Koala retrovirus-A (KoRV-A) is spreading through wild koalas in a north-to-south wave while transducing the germ line, modifying the inherited genome as it transitions to an endogenous retrovirus. Previously, we found that KoRV-A is expressed in the germ line, but unspliced genomic transcripts are processed into sense-strand PIWI-interacting RNAs (piRNAs), which may provide an initial "innate" form of post-transcriptional silencing. Here, we show that this initial post-transcriptional response is prevalent south of the Brisbane River, whereas KoRV-A expression is suppressed, promoters are methylated, and sense and antisense piRNAs are equally abundant in a subpopulation of animals north of the river. These animals share a KoRV-A provirus in the MAP4K4 gene's 3' UTR that is spreading through northern koalas and produces hybrid transcripts that are processed into antisense piRNAs, which guide transcriptional silencing. We speculate that this provirus triggers adaptive transcriptional silencing of KoRV-A and is sweeping to fixation.

Background Koala Retrovirus-A is a gamma-retrovirus that is spreading across wild koala populations through horizontal and vertical transmission, contributing significantly to genomic diversity across and even within koala populations. Previous studies have estimated that KoRV-A initially integrated into the koala genome less than 50,000 years ago, but the precise origins and the patterns of spread after its endogenization remain unclear. Results In this study, we analyzed germline insertions of KoRV-A using whole-genome sequencing data from 405 wild koalas, representing nearly the species’ entire geographic range. Our findings reveal an evolutionary trajectory for KoRV-A, suggesting that the initial endogenization might occur near Coffs Harbour on the Mid-north coast of NSW around the middle of the koala’s range. As KoRV-A spread, certain subtypes emerged and became prevalent, two of which recombined with an ancient endogenous retrovirus, PhER, resulting in distinct recombination variants in northern and southern koala populations. Additionally, we identified a geographic barrier north of Sydney, which may have slowed the southward spread of KoRV-A into Sydney and beyond. Conclusions Our study proposes a comprehensive evolutionary pathway for KoRV-A, beginning with its initial endogenization near Coffs Harbour and highlighting barriers and diversification events that have shaped its distribution and impact on koala populations.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Correspondence20 March 2014free access Antisense piRNA amplification, but not piRNA production or nuage assembly, requires the Tudor-domain protein Qin Zhao Zhang Zhao Zhang Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Birgit S Koppetsch Birgit S Koppetsch Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Jie Wang Jie Wang Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Cindy Tipping Cindy Tipping Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Zhiping Weng Zhiping Weng Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author William E Theurkauf Corresponding Author William E Theurkauf Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Phillip D Zamore Corresponding Author Phillip D Zamore Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Zhao Zhang Zhao Zhang Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Birgit S Koppetsch Birgit S Koppetsch Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Jie Wang Jie Wang Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Cindy Tipping Cindy Tipping Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Zhiping Weng Zhiping Weng Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author William E Theurkauf Corresponding Author William E Theurkauf Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Phillip D Zamore Corresponding Author Phillip D Zamore Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Author Information Zhao Zhang1, Birgit S Koppetsch2, Jie Wang3, Cindy Tipping1, Zhiping Weng3, William E Theurkauf 2 and Phillip D Zamore 1 1Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA 2Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA 3Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA *Corresponding authors. Tel: 1 508 856 2191; E-mails: [email protected], [email protected] The EMBO Journal (2014)33:536-539https://doi.org/10.1002/embj.201384895 Comment on: Anand and Kai 2012, EMBO J 31: 870–882 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Qin is required for transposon silencing by the PIWI-interacting RNA (piRNA) pathway (Zhang et al, 2011; Anand & Kai, 2012). Initial descriptions of qin mutants led to conflicting explanations for the role of Qin in piRNA biogenesis. One study suggested that loss of Qin causes the accumulation of sense piRNAs instead of antisense without altering total piRNA levels or perturbing the localization of Aub and Ago3 to the perinuclear nuage (Zhang et al, 2011). A second report concluded that both piRNAs and nuage were lost from the germline in qin mutants, leading to a complete failure of the piRNA pathway (Anand & Kai, 2012). We re-analyzed the qin alleles used in the two studies: qin1, qin2 (Zhang et al, 2011) and qinkumo (Anand & Kai, 2012). These analyses corroborate our original findings that the fundamental defect in qin mutants is not a loss of piRNAs, but rather the replacement of heterotypic Aub:Ago3 Ping-Pong with non-productive, homotypic Aub:Aub Ping-Pong. Our data suggest that the phenotypes reported for qinkumo homozygotes are caused by a secondary mutation unlinked to qin. Compared with genotypically matched w1118 and qinkumo/TM3 controls, homozygous qinkumo mutant ovaries are small, with few egg chambers beyond stage 10 (Supplementary Fig S1). In contrast, qin1, qin2, qinkumo in trans to a complete deletion of the qin locus (Df(3R)Excel6180; henceforth, Df), as well as qin1/qinkumo and qin2/qinkumo, all had normal ovary size and shape. Figure 1. Without Qin, Ago3, Aub and Vasa still reside in nurse cell nuage RNA-seq data for wild-type and qin mutant ovaries. Ago3 and Aub immunostaining or live EGFP-Vasa image in qin mutants. EGFP-Vasa fusion protein was expressed from a transgene using the vasa promoter. Download figure Download PowerPoint qinkumo/qinkumo females laid almost no eggs (one egg per female on day 2, and none thereafter), yet qinkumo/Df females each laid approximately 50 eggs per day. Typically, the phenotype of a strong mutant allele remains the same or worsens in trans to a deficiency, but qinkumo/qinkumo was more severe than qinkumo/Df. Potential explanations include (i) qinkumo is a neomorph; (ii) the Df(3R)Excel6180 deficiency fails to uncover the entire qin gene; and (iii) qinkumo contains a secondary mutation unlinked to qin. Our data support the idea that qinkumo is a null mutation and that Df(3R)Excel6180 removes all of qin: RNA-seq detected no qin mRNA in qinkumo/Df ovaries (Fig 1A). The qin1 allele results from a piggyBac transposon insertion and produces a truncated mRNA 4432 nt long. As anticipated, qin1/Df ovaries produced a approximately 4400 nt RNA less than half as abundant as qin mRNA in w1118 (12 versus 32 rpkm). The qin deficiency extends beyond the 5′ end of qin, disrupting the upstream gene CG7694: CG7694 mRNA abundance was 12 rpkm in w1118 but only 4.2 rpkm in qin1/Df and 3.6 rpkm in qinkumo/Df. We conclude that both qinkumo and Df(3R)Excel6180 are null alleles of qin and that a secondary mutation unlinked to qin is present on the qinkumo chromosome. By immunofluorescence antibody staining, Ago3 and Aub were present in nuage in all genotypes tested except qinkumo/qinkumo (Fig 1B). For example, qinkumo/Df, Ago3 was correctly localized to perinuclear foci in 62 of 67 nurse cells among 12 separate egg chambers, compared with 66 of 70 in 16 separate egg chambers from qinkumo/TM3 flies and 96 of 102 in 20 separate egg chambers from w1118 ovaries; Aub was present in perinuclear puncta typical of nuage in 50 of 57 nurse cells from nine qinkumo/Df egg chambers compared to 61 of 68 nurse cells from nine qinkumo/TM3 egg chambers and 90 of 98 from 20 w1118 egg chambers. Aub localizes to the posterior end of the oocyte in late-stage egg chambers, and this localization was preserved in qin mutants (Supplementary Fig