify the target RNAs to be repressed. In RNA interference (RNAi), siRNAs are produced from long exogenous double-stranded RNA (dsRNA). In contrast, ~22-nt miRNAs are endonucleolytically processed from endogenous RNA polymerase II transcripts. Dicer ribonuclease III (RNase III) enzymes produce both siRNAs and miRNAs. In flies, Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)–Loquacious (Loqs) complex produces miRNAs. After their production, small silencing RNAs bind Argonaute proteins to form the functional RNA silencing effector complexes believed to mediate all RNA silencing processes (Vagin, 2006 and references therein).

In Drosophila, processive dicing of long dsRNA and the accumulation of sense and antisense siRNAs without reference to the orientation of the target mRNA are hallmarks of RNAi in vitro. Total small RNA was prepared from the heads of adult males expressing a dsRNA hairpin that silences the white gene via the RNAi pathway. white silencing requires Dcr-2, R2D2, and Ago2. siRNAs were detected with a microarray containing TM (melting temperature)–normalized probes, 22 nt long, for all sense and antisense siRNAs that theoretically can be produced by dicing the white exon 3 hairpin. Both sense and antisense white siRNAs were detected in wild-type flies but not in dcr-2L811fsX homozygous mutant flies. The Dcr-2–dependent siRNAs were produced with a periodicity of ~22 nt, consistent with the phased processing of the dsRNA hairpin from the end formed by the 6-nt loop predicted to remain after splicing of its intron-containing primary transcript (Vagin, 2006).

Drosophila repeat-associated small interfering RNAs (rasiRNAs) can be distinguished from siRNAs by their longer length, 24 to 29 nt. rasiRNAs have been proposed to be diced from long dsRNA triggers, such as the ~50 copies of the bidirectionally transcribed Suppressor of Stellate [Su(Ste)] locus on the Y chromosome that in testes silence the ~200 copies of the protein-coding gene Stellate (Ste) found on the X chromosome (Vagin, 2006).

Microarray analysis of total small RNA isolated from fly testes revealed that Su(Ste) rasiRNAs detectably accumulate only from the antisense strand, with little or no phasing. As expected, Su(Ste) rasiRNAs were not detected in testes from males lacking the Su(Ste) loci (cry1Y). Su(Ste) rasiRNAs were also absent from armitage (armi) mutant testes, which fail to silence Ste and do not support RNAi in vitro. armi encodes a non–DEAD-box helicase homologous to the Arabidopsis thaliana protein SDE3, which is required for RNA silencing triggered by transgenes and some viruses, and depletion by RNAi of the mammalian Armi homolog Mov10 blocks siRNA-directed RNAi in cultured human cells. Normal accumulation of Su(Ste) rasiRNA and robust Ste silencing also require the putative helicase Spindle-E (Spn-E), a member of the DExH family of adenosine triphosphatases (Vagin, 2006).

The accumulation in vivo of only antisense rasiRNAs from Su(Ste) implies that sense Su(Ste) rasiRNAs either are not produced or are selectively destroyed. Either process would make Ste silencing mechanistically different from RNAi. In support of this view, mutations in the central components of the Drosophila RNAi pathway—dcr-2, r2d2, and ago2—did not diminish Su(Ste) rasiRNA accumulation. Deletion of the Su(Ste) silencing trigger (cry1Y) caused a factor of ~65 increase in Ste mRNA, but null or strong hypomorphic mutations in the three key RNAi proteins did not (Vagin, 2006).

Fly Argonaute proteins can be subdivided into the Ago (Ago1 and Ago2) and Piwi [Aubergine (Aub), Piwi, and Ago3] subfamilies. Unlike ago1 and ago2, the aub, piwi, and ago3 mRNAs are enriched in the germline. Aub is required for Ste silencing and Su(Ste) rasiRNA accumulation. In aubHN2/aubQC42 trans-heterozygous mutants, Su(Ste) rasiRNAs were not detected by microarray or Northern analysis, and Su(Ste)-triggered silencing of Ste mRNA was lost completely. Even aubHN2/+ heterozygotes accumulated less of the most abundant Su(Ste) rasiRNA than did the wild type. That the Ago subfamily protein Ago2 is not required for Ste silencing, whereas the Piwi subfamily protein Aub is essential for it, supports the view that Ste is silenced by a pathway distinct from RNAi. Intriguingly, Su(Ste) rasiRNAs hyperaccumulated in piwi mutant testes, where Ste is silenced normally (Vagin, 2006).

Mutations in aub also cause an increase in sense, but not antisense, Su(Ste) RNA; these results suggest that antisense Su(Ste) rasiRNAs can silence both Ste mRNA and sense Su(Ste) RNA, but that no Su(Ste) rasiRNAs exist that can target the antisense Su(Ste) transcript. The finding that Su(Ste) rasiRNAs are predominantly or exclusively antisense is essentially in agreement with the results of small RNA cloning experiments, in which four of five Su(Ste) rasiRNAs sequenced were in the antisense orientation, but is at odds with earlier reports detecting both sense and antisense Su(Ste) rasiRNAs by non-quantitative Northern hybridization (Vagin, 2006).

Is germline RNA silencing of selfish genetic elements generally distinct from the RNAi and miRNA pathways? The expression of a panel of germline-expressed selfish genetic elementswas examined in mutants defective for eight RNA silencing proteins: three long terminal repeat (LTR)-containing retrotransposons (roo, mdg1, and gypsy); two non-LTR retrotransposons (I-element and HeT-A, a component of the Drosophila telomere), and a repetitive locus (mst40). All selfish genetic elements tested behaved like Ste: Loss of the RNAi proteins Dcr-2, R2D2, or Ago2 had little or no effect on retrotransposon or repetitive element silencing. Instead, silencing required the putative helicases Spn-E and Armi plus one or both of the Piwi subfamily Argonaute proteins, Aub and Piwi. Silencing did not require Loqs, the dsRNA-binding protein required to produce miRNAs (Vagin, 2006).

