RNA degradation sculpts the maternal transcriptome during Drosophila oogenesis (original) (raw)
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The EMBO Journal, 1999
A.Bashirullah, S.R.Halsell and R.L.Cooperstock contributed equally to this work Maternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis-acting elements in the 3Ј-untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.
Molecular Cell, 2013
The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two interrelated branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborate pathway centered on the three gonad-specific Argonaute proteins (Piwi, Aubergine, and Argonaute 3). While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals key factors of piRNA-mediated transposon silencing, including the piRNA biogenesis factors CG2183 (GASZ) and Deadlock. Our data uncover a previously unanticipated set of factors preferentially required for repression of different transposon types.
Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference
PLoS ONE, 2014
In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.
Development, 2005
Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans,apoptosis occurring through a specific `physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased,brood size is modestly reduced, and early...
Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary
Cell, 2009
In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicerdependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.
Regulation of Maternal Transcript Destabilization During Egg Activation in Drosophila
2009
In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation.
Cells, 2020
DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Belle is involved both in translational activation and repression of target mRNAs in different tissues; however, direct targets of Belle in the testes are essentially unknown. Here we showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of β-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail we performed cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis and identified multiple mRNAs ...
Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila
Nature Communications, 2016
The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earlie...
Expression of the Sex-lethal gene is controlled at multiple levels during Drosophila oogenesis
Development (Cambridge, England), 1993
In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal (Sxl) gene is required for proper differentiation of female germ cells. To investigate its role in germ-line development, we have examined the expression of Sxl in wild-type ovaries and ovaries that are defective in early steps of germ cell differentiation. As in the soma, the basic mechanism for on/off regulation of Sxl relies on sex-specific processing of its transcripts in germ cells. One class of female-sterile mutations, which includes fs(1)1621 and the tumorous-ovary-producing allele of the ovarian tumor gene, otu1, is defective in the splicing process. These mutants have germ lines with high amounts of Sxl RNA spliced in the male mode and a severe reduction of protein levels in the germ cells. Another class of female-sterile mutations produces a phenotype similar to that seen in fs(1)1621 and otu1 but appears to express normal levels of Sxl protein in the germ cells. However, this...