S2). In contrast, qinkumo homozygotes showed mislocalized Ago3 and Aub as previously reported (Fig 1B; Anand & Kai, 2012). To provide an independent test of whether qinkumo/Df disrupts nuage, we monitored the localization of GFP-Vasa, a nuage marker, in live nurse cells (Fig 1B). We detected no disruption of the localization of GFP-Vasa in qin mutants. We conclude that loss of Qin does not affect nuage structure in unfixed, living nurse cells. piRNA levels in qin1/Df ovaries are indistinguishable from controls (Zhang et al, 2011). We used small RNA sequencing to measure piRNA abundance in qin2/Df and qinkumo/Df ovaries. Compared to heterozygotes, the abundance of total transposon-derived, 23–29 nt small RNAs in qin2/Df and qinkumo/Df changed < 4% (Fig 2A; Supplementary Tables S1 and S2). Among the 93 transposon families with > 100 ppm piRNA reads in qin1/TM6B ovaries (Zhang et al, 2011), there was no significant change in median piRNA abundance relative to w1118 controls for qin1/Df (P = 0.57, Wilcoxon test), qin2/Df (P = 0.13), or qinkumo/Df (P = 0.33; Fig 2B; for additional analyses by transposon families see http://www.umassmed.edu/uploadedFiles/zamore/Transposon_buckets.zip). However, the fraction of piRNAs with the same orientation as the corresponding transposon sense mRNA increased: the median sense fraction (i.e. sense piRNAs/all piRNAs) among 93 transposon families was 0.25 for w1118 ovaries but 0.41 for qin1/Df mutants (P = 5.2 × 10−7, Wilcoxon test), 0.37 for qin2/Df (P = 2.4 × 10−4), and 0.38 for qinkumo/Df (P = 1.1 × 10−5; Fig 2B). Figure 2. piRNA abundance and Ping-Pong efficiency are unaltered in qin mutant ovaries piRNA length distribution. Blue, sense piRNAs; red, antisense. Box plots reporting the change in abundance of all piRNAs mapping to transposons. qin1/Df, qin2/Df and qinkumo/Df, but not qinkumo/qinkumo, affect piRNA production similarly. Group 1: transposon families with piRNAs amplified by the Ping-Pong pathway and more antisense piRNAs bound to Aub and more sense piRNAs bound to Ago3. Group 2: transposon families with piRNAs amplified by the Ping-Pong pathway and more sense piRNAs bound to Aub and more antisense piRNAs bound to Ago3. Group 3: transposon families expressed in the somatic follicle cells, predominantly antisense primary piRNAs, and little Ping-Pong amplification. Box plots reporting Ping-Pong Z-score by transposon family. Z-score = 1.96 corresponds to P-value = 0.05. Download figure Download PowerPoint We also measured piRNA abundance in qinkumo/qinkumo and qinkumo/TM3 ovaries (Supplementary Tables S1 and S2). qinkumo homozygous mutant flies, like other qin loss-of-function mutations, produced amounts of piRNAs similar to their qinkumo/TM3 siblings (Fig 2A). Our analysis of previously published deep sequencing data from homozygous qinkumo ovaries (Anand & Kai, 2012) also led us to conclude that there was no change in total piRNA production (Fig 2A). Moreover, the effects of qin1/Df and qinkumo/Df on piRNA production were highly correlated (r = 0.94), but less well correlated with qinkumo/qinkumo (r = 0.85, P-value < 2.2 × 10−16; Fig 2C and Supplementary Fig S3A). All qin allelic combinations showed significant (Z > 46; P-value < 2.2 × 10−16) Ping-Pong amplification as measured by comparing piRNA pairs overlapping by 10 bp to other lengths of overlap (Fig 2D and Supplementary Fig S3B). Finally, we reached these same conclusions when normalizing the data by two alternative strategies—microRNA abundance and non-coding RNA abundance (Supplementary Figs S4, S5 and S6). We conclude that Qin is not required to maintain overall piRNA levels or for Ping-Pong amplification. We used RNA-seq to measure transcript abundance in w1118, qin1/Df, and qinkumo/Df ovaries. Without Qin, RNA sequences mapping uniquely to the 42AB cluster, which is the longest piRNA cluster in flies and produces approximately 30% of all ovary piRNAs (Brennecke et al, 2007), increased from 1.5 rpkm in w1118 flies to 2.0 rpkm in qinkumo/Df and 2.5 in qin1/Df flies (Supplementary Fig S7A). We note that our result disagrees with the finding that transcripts from the 42AB cluster declined in qinkumo