The null allele dcr-1Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1Q1147X/dcr-1Q1147X adult females. Therefore, clones of dcr-1Q1147X/dcr-1Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovoD1. RNA levels, relative to rp49 mRNA, were measured for three retrotransposons (roo, HeT-A, and mdg1) and one repetitive sequence (mst40) in dcr-1/dcr-1 recombinant ovary clones and in ovoD1/TM3 and dcr-1/ovoD1 nonrecombinant ovaries. The ovoD1 mutation blocks oogenesis at stage 4, after the onset of HeT-A and roo rasiRNA production. Retrotransposon or repetitive sequence transcript abundance was unaltered or decreased in dcr-1/dcr-1 relative to ovoD1/TM3 and dcr-1/ovoD1 controls. It is concluded that Dcr-1 is dispensable for silencing these selfish genetic elements in the Drosophila female germline (Vagin, 2006).

roo is the most abundant LTR retrotransposon in flies. roo silencing was analyzed in the female germline with the use of microarrays containing 30-nt probes, tiled at 5-nt resolution, for all ~18,000 possible roo rasiRNAs; the data was corroborated at 1-nt resolution for those rasiRNAs derived from LTR sequences. As observed for Su(Ste) but not for white RNAi, roo rasiRNAs were nonhomogeneously distributed along the roo sequence and accumulated primarily from the antisense strand. In fact, the most abundant sense rasiRNA peak corresponded to a set of probes containing 16 contiguous uracil residues, which suggests that these probes nonspecifically detected fragments of the mRNA polyadenylate [poly(A)] tail. Most of the remaining sense peaks were unaltered in armi mutant ovaries, in which roo expression is increased; this result implies that they do not contribute to roo silencing. No phasing was detected in the distribution of roo rasiRNAs (Vagin, 2006).

As for Su(Ste), wild-type accumulation of antisense roo rasiRNA required the putative helicases Armi and Spn-E and the Piwi subfamily Argonaute proteins Piwi and Aub, but not the RNAi proteins Dcr-2, R2D2, and Ago2. Moreover, accumulation of roo rasiRNA was not measurably altered in loqs f00791, an allele that strongly disrupts miRNA production in the female germline (Vagin, 2006).

Loss of Dcr-2 or Dcr-1 did not increase retrotransposon or repetitive element expression, which suggests that neither enzyme acts in rasiRNA-directed silencing. Moreover, loss of Dcr-2 had no detectable effect on Su(Ste) rasiRNA in testes or roo rasiRNA in ovaries. The amount of roo rasiRNA and miR-311 was measured in dcr-1/dcr-1 ovary clones generated by mitotic recombination. Comparison of recombinant (dcr-1/dcr-1) and nonrecombinant (ovoD1/TM3 and dcr-1/ovoD1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1Q1147X mutation. Pre–miR-311 increased and miR-311 declined by a factor of ~3 in the dcr-1/dcr-1 clones, consistent with about two-thirds of the tissue corresponding to mitotic dcr-1/dcr-1 recombinant cells. Yet, although most of the tissue lacked dcr-1 function, improved, rather than diminished, silencing was observed for the four selfish genetic elements examined. Moreover, the dsRNA-binding protein Loqs, which acts with Dcr-1 to produce miRNAs, was also dispensable for roo rasiRNA production and selfish genetic element silencing. Although the possibility that dcr-1 and dcr-2 can fully substitute for each other in the production of rasiRNA in the ovary cannot be excluded, biochemical evidence suggests that none of the three RNase III enzymes in flies—Dcr-1, Dcr-2, and Drosha—can cleave long dsRNA into small RNAs 24 to 30 nt long (Vagin, 2006).

Animal siRNA and miRNA contain 5' phosphate and 2',3' hydroxy termini. Enzymatic and chemical probing was used to infer the terminal structure of roo and Su(Ste) rasiRNAs. RNA from ovaries or testes was treated with calf intestinal phosphatase (CIP) or CIP followed by polynucleotide kinase plus ATP. CIP treatment caused roo and Su(Ste) rasiRNA to migrate more slowly in polyacrylamide gel electrophoresis, consistent with the loss of one or more terminal phosphate groups. Subsequent incubation with polynucleotide kinase and ATP restored the original gel mobility of the rasiRNAs, indicating that they contained a single 5' or 3' phosphate before CIP treatment. The roo rasiRNA served as a substrate for ligation of a 23-nt 5' RNA adapter by T4 RNA ligase, a process that requires a 5' phosphate; pretreatment with CIP blocked ligation, thus establishing that the monophosphate lies at the 5' end. The rasiRNA must also contain at least one terminal hydroxyl group, because it could be joined by T4 RNA ligase to a preadenylated 17-nt 3' RNA adapter. Notably, the 3' ligation reaction was less efficient for the roo rasiRNA than for a miRNA in the same reaction (Vagin, 2006).

RNA from ovaries or testes was reacted with NaIO4, then subjected to ß-elimination, to determine whether the rasiRNA had either a single 2' or 3' terminal hydroxy group or had terminal hydroxy groups at both the 2' and 3' positions, as do animal siRNA and miRNA. Only RNAs containing both 2' and 3' hydroxy groups react with NaIO4; ß-elimination shortens NaIO4-reacted RNA by one nucleotide, leaving a 3' monophosphate terminus, which adds one negative charge. Consequently, NaIO4-reacted, ß-eliminated RNAs migrate faster in polyacrylamide gel electrophoresis than does the original unreacted RNA. Both roo and Su(Ste) rasiRNA lack either a 2' or a 3' hydroxyl group, because they failed to react with NaIO4; miRNAs in the same samples reacted with NaIO4. Together, these results show that rasiRNAs contain one modified and one unmodified hydroxyl. Because T4 RNA ligase can make both 3'-5' and 2'-5' bonds, the blocked position cannot currently be determined. Some plant small silencing RNAs contain a 2'-O-methyl modification at their 3' terminus (Vagin, 2006).

