Small RNAs from Drosophila melanogaster WT and Tailor mutant S2 cells and ovaries
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE67984 | GEO | 2016/04/15
SECONDARY ACCESSION(S): PRJNA281580
REPOSITORIES: GEO
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Project description:Uridylation of diverse RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms and mammals. Here, we report the first uridylyltransferase that acts on small RNAs in Drosophila, which we refer to as Tailor. Tailor is the source for the majority of 3´ end-modifications in microRNAs and predominantly targets precursor-hairpins. Uridylation modulates the characteristic two-nucleotide 3´ overhangs of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Furthermore, Tailor preferentially uridylates mirtron-hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron-selectivity is explained by unique primary sequence specificity of Tailor, selecting RNA substrates ending with a 3´ guanosine, a feature not previously observed for TUTases. In contrast to mirtrons, conserved Drosophila pre-miRNAs are significantly depleted in 3´ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to pre-miRNA uridylation shapes the nucleotide composition of pre-miRNA 3´ ends. Hence, hairpin-uridylation may serve as a barrier for the de novo creation of miRNAs in Drosophila.
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Project description:Uridylation of hairpin-RNAs by Tailor confines the emergence of miRNAs in Drosophila
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Project description:Uridylation of diverse RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms and mammals. Here, we report the first uridylyltransferase that acts on small RNAs in Drosophila, which we refer to as Tailor. Tailor is the source for the majority of 3´ terminal uridine-modifications in small RNAs and predominantly targets precursor-miRNAs. Uridylation modulates the characteristic two-nucleotide 3´ overhangs of miRNA hairpins, which regulates pre-miRNA processing by Dicer-1. Furthermore, Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron-selectivity is explained by unique primary sequence specificity of Tailor, selecting RNA substrates ending with a 3´ guanosine, a feature not previously observed for terminal uridylyltransferases. In contrast to mirtrons, conserved Drosophila pre-miRNAs are significantly depleted in 3´ guanosine, thereby escaping regulatory uridylation.Our data support the hypothesis that evolutionary adaptation to pre-miRNA uridylation shapes the nucleotide composition of pre-miRNA 3´ ends and may serve as a barrier for the de novo creation of miRNAs in Drosophila.
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Project description:Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.
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Project description:Insect ovaries are made up of tubular egg producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the Drosophila larval ovary at the beginning, middle and end of terminal filament formation using RNA-seq. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development.
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Project description:Small RNAs from Drosophila melanogaster WT and Tailor mutant S2 cells and ovaries
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Project description:The Piwi-interacting RNA (piRNA)-interacting Piwi protein is involved in transcriptional silencing of transposable elements in ovaries of Drosophila melanogaster. Here we characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. Our results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. We found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. We also show that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but we noted an increase in small nuclear spliceosomal RNAs abundance and propose Piwi involvement in their post-transcriptional regulation. Our work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression.
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