), the authors note that in Fig. 4 A, the staining for Vasa in spindle-E mutant ovaries was incorrect. Consistent with Findley et al. (4), Vasa perinuclear localization is unaffected in spindle-E mutant ovaries. This does not change the major conclusion that the nuage functions to maintain genome stability by repressing the expression of the selfish genetic elements via rasiRNA-mediated gene silencing. Rather, Spindle-E may function at the same hierarchical level or downstream of Vasa to regulate the localization of Aubergine, Krimper, and Maelstrom. The corrected figure and legend appear below. Fig. 4. Nuage foci are mislocalized in the germ-line-specific RNA-silencing component mutants. Ovaries from different mutant flies were immunostained for the nuage components. Homozygous mutant alleles or allelic combinations were used for all the mutants, except for dcr-1, where clonal analysis was employed. (Scale bar: 10 m.) (A) Localization of the nuage components at the perinuclear regions of the germ-line cells reflects a hierarchical assembly. The nuage components, AUB, KRIMP, and MAEL, depend on SPN-E and VAS to localize normally to the perinuclear regions; KRIMP and MAEL depend on SPN-E, VAS, and AUB to localize to the nuage; and MAEL depends on SPN-E, VAS, AUB, and KRIMP to localize normally. (B) Nuage localization is unaffected in the conventional dicing enzyme mutants, dcr-1 and dcr-2. The nuage is an electron-dense perinuclear structure that is known to be a hallmark of animal germ-line cells. Although the conservation of the nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germ line still remain unknown. Here, we report a nuage component, Krimper (KRIMP), in Drosophila melanogaster and show that it ensures the repression of the selfish genetic elements in the female germ line. The Krimp loss-of-function allele exhibited female sterility, defects in karyosome formation and oocyte polarity, and precocious osk translation. These phenotypes are commonly observed in the other nuage component mutants, vasa (vas) and maelstrom (mael), and the RNAsilencing component mutants, spindle-E (spn-E) and aubergine (aub), suggesting a shared underlying defect that uses RNA silencing. Moreover, we demonstrated that the localization of the nuage components depends on both SPN-E and AUB and that the selfish genetic elements were derepressed to different extents in the nuage component mutants, as well as in aub and armitage (armi) mutants. In the nuage component mutants, vas, krimp, and mael, the levels of roo, I-element, and HeT-A repeat-associated small interfering RNAs were greatly reduced. Hence, our data suggest that the nuage functions as a specialized center that protects the genome in the germ-line cells via gene regulation mediated by repeat-associated small interfering RNAs.Krimper ͉ repeat-associated small interfering RNA

Nuage, a well-conserved perinuclear organelle found in germline cells, is thought to mediate retroelement repression in Drosophila melanogaster by regulating the production of Piwi-interacting RNAs (piRNAs). In this study, we present evidence that the nuage–piRNA pathway components can be found in cytoplasmic foci that also contain retroelement transcripts, antisense piRNAs, and proteins involved in messenger RNA (mRNA) degradation. These mRNA degradation proteins, decapping protein 1/2 (DCP1/2), Me31B (maternal expression at 31B), and pacman (PCM), are normally thought of as components of processing bodies. In spindle-E (spn-E) and aubergine (aub) mutants that lack piRNA production, piRNA pathway proteins no longer overlap the mRNA degradation proteins. Concomitantly, spn-E and aub mutant ovaries show an accumulation of full-length retroelement transcripts and prolonged stabilization of HeT-A mRNA, supporting the role of piRNAs in mediating posttranscriptional retroelement silencing. HeT-A mRNA is derepressed in mRNA degradation mutants twin, dcp1, and ski3, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates.

Nuage is a germline-unique perinuclear structure conserved throughout the animal kingdom. Maelstrom (Mael) is an unusual nuage component, as it is also found in the nucleus. Mael contains a High Mobility Group box, known to mediate DNA binding. We show that Mael nuclear function is required for proper differentiation in the Drosophila germline stem cell (GSC) lineage. In mael mutant testes, transit-amplifying cysts fail to differentiate into primary spermatocytes, instead breaking down into ectopic GSCs and smaller cysts, due to a depletion of Bag-of-marbles (Bam) protein. Mael regulates Bam via repression of miR-7. Mael binds the miR-7 promoter and is required for the local accumulation of HP1 and H3K9me3. miR-7 targets bam directly at its 3'UTR, and a reduction in miR-7 expression can rescue germline differentiation defects found in mael mutants by alleviating Bam repression. We propose that Mael ensures proper differentiation in the GSC lineage by repressing miR-7.

SUMMARYMobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.

Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.

Complete genomic reprogramming happens twice during the life of a genome, once during the formation of gametes in their parents and once after their union at fertilization. For that matter complete genomic reprogramming happens twice in the same parental cell, the oocyte, when it is forming and after it matures and receives the paternal gamete. Control of these processes in this unique single cell is epigenetic, and our understanding of it is based on information gleaned from imprinting, X chromosome inactivation, and activation and silencing of endogenous retroviruses (ERV). Activation of ERVs is attributed to demethylation of chromatin and DNA, whereas silencing requires methylation, attributed to the nuage proteins, which engage the Piwi-interacting RNA pathway and other posttranscriptional mechanisms. This reprogramming process has evolved throughout speciation because in mammals, but not fish, flies and worms, nuage-component muations affect male and female gametogenesis differentially. Transcription of ERVs is derepressed in both sexes in nuage-mutant mice, but whereas males are sterile, females are fertile. Using a proteomic approach we now report molecular interactions between nuage proteins and components of the oocyte cytoplasmic lattice and speculate how this interaction could preserve ERV/host chimeric gene products affecting female fertility.

Nuage, a well conserved perinuclear organelle found in germline cells, is thought to mediate the repression of retroelements in Drosophila melanogaster by regulating the production of Piwiinteracting RNAs (piRNAs). Here, we present evidence that the nuage/piRNA pathway components can be found in cytoplasmic foci that also contain retroelement transcripts, anti-sense piRNAs and proteins involved in mRNA degradation. These mRNA degradation proteins, Decapping Proteins 1/2, Me31B and Pacman, are normally thought of as components of the Processing bodies. In spn-E and aub mutants where the production of piRNAs is compromised, piRNA pathway proteins no longer overlap the mRNA degradation proteins. Concomitantly, spn-E and aub mutant ovaries show a substantial accumulation of the full-length retroelement transcripts and prolonged stabilization of HeT-A mRNA, supporting a role of piRNAs in mediating post-transcriptional retroelement silencing. HeT-A mRNA is de-repressed in the canonical mRNA degradation mutants, indicating that these enzymes aid in removal of the full-length transcripts and/or decay intermediates. We have characterized small RNA expression profiles in differentiating mouse Embryonic Stem (ES) cell as a model for early mammalian development. High-throughput 454 pyrosequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs largely dominated the small RNA repertoire and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised Partitioning Around Meloids (PAM) analysis revealed that differentiating ES cell miRNAs fall into three statistically significant expression clusters with highly contrasted patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several microRNA genomic clusters. Notably this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male, but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. MicroRNAs (miRNAs) are a class of small RNAs that bind to specific mRNAs targets, directing their degradation and/or repressing their translation, which have been implicated in the control o...

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