Drosophila and mammalian siRNA and miRNA function through members of the Ago subfamily of Argonaute proteins, but Su(Ste) and roo rasiRNAs require at least one member of the Piwi subfamily for their function and accumulation. To determine whether roo rasiRNAs physically associate with Piwi and Aub, ovary lysate were prepared from wildtype flies or transgenic flies expressing either myc-tagged Piwi or green fluorescent protein (GFP)–tagged Aub protein; they were immunoprecipitated with monoclonal antibodies (mAbs) to myc, GFP, or Ago1; and then the supernatant and antibody-bound small RNAs were analyzed by Northern blotting. Six different roo rasiRNAs were analyzed. All were associated with Piwi but not with Ago1, the Drosophila Argonaute protein typically associated with miRNAs; miR-8, miR-311, and bantam immunoprecipitated with Ago1 mAb. No rasiRNAs immunoprecipitated with the myc mAb when lysate was used from flies lacking the myc-Piwi transgene (Vagin, 2006).

Although aub mutant ovaries silenced roo mRNA normally, they showed reduced accumulation of roo rasiRNA relative to aub/+ heterozygotes, which suggests that roo rasiRNAs associate with both Piwi and Aub. The supernatant and antibody-bound small RNAs were analyzed after GFP mAb immunoprecipitation of ovary lysate from GFP-Aub transgenic flies and flies lacking the transgene. roo rasiRNA was recovered only when the immunoprecipitation was performed with the GFP mAb in ovary lysate from GFP-Aub transgenic flies. The simplest interpretation of these data is that roo rasiRNAs physically associate with both Piwi and Aub, although it remains possible that the roo rasiRNAs are loaded only into Piwi and that Aub associates with Piwi in a stable complex. The association of roo rasiRNA with both Piwi and Aub suggests that piwi and aub are partially redundant, as does the modest reduction in roo silencing in piwi but not in aub mutants. Alternatively, roo silencing might proceed through Piwi alone, but the two proteins could function in the same pathway to silence selfish genetic elements (Vagin, 2006).

These data suggest that in flies, rasiRNAs are produced by a mechanism that requires neither Dcr-1 nor Dcr-2, yet the patterns of rasiRNAs that direct roo and Ste silencing are as stereotyped as the distinctive siRNA population generated from the white hairpin by Dcr-2 or the unique miRNA species made from each pre-miRNA by Dcr-1. A key challenge for the future will be to determine what enzyme makes rasiRNAs and what sequence or structural features of the unknown rasiRNA precursor lead to the accumulation of a stereotyped pattern of predominantly antisense rasiRNAs (Vagin, 2006).

The Ago2-RISC-assembly pathway; Hen1 modifies germline piRNAs and single-stranded siRNAs in RISC

Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways. In the RNA interference (RNAi) pathway, small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons. This study reports that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs and mouse and rat piRNAs. Plant small-RNA methylation is catalyzed by the protein HEN1. Drosophila melanogaster Hen1 (DmHen1), the Drosophila homolog of HEN1, termed Pimet (piRNA methyltransferase) by Saito (2007) in a parallel study, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs, which derive from double-stranded precursors, and piRNAs which do not. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after the Argonaute-bound siRNA duplex is converted to single-stranded RNA (Horwich, 2007; Saito, 2007).

The 3' termini of two types of highly abundant piRNAs were examined in the germline of flies heterozygous or homozygous for hen1f00810. In testes, the Suppressor of Stellate [Su(Ste)] locus produces 24-27 nucleotide rasiRNAs, a subclass of piRNAs that directs silencing of the selfish genetic element Stellate. Su(Ste) rasiRNAs, like other Drosophila piRNAs, are modified at their 3' termini and therefore do not react with NaIO4. In contrast, Su(Ste) rasiRNAs from hen1f00810/hen1f00810 mutant testes reacted with NaIO4 and could therefore be β-eliminated to remove the last nucleotide of the RNA, thereby increasing their gel mobility and indicating that in the absence of DmHen1 protein, they are not modified. Similarly, rasiRNAs that guide silencing of roo, the most abundant retrotransposon in Drosophila melanogaster, were not modified in hen1f00810 homozygous ovaries. The Su(Ste) and roo rasiRNAs were also shorter in the hen1f00810 homozygotes. In contrast, the length and amount of miR-8, which is expressed in both the male and female germline, was unaltered in hen1f00810 homozygotes. For both Su(Ste) and roo, rasiRNAs were on average shorter and less modified even in hen1f00810 heterozygotes, compared to the wild-type, suggesting that the abundance of DmHen1 protein limits the stability or production of piRNAs in of piRNAs in flies (Horwich, 2007).

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

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).

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 zucHm27/+ and transheterozygous zucHm27/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 zucHm27/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 squHE47/squPP32 mutant females as compared to the control squHE47/+ 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).

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).

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, 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. 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. 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. However, the results reported here together with previous data 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. 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. 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. 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. 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, 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).

Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons

The canalization concept describes the resistance of a developmental process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways, indicate that it is a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. This study offers an additional, perhaps alternative, explanation for proposals of a concrete mechanism underlying canalization. This study shows that, in Drosophila, functional alterations of Hsp90 affect the Piwi-interacting RNA (piRNA; a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the induction of morphological mutants. This indicates that Hsp90 mutations can generate new variation by transposon-mediated 'canonical' mutagenesis (Specchia, 2010).

