Project description:Drosophila Piwi-family proteins have been implicated in transposon control. Here, we examine piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein and find that Piwi and Aubergine bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. Our data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5’ end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs, may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci. Keywords: small RNA libraries from Drosophila ovaries
Project description:Species of the genus Drosophila have served as favorite models in speciation studies, however genetic factors of the interspecific hybrid sterility are underinvestigated to date. Here we performed the analysis of reproductive incompatibilities of hybrid females in crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis, molecular, cellular and genetic approaches we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis and functional defects of oogenesis in hybrids. A premature GSC loss was a most prominent defect of oogenesis in hybrid ovaries. Owing differential expression of genes encoding components of the piRNA pathway rhino and deadlock, functional RDCmel complex in hybrid ovaries was not assembled. At the same time the activity of RDCsim complex was maintained in hybrids, independently from the genomic origin of piRNA clusters. Despite identification of a cohort of overexpressed TEs in hybrid ovaries we found no evidences that their activity can be considered as the main cause of hybrid sterility. We revealed complex pattern of Vasa protein expression in hybrid germline, including partial AT-chX piRNA targeting of vasasim allele and significant developmental delay of vasamel expression. We came to the conclusions that complex multi-locus genetic changes between the species were responsible for hybrid sterility phenotype.
Project description:Species of the genus Drosophila have served as favorite models in speciation studies, however genetic factors of the interspecific hybrid sterility are underinvestigated to date. Here we performed the analysis of reproductive incompatibilities of hybrid females in crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis, molecular, cellular and genetic approaches we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis and functional defects of oogenesis in hybrids. A premature GSC loss was a most prominent defect of oogenesis in hybrid ovaries. Owing differential expression of genes encoding components of the piRNA pathway rhino and deadlock, functional RDCmel complex in hybrid ovaries was not assembled. At the same time the activity of RDCsim complex was maintained in hybrids, independently from the genomic origin of piRNA clusters. Despite identification of a cohort of overexpressed TEs in hybrid ovaries we found no evidences that their activity can be considered as the main cause of hybrid sterility. We revealed complex pattern of Vasa protein expression in hybrid germline, including partial AT-chX piRNA targeting of vasasim allele and significant developmental delay of vasamel expression. We came to the conclusions that complex multi-locus genetic changes between the species were responsible for hybrid sterility phenotype.
Project description:H3K27me3 profiles using Cleavage under targets and Release using nuclease (Cut&Run) in control and KD Drosophila melanogaster ovaries. We examined the impact on chromatin profiles in Drosophila melanogaster ovaries in which the lid, the Sin3a, the Snr1 or the mod(mdg4) gene have been selectively knocked down by tissue-specific shRNA expression. We additionally explored H3K27me3 and H3K9me3 in control and dhd mutant ovaries either carrying or not a transgene.
Project description:Drosophila Piwi-family proteins have been implicated in transposon control. Here, we examine piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein and find that Piwi and Aubergine bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. Our data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5’ end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs, may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci. Keywords: small RNA libraries from Drosophila ovaries small RNAs (23-29nt) were isolated from total ovarian RNA or from immunopreciptated Piwi/Aubergine/Ago3 complexes. cDNA libraries were constructed after Pfeffer et al. 2005 (Nat. Methods) and sequenced at 454 Life Sciences. The used strain is OregonR. Only sequences matching the Release5 genome assembly (www.fruitfly.org) are considered.
Project description:Here we perform transcriptional profiling of late third instar larval ovaries and gonadal fatbody from Drosophila melanogaster w1118 flies. We designed this experiment to investigate the effect of protease treatment on dissected ovaries. We profiled ovaries without protease treatment with some fatbody still attached, fatbody alone dissected from around the ovaries, ovaries cleaned of fatbody using a papain/collagenase cocktail, and ovaries dissociated using a papain/collagenase cocktail. We then constructed polyA+ libraries and performed 50 bp stranded single end RNA-Seq.