homozygous ovaries as measured by qRT-PCR (Anand & Kai, 2012). Among the 142 previously defined piRNA clusters (Brennecke et al, 2007), the steady-state abundance of transcripts from six clusters increased significantly in qin1/Df ovaries (>5-fold; q < 0.05); just one decreased significantly (>2-fold; q < 0.05; Supplementary Fig S7B). In qinkumo/Df ovaries, the transcript abundance for 11 clusters increased significantly (>5-fold; q < 0.05); none decreased significantly (Supplementary Fig S7B). Both qin1/Df and qinkumo/Df ovaries suffered increased transposon expression, as measured by RNA-seq (Supplementary Fig S7B). Of the 93 transposon families we examined, the steady-state RNA abundance of 13 families increased significantly (>6-fold; q < 0.05) in qin1/Df, compared with w1118. Similarly, in qinkumo/Df ovaries the steady-state RNA abundance of 12 transposon families increased significantly (>4-fold; q < 0.05). Expression of ten transposon families increased significantly in both qin1/Df and qinkumo/Df ovaries (q < 0.05), including eight of the 11 transposons whose abundance was reported to increase significantly when measured using both whole-genome tiling microarrays and qRT-PCR (Zhang et al, 2011). Transposon expression in qin1/Df and qinkumo/Df were highly correlated (r = 0.95; P-value < 2.2 × 10−16). We conclude that loss of qin in the fly ovary does not affect nuage assembly or overall piRNA abundance. Instead, loss of Qin leads to an increase in sense piRNAs and a decrease in antisense piRNAs. The result presented here, together with those reported previously (Zhang et al, 2011) are consistent with the loss of heterotypic Aub:Ago3 Ping-Pong in qin mutants. Without Qin, piRNA cluster transcripts accumulate, rather than decline. Thus, when Aub:Aub Ping-Pong predominates (Zhang et al, 2011), Ping-Pong amplification appears to consume cluster transcripts less efficiently, consistent with a role for Qin in piRNA precursor processing. Understanding how Qin couples Aub with Ago3 to efficiently generate piRNAs and silence transposons remains a challenge for future studies. Accession numbers Sequence data generated in this study are available via the NCBI trace archives (http://www.ncbi.nlm.nih.gov/Traces/) using accession number SRP024291 Acknowledgements We thank Toshie Kai for sharing qinkumo flies, and Paul Lasko for sharing EGFP-Vasa flies. We thank Shikui Tu, Xiaopeng Zhu and members of our laboratories for advice, suggestions, and critical comments on the manuscript. This work was supported in part by National Institutes of Health grant HD049116 to WET, ZW, and PDZ and GM62862 and GM65236 to PDZ. Author contributions ZZ, WET and PDZ conceived the experiments. BSK performed the immuno-staining, ZZ conducted the experiments. JW mapped the sequence reads. ZZ analyzed the sequencing data with guidance from JW and ZW. CT helped with fly fertility test. ZZ and PDZ wrote the manuscript in consultation with all authors. Conflict of interest The authors declare that they have no conflicts of interest. Supporting Information Supplementary Figure S1 (application/PDF, 1.6 MB) Supplementary Figure S2 (application/PDF, 5 MB) Supplementary Figure S3 (application/PDF, 1.5 MB) Supplementary Figure S4 (application/PDF, 1.2 MB) Supplementary Figure S5 (application/PDF, 1.2 MB) Supplementary Figure S6 (application/PDF, 1.2 MB) Supplementary Figure S7 (application/PDF, 1.3 MB) Supplementary Table S1 (application/PDF, 121.8 KB) Supplementary Table S2 (application/PDF, 120.9 KB) Supplementary Table S3 (application/PDF, 96.7 KB) Supplementary Materials and Methods (Word document, 19.3 KB) References Anand A, Kai T (2012) The tudor domain protein kumo is required to assemble the nuage and to generate germline piRNAs in Drosophila. EMBO J 31: 870–882Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128: 1089–1103CrossrefCASPubMedWeb of Science®Google Scholar Zhang Z, Xu J, Koppetsch BS, Wang J, Tipping C, Ma S, Weng Z, Theurkauf WE, Zamore PD (2011) Heterotypic piRNA ping-pong requires Qin, a protein with both E3-ligase and tudor domains. Mol Cell 44: 572–584CrossrefCASPubMedWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 