In Drosophila, primary spermatocytes of Hsp90 mutant males exhibit crystalline aggregates, usually absent in wild-type testes, the formation of which is due to the transcriptional activation, in male germ line, of the repeated Stellate (Ste) elements. Such elements encode a protein, similar to the β-subunit of casein kinase 2 (CK2), that is the main component of the crystalline aggregates. To test the specificity of Hsp90 mutations in inducing crystalline aggregates, spermatocytes were analyzed of wild-type males treated with the Hsp90 inhibitor geldanamycin, and crystalline aggregates and a significant amount of Stellate transcript were found (Specchia, 2010).

The silencing of the Stellate sequences is mediated by RNA interference (RNAi) mechanism and mutations in genes involved in RNAi, such as aubergine, armitage and spindle E (also called homeless), activate Stellate in testes of mutant males. It has been shown that Stellate is repressed by a piRNA-mediated mechanism that is specific for repetitive sequences and transposon silencing. Consistently, it was found that Hsp90 mutations affect the biogenesis of piRNAs specific for Stellate and transposons. These results prompted a test, in ovaries and testes, for possible effects of Hsp90 mutations on the expression of long terminal repeat (LTR) springer, opus, roo and aurora, non-LTR I elements that transpose by an RNA intermediate, and invert repeat (IR) Bari1, an element that transposes by a DNA intermediate. Homozygous hsp83scratch and trans-heterozygous hsp83scratch/hsp83e4A mutants were analyzed. Hsp83 is the denomination of Hsp90 in Drosophila; hsp83scratch is a viable hypomorphic male sterile mutation and hsp83e4A is a lethal amorphic mutation. The expression of the same transposons was also tested in ovaries and testes of wild-type Oregon-R (Ore-R) flies treated with geldanamycin. In mutant ovaries and testes the amount of transcripts increases significantly, although differentially, for all the transposons. The increase is more abundant in trans-heterozygous than in homozygous mutants and in ovaries than in testes. Treatment with geldanamycin induces a significant increase of all the transposon transcripts, except for the I element, but only in testes (Specchia, 2010).

Southern blot analysis was used to look at the effect of Hsp90 mutations on transposon mobility. To compare genomes homogeneously, DNA was extracted from single heterozygous flies (the parents) and from single homozygous flies of F1 progeny (males and females); all the DNA genomic samples were digested with HindIII. Hybridization patterns were examined with aurora, I and Bari1, where significant differences between parents and F1 progeny are evident for each element. New bands in the DNA of the F1 progeny were found compared to the parental DNA, thus suggesting a mutation-induced mobilization of these elements. Similar results were obtained with springer, opus and roo. To ensure that the effects on transposons are due exclusively to the homozygosis of the hsp83scratch mutation and not to the genetic background of the mutagenized III chromosome, the same experiments were done with aurora, I and Bari1 in trans-heterozygous hsp83scratch/hsp83e4A mutants with similar results. In addition, no significant mobilization or increased transcription of the same elements were found in a homozygous hsp83rev22 revertant strain (Specchia, 2010).

Because active transposons are mutagenic, these data suggest that the phenotypic variation observed in Hsp90 mutants could be due to de novo mutations produced by activated transposons. To test this possibility, 3,220 hsp83scratch homozygous flies were screened and 30 flies were found with morphological abnormalities, corresponding to a frequency of about 1%. This is similar to the frequency previously reported in some mutant stocks. Also 3,220 flies from an Ore-R stock were analyzed without finding any morphological abnormalities. Among the abnormalities observed, there was a fly resembling the dominant mutation Scutoid. Because the Scutoid phenotype is caused by mutations in the no ocelli (noc) gene, this gene was molecularly analysed in the phenotypic variant. Two pairs of primers were chosed that in the wild-type gene amplify two DNA fragments of 2,100bp and 3,400bp. The 2,100-bp fragment is present in both the Sco-like mutant and in Ore-R, whereas the 3,400-bp fragment is detected only in the wild type. Inverse polymerase chain reaction (PCR) analysis showed that the noc gene is interrupted at nucleotide 1394 of the cDNA sequence by an I element-like sequence. Consistent with this observation is the amplification of a DNA fragment obtained by using an upper primer (the position of the noc gene) and a lower primer corresponding to the I element at the 1243 nucleotide of the M14954 sequence. After sequencing it was found that this fragment encodes a Noc truncated protein, which cannot function as a transcriptional factor owing to the loss of the zinc-finger domain. The noc sequence was also analyzed in the DNA extracted from about 1,000 phenotypically wild-type flies collected during the screening, and only the normal noc sequence was found. This indicates that the Scutoid phenotype that was found was caused by a de novo mutation instead of being the expression of a pre-existing cryptic mutation. The impairment of piRNA biogenesis by Hsp83 indicated that other mutations affecting transposon activity might also induce phenotypic variation. To test this possibility, a screen was made for morphological variants in a stock carrying spindle Ec00786, a male sterile mutation at the spindle E gene. It has helicase activity, de-represses Stellate sequences and transposon, and is involved in piRNA biogenesis. 1,500 flies were screened, and 19 morphological variants were found, a similar rate to that found in some Hsp90 mutants strains (1%-2%). The transposons are activated in the germ line of male and female spindle E mutants. This further indicates that the expression of morphological variability could be related to the disruption of the piRNA silencing mechanism (Specchia, 2010).

These data clearly show a novel function of Hsp90 with important implications for the current hypothesis about this protein as a capacitor of morphological evolution. Hsp90 is involved in stress responses and the expression of wide ranging morphological changes in Drosophila and other organisms. A reduced amount of Hsp90 can induce abnormal developmental phenotypic variations; these morphogenetic changes can become fixed and stably transmitted even if wild-type Hsp90 function is restored in subsequent generations. The current interpretation is that Hsp90 buffers pre-existing genetic variation that is not expressed and accumulates in neutral conditions; its mutations will then induce the expression of this variation (Rutherford, 1998). This stress-sensitive storage and release of genetic variation by Hsp90 would favour adaptive evolution. The hypothesis of pre-existing genetic variation has been based on the observation that, when flies carrying Hsp83 alleles were outcrossed to different laboratory stocks, the observed defects were typical for each outcross, thus suggesting that the defects depended on specific genetic backgrounds (Rutherford, 1998). Those data could be also explained in the light of the current results. There is evidence that different genetic backgrounds may induce different transposon insertions (Specchia, 2010).

The demonstration that Hsp90 is involved in the control of transcription and mobilization of transposable elements in germ cells by affecting piRNA biogenesis strongly suggests that the reduction of Hsp90 causes a stress-response-like activation and transposition of mobile elements affecting piRNA silencing. This in turn would induce de novo gene mutations that affect development pathways and that can be expressed and fixed across subsequent generations. This explanation agrees with the suggestion that transposable element activity could be a response to stress. The results do not exclude, however, that Hsp90 could be both a buffering factor as well as a suppressor of transposable element (TE)-induced mutations (Specchia, 2010).

These data indicate an additional, if not alternative, mechanism to the canalization and assimilation hypothesis based on a link between stress and transposon activity through piRNA-mediated silencing. This mechanism also potentially provides another molecular interpretation with respect to the vague capacitor concept. It is not yet known if Hsp90 also has a direct role in piRNA biogenesis, or is only involved in triggering the stress response leading to transposon activation (Specchia, 2010).

The Drosophila fragile X mental retardation protein participates in the piRNA pathway

RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. This study demonstrates for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. These data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system (Bozzetti, 2015).

dFmr1 is a translational regulator and its role in the miRNA pathway is widely accepted. This study provides several lines of evidence that dFmr1 can be considered as a ‘bona fide’ member of the piRNA pathway that keeps repetitive sequences and transposons silenced. First, dFmr1 mutant testes display crystalline aggregates, as do other mutants of the piRNA pathway. Second, the levels of cry (Suppressor of Stellate)-specific and transposon-specific piRNAs dramatically decrease in dFmr1 mutant testes. Third, as a consequence of this decrease, the Ste RNA is produced and, in addition, transposons are expressed at higher levels than in wt animals. Fourth, dFmr1 mutant animals display fertility defects, a phenotype shown by several mutations affecting the piRNA pathway. The fact that earlier screens did not identify dFmr1 as a member of the somatic piRNA pathway could be due to the heterogeneous phenotypes observed with the somatic transposons (this study) and/or to the material used for those assays. The crySte system thus proves very efficient for identifying new members of this important pathway (Bozzetti, 2015).

The movement of transposable elements is one of the molecular causes of DNA instability and sterility. Considering that human patients mutant for FMRP also display defects in male and female gonads, it will be interesting to characterize the activity of transposons and repetitive sequences in the gonads of mice or humans that are mutant for the FMRP pathway, although there might be no observable defects in mammals because they express three members of the FMRP family versus the single ortholog in fly. Finally, mutations affecting the piRNA pathway might also induce gonadal defects in humans (Bozzetti, 2015).

Until now, the members of the piRNA pathway controlling the crySte interaction, including Aub, have been described as being required in the male germline. Surprisingly, the conditional dFmr1 rescue and KD experiments demonstrate that dFmr1 controls the piRNA pathway both in the germline and in the somatic cells of the gonad, which raises questions as to the somatic contribution of other members of the piRNA pathway in the male gonad. The phenotypes induced by somatic Aub expression also suggest that the hub expresses one or more AGO proteins that are involved in the somatic piRNA-mediated Ste silencing and that interact with dFmr1; however, the only other protein of the Piwi clade present in the somatic tissue, Piwi, does not participate in Ste silencing. Based on preliminary data, this study proposes that AGO1 might be one such protein. First, AGO1/+ testes display Ste-made crystals, as do testes expressing UAS-AGO1 RNAi driven by the upd-Gal4 driver. Second, aubsting rescues the AGO1-mediated crystal phenotype. Third, AGO1 and dFmr1 interact biochemically and are known to interact genetically in the ovaries to control germline stem cell maintenance, as well as in the nervous system, where they modulate synaptic plasticity. Taken together, these data suggest that AGO1 contributes to the piRNA pathway that controls the cry–Ste system in the somatic part of the gonad (Bozzetti, 2015).

The finding that Aub somatic expression affects the NMJ and counteracts the AGO1 loss of function phenotype is also unexpected. Recent work has documented the activation of piRNA pathway in the nervous system in flies, mice, humans and molluscs and it has been proposed that synaptic plasticity, cognitive functions and neurodegeneration might involve the control of genome stability, even though the precise mode of action and impact of this pathway are not completely understood. Because Aub is not required in the larval somatic tissues, its ectopic expression could affect the NMJ by replacing AGO1 in its known role on the miRNA pathway. However, AGO1 might also affect the NMJ through the piRNA pathway, much in the same way as AGO1 loss of function affects a piRNA pathway in the gonad. Even though AGO1 has been previously described as being exclusively involved in the miRNA pathway, some degree of overlapping between different RNAi pathways has been recently described: (1) the double-stranded-RNA-binding protein Loquacious (Loqs) is involved in the miRNA pathway and in the endogenous siRNA pathway, (2) AGO1 and AGO2 can compete for binding with miRNAs, and (3) ectopic expression of Aub in the soma competes for the siRNAs pathway mediated by AGO2. In addition, miRNAs have been demonstrated to have a role on easi-RNA biogenesis in plants. In a similar manner, AGO1 could act on piRNAs through its activity on the miRNA pathway. Although future studies will clarify the connection between AGO1 and the piRNA pathway, the present data provide novel perspectives in the field and could have a broad relevance to diseases affecting cognitive functions (Bozzetti, 2015).