33,Issue 6,18 March 2014Cover: Into the wild – This long‐exposure photograph was taken just after sunset on the coast of the Northwest Highlands of Scotland. The photographer, Christoph Kurze, is a PhD student at the Martin Luther University of Halle‐Wittenberg, Germany, where he studies evolutionary ecology with a focus on host‐parasite co‐evolution using the honeybee‐Nosema model system. In his leisure time, Christoph enjoys spending time outdoors, seeking to capture some of Nature's beauty in his photographs. The image is one of the runners‐up of the recently concluded EMBO Journal Cover Contest 2014. Visit covercontest.embo.org for a small gallery of other high‐scoring images and the two first‐prize winners. If you send us an email at [email protected], we will be happy to notify you once the 2015 contest opens. Volume 33Issue 618 March 2014In this issue FiguresReferencesRelatedDetailsLoading ...

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster—expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.

Progression through the G(2)/M transition following DNA damage is linked to cytokinesis failure and mitotic death. In four different transformed cell lines and two human embryonic stem cell lines, we find that DNA damage triggers mitotic chromatin decondensation and global phosphorylation of histone H2AX, which has been associated with apoptosis. However, extended time-lapse studies in HCT116 colorectal cancer cells indicate that death does not take place during mitosis, but 72% of cells die within 3 days of mitotic exit. By contrast, only 11% of cells in the same cultures that remained in interphase died, suggesting that progression through mitosis enhances cell death following DNA damage. These time-lapse studies also confirmed that DNA damage leads to high rates of cytokinesis failure, but showed that cells that completed cytokinesis following damage died at higher rates than cells that failed to complete division. Therefore, post-mitotic cell death is not a response to cytokinesis failure or polyploidy. We also show that post-mitotic cell death is largely independent of p53 and is only partially suppressed by the apical caspase inhibitor Z-VAD-FMK. These findings suggest that progression through mitosis following DNA damage initiates a p53- and caspase-independent cell death response that prevents propagation of genetic lesions.

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster,but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle.

Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of gamma-H2Av foci characteristic of DNA damage. We conclude that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline.

The 13 syncytial cleavage divisions that initiate Drosophilaembryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. We show that mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. We also show that grp mutant embryos accumulate DNA double-strand breaks and that DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. We conclude that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT.

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is 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. Our results suggest that armi is required for RISC maturation.

Microtubules and the plus-end-directed microtubule motor Kinesin I are required for the selective accumulation of oskar mRNA at the posterior cortex of the Drosophila melanogaster oocyte, which is essential to posterior patterning and pole plasm assembly. We present evidence that microtubule minus ends associate with the entire cortex, and that Kinesin and microtubules are not required for oskar mRNA association with the posterior pole, but prevent ectopic localization of this transcript and the pole plasm proteins Oskar and Vasa to other cortical regions. Cortical binding of oskar mRNA seems to be dependent on the actin cytoskeleton. We conclude that most of the actin-rich oocyte cortex can support pole plasm assembly, and propose that Kinesin restricts pole plasm formation to the posterior by moving oskar mRNA away from microtubule-rich lateral and anterior cortical regions.