Expression, genetic and biochemical data indicate that Aub and dFmr1 interact directly. dFmr1 has been proposed to bind specific cargo RNAs and the human FMRP binds small RNA, in addition to mRNAs. Similarly, the Aub–dFmr1 interaction might allow the targeting of piRNAs to the transcripts of repetitive sequences and transposable elements, dFmr1 providing the molecular link between small RNAs and AGO proteins of the RISC (Bozzetti, 2015).

The Aub and dFmr1 proteins colocalize and likely interact in the piRNA pathway in a specific stage of testis development and also have additional functions that are independent from each other. Typically, dFmr1 accumulates at high levels in more differentiated cells of the testis, where Aub is not detectable, likely accounting for the axoneme phenotype described in dFmr1 testes. In the future, it will be interesting to analyze whether the other genes involved in the piRNA pathway in testis are also required at specific stages, as also recently found in the ovary (Bozzetti, 2015).

Finally, FMRP proteins work in numerous molecular networks, show complex structural features (TUDOR, KH, NLS, NES RGG domains) and are characterized by widespread expression and subcellular localization (cytoplasm, nucleus, axons, dendrites, P bodies), providing versatile platforms that control mRNA and small RNA metabolism (e.g. translation, degradation and transport). Understanding whether FMRP proteins interact with other members of the piRNA pathway, whether this interaction is modulated physiologically and how does the interaction with this pathway compare with that observed with other AGO proteins will clarify the role and mode of action this family of proteins in small RNA biogenesis and metabolism (Bozzetti, 2015).

The biogenesis of the piRNAs requires two pathways. The primary pathway involves Piwi and predominantly occurs in the somatic tissues. The ping-pong pathway involves Aub, as well as AGO3, and predominantly occurs in the germline, where Aub is thought to bind an antisense piRNA, to cleave the sense transcript from an active transposon and to produce a sense piRNA that is loaded onto AGO3. The AGO3–piRNA complex binds complementary transcripts from the piRNA cluster, producing the so-called secondary piRNAs by an amplification loop. Although the piRNA pathways have emerged as a very important tool to understand the role of RNA metabolism in physiological and pathological conditions, the relationship and interactions among the involved proteins are not simple to interpret, mostly because not all the players have been characterized. Moreover, recent data support the hypothesis that the somatic and the germline piRNA pathways share components: for example, shutdown (shu), vreteno (vret) and armitage (arm) affect primary as well as ping-pong pathways in ovaries. Results from this study call for a role of dFmr1 in both piRNA pathways at least in testes. Based on the alignment of the human, mouse and fly FMRP family members, dFmr1 might participate in piRNA biogenesis as a Tudor domain (TDRD) containing protein (Bozzetti, 2015).

TDRDs are regions of about 60 amino acids that were first identified in a Drosophila protein called Tudor. In the recent years, the requirement of TDRD proteins in piRNA biogenesis and metabolism has become evident. Typically, the founding member of the family, Tudor, binds AGO proteins and helps them interact with specific piRNAs. Among the different TDRD proteins, fs(1)Yb works in the primary pathway; Krimper, Tejas, Qin/Kumo, and PAPI work in the ping-pong pathway; and Vret works in both systems. PAPI, the only TDRD protein that has a modular structure closely related to dFmr1 (two KH domains and one TDRD), interacts with the di-methylated arginine residues of AGO3 and controls the ping-pong cycle in the nuage. At least during the early stages of testis development, dFmr1 might interact with Aub in a similar way. Given that TDRDs are involved in the interactions between proteins and in the formation of ribonucleoprotein complexes, future studies will assess whether RNAs mediate the Aub–dFmr1 interaction (Bozzetti, 2015).

In conclusion, the discovery of dFmr1 as a player in the piRNA pathway highlights the importance of the fly model. Data from this study also adds a new perspective to understanding the role and mode of action of this protein family and the physiopathological mechanisms underlying the Fragile X syndrome (Bozzetti, 2015).

Protein Interactions

In vitro experiments have shown that Stellate can interact with the catalytic ß subunit of casein kinase 2 enzyme, altering CK2 activity (Bozzetti, 1995).


Males of Drosophila melanogaster lacking the Y chromosome-linked Su(Ste) locus show multiple meiotic alterations, including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, the protein encoded by the Stellate sequences was expressed in bacteria and an antibody against the fusion protein was produced. Immunostaining of Su(Ste) minus testes has clearly shown that the Stellate protein is a major component of the crystals. The Ste protein is absent in XY males but is a major component of the crystalline aggregates in Su(Ste) mutant males. (Bozzetti, 1995).

Effects of Mutation or Deletion

Drosophila melanogaster males deficient for the crystal (cry) locus of the Y chromosome that carries between 15 and 60 copies of the X-linked Stellate (Ste) gene are semisterile, have elevated levels of nondisjunction, produce distorted sperm genotype ratios (meiotic drive), and evince hyperactive transcription of Ste in the testes. Ste seems to be the active element in this system, and it has been proposed that the ancestral Ste gene was 'selfish' and increased in frequency because it caused meiotic drive. This hypothetical evolutionary history is based on the idea that Ste overexpression, and not the lack of cry, causes the meiotic drive of cry- males. To test whether this is true, a Ste-deleted X chromosome was constructed and the phenotype of Ste-/cry- males was examined. If hyperactivity of Ste were necessary for the transmission defects seen in cry- males, cry- males completely deficient for Ste would be normal. Although it is impossible to construct a completely Ste- genotype, it was found that Ste-/cry- males have exactly the same phenotype as Ste+/cry- males. The deletion of all X chromosome Ste copies not only does not eliminate meiotic drive and nondisjunction, but it also does not even reduce them below the levels produced when the X carries 15 copies of Ste (Belloni, 2002).

Meiotic drive was proposed originally as an evolutionary mechanism, but it has been difficult to demonstrate that any example of drive serves this role. Hurst (1992 and 1996) has suggested that the cry-Ste system could be an evolutionarily important case. Paraphrasing his reasoning:

  1. Transcription of Ste is induced in the testes of cry- males. Hence the element whose frequency would be increased by drive is the active element in the system, and presumably the one that causes the drive (Belloni, 2002).

  2. X/O males of the sister species D. simulans do not have protein accretions in their testes; hence D. simulans does not have a Ste-suppressing cry locus in its Y chromosome, leading to the natural presumption that Ste is the older of the two elements (Belloni, 2002).

  3. Because there is no recombination between the X and Y chromosomes, an increase in the population frequency of Ste would have a strong effect on the evolution of the entire chromosome (Belloni, 2002).

This is an attractive hypothesis, but its correctness depends on the validity of the first two points. It is not obvious, however, that hyperactivity of Ste necessarily implies that it is the element that causes the drive, nor does the lack of crystals in spermatocytes of D. simulans XO males necessarily mean that cry is not present somewhere in that species' genome (Belloni, 2002).

A Ste-deleted X chromosome was created to test the first of these premises. There is not the slightest hint that Ste is necessary for the meiotic drive that occurs in cry- males. It is the absence of cry, and not hyperactivation of Ste, that causes meiotic drive. To get around this conclusion would require that the Ste activity induced in the Stellate depleted BSY-born Stellate copies is sufficient to provoke drive and that there is no response to doubling its Ste copy number and testes-specific Ste transcription, even though there is a linear response to further increases in Ste copy number. For now, it seems preferable to think that deletion of cry triggers the downstream problems of sperm function, but that the downstream targets, if they include Ste at all, must include other elements as well. There is no lack for candidates: the cis-responsive element that has been identified in translocation-provoked meiotic drive, recessive autosomal mutants that accumulate Ste protein, or the transposable elements that are reported to respond to cry deletion (Belloni, 2002).

If the role of cry+ is not merely the suppression of a parasitic Ste element, what does it do? Perhaps cry+ and the other genes whose mutants mimic cry- form part of a general gene-silencing system or part of a system aimed particularly at silencing repeated elements that must act before spermiogenesis can proceed. If so, meiotic drive is not the result of a normal checkpoint control that eliminates aneuploid products, but is instead one symptom of the dysgenesis produced by the testicular expression of genes (Ste and others) that should have been shut off by the end of meiosis. Working out the cellular pathway that, disrupted, leads to meiotic drive remains a problem yet to be resolved (Belloni, 2002).

Construction of the Ste-deleted chromosomes also allowed the direct measurement of the activity of the transposed heterochromatic block of Ste copies present in the BSY chromosome. Testes-specific transcription of those copies is induced by deleting cry, but the heterochromatic copies appear to be substantially less active in the testes than are the euchromatic copies. While not directly relevant to the question that led to these experiments, the tightly regulated level of the 1400-nt ubiquitous transcript (independent of cry, independent of copy number, and independent of heterochromatic or euchromatic origin) needs to be considered as attempts are made to understand the findings that implicate an iRNA in the regulation of Ste. How, in the very same tissue, is production of one Ste transcript inhibited by cry+, while the other is not only not inhibited, but is produced in a copy-number-compensated fashion (Belloni, 2002)?

At the same time as these experiments were undertaken, Palumbo and Bozzetti (personal communication to Belloni, 2002) independently started a species survey of Ste and cry sequences. That project examines the second of the key assumptions in the evolutionary hypothesis, and their results are no more coherent with the selfish evolution of Ste than are those reported here. For example, although there is no crystal production in spermatocytes of D. simulans XO males, the D. simulans Y chromosome does carry Ste-like repeats (Belloni, 2002).

Numerous situations in D. melanogaster cause distorted sperm recovery, including rDNA deletions, segregation of some translocations, segregation of univalents, compound autosomes, deletion of cry, and mutation of other genes that interact in the cry-Ste system. Hence, meiotic drive, in the sense of distorted sperm recovery rather than in the sense of an evolutionary force, seems to be a general downstream response to meiotic problems. Whether any situations that activate this process are of evolutionary importance remains to be seen (Belloni, 2002).

Repeated elements are remarkably important for male meiosis and spermiogenesis in Drosophila melanogaster. Pairing of the X and Y chromosomes is mediated by the ribosomal RNA genes of the Y chromosome and X chromosome heterochromatin, and spermiogenesis depends on the fertility factors of the Y chromosome. Intriguingly, a peculiar genetic system of interaction between the Y-linked crystal locus and the X-linked Stellate elements seem to be also involved in male meiosis and spermiogenesis. Deletion of the crystal element of the Y, via an interaction with the Stellate elements of the X, causes meiotic abnormalities, gamete-genotype dependent failure of sperm development (meiotic drive), and deposition of protein crystals in spermatocytes. The current hypothesis is that the meiotic abnormalities observed in cry- males is due to an induced overexpression of the normally repressed Ste elements. An implication of this hypothesis is that the strength of the abnormalities would depend on the amount of the Ste copies. To test this point the relationship of Ste copy number and organization to meiotic behavior in cry- males has been genetically and cytologically examined. Heterochromatic as well as euchromatic Ste repeats are functional and the abnormality in chromosome condensation and the frequency of nondisjunction are related to Ste copy number. Moreover, meiosis is disrupted after synapsis and cry-induced meiotic drive is probably not mediated by Ste (Palumbo, 1994).

The sting mutation (see aubergine), caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry- males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced (Schmidt, 1999).


Aravin, A. A., et al. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline Curr. Biol. 11: 1017-1027. 11470406

Aravin, A. A., Klenov, M. S., Vagin, V. V., Bantignies, F., Cavalli, G. and Gvozdev, V A. (2004). Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line. Mol. Cell. Biol. 24(15): 6742-50. Medline abstract: 15254241

Balakireva, M. D., et al. (1992). Structural organization and diversification of Y-linked sequences comprising Su(Ste) genes in Drosophila melanogaster. Nucleic Acids Res. 20: 3731-3736. 1322529

Belloni, M., et al. (2002). Does Stellate cause meiotic drive in Drosophila melanogaster? Genetics 161: 1551-1559. 12196400

Bozzetti, M.P., Specchia, V., Cattenoz, P.B., Laneve, P., Geusa, A., Sahin, H.B., Di Tommaso, S., Friscini, A., Massari, S., Diebold, C. and Giangrande, A. (2015). The Drosophila fragile X mental retardation protein participates in the piRNA pathway. J Cell Sci 128: 2070-2084. PubMed ID: 25908854

Bozzetti, M. P., et al. (1995). The Ste Locus, a component of the parasitic cry-Ste System of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the ß subunit of Casein kinase 2. Proc. Natl. Acad. Sci. 92: 6067-6071. 7597082

Dernburg, A. F., Zalevsky, J., Colaiacovo, M. P. and Villeneuve, A. M. (2000). Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev. 14: 1578-1583. 10887151

Forstemann, K., Tomari, Y., Du, T., Vagin, VV., Denli, A. M., Bratu, D. P., Klattenhoff, C., Theurkauf, W. E. and Zamore, P. D. (2005). Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3(7): e236. 15918770

Horwich, M. D., et al. (2007). The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17: 1265-1272. Medline abstract: 17604629

Hurst, L. D. (1992). Is Stellate a relict meiotic driver? Genetics 130: 229-230. 1732164

Hurst, L. D. (1996). Further evidence consistent with Stellate's involvement in meiotic drive. Genetics 142: 641-643. 8852860

Kalmykova, A. I., Shevelyov, Y. Y., Dobritsa, A. A. and Gvozdev, V. A. (1997a) Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome. Proc. Natl. Acad. Sci. 94: 6297-6302. 9177211

Kalmykova, A. I., Dobritsa, A. and Gvozdev, V. (1997b) The Su(Ste) repeat in the Y chromosome and betaCK2tes gene encode predicted isoforms of regulatory beta-subunit of protein kinase CK2 in Drosophila melanogaster FEBS Lett. 416: 164-166. 9369205

Kalmykova, A. I., Dobritsa, A..A. and Gvozdev, V. A. (1998). Su(Ste) diverged tandem repeats in a Y chromosome of Drosophila melanogaster are transcribed and variously processed. Genetics 148: 243-249. 9475736

Kalmykova, A. I., et al. (2002). CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster. Eur. J. Biochem. 269(5): 1418-27. 11874456

Kogan, G. L., Epstein, V. N., Aravin, A. A. and Gvozdev, V. A. (2000). Molecular evolution of two paralogous tandemly repeated heterochromatic gene clusters linked to the X and Y chromosomes of Drosophila melanogaster. Mol. Biol. Evol. 17: 697-702. 10779530

Livak, K. J. (1984). Organization and mapping of a sequence on the Drosophila melanogaster X and Y chromosomes that is transcribed during spermatogenesis. Genetics 107: 611-634. 6430749

Livak, K. J. (1990). Detailed structure of the Drosophila melanogaster stellate genes and their transcripts. Genetics. 124(2): 303-16. 1689686

Malinskya, S., Buchetona, A. and Busseau, I. (2000). New insights on homology-dependent silencing of I factor activity by transgenes containing ORF1 in Drosophila melanogaster. Genetics 156: 1147-1155. 11063690

Mette, M. F., Aufsatz, W., van Der Winden, J., Matzke, M. A. and Matzke, A. J. (2000). Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19: 5194-5201. 11013221

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1997). Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent. Cell 90: 479-490. 9267028

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1999). Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences. Cell 99: 35-46. 10520992

Palumbo, G., Bonaccorsi, S., Robbins, L. G. and Pimpinelli, S. (1994). Genetic analysis of Stellate elements of Drosophila melanogaster Genetics 138: 1181-1197. 7896100

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

Tulin, A. V., et al. (1997). Heterochromatic Stellate gene cluster in Drosophila melanogaster: structure and molecular evolution. Genetics 146(1): 253-62. 9136015

Saito, K., Ishizuka, A., Siomi, H., Siomi, M. C. (2005). Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol. 3(7): e235. 15918769

Saito, K., Sakaguchi, Y., Suzuki, T., Suzuki, T., Siomi, H. and Siomi, M. C. (2007). Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends. Genes Dev. 21(13): 1603-8. Medline abstract: 17606638

Schmidt, A., Palumbo, G., Bozzetti, M. P., Tritto, P., Pimpinelli, S. and Schafer, U. (1999). Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster Genetics 151: 749-760. 9927466

Specchia, V., Piacentini, L., Tritto, P., Fanti, L., D'Alessandro, R., Palumbo, G., Pimpinelli, S. and Bozzetti, M. P. (2010). Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons. Nature 463: 662-665. PubMed ID: 20062045

Stapleton, W., Das, S. and McKee, B. D. (2001). A role of the Drosophila homeless gene in repression of Stellate in male meiosis. Chromosoma 110(3): 228-40. 11513298

Tomari, Y., Du, T., Haley, B., Schwarz, D. S., Bennett, R., Cook, H. A., Koppetsch, B. S., Theurkauf, W. E. and Zamore, P. D. (2004). RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116(6): 831-41. 15035985

Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. and Zamore, P. D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313(5785): 320-4. 16809489

Stellate: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 16 January 2008

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.