Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA215941 | ENA |
REPOSITORIES: ENA
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Project description:Pumilio (PUM) is a Drosophila member of a conserved family of sequence-specific RNA-binding proteins that have been shown to regulate mRNA stability and/or translation in a variety of organisms. PUM has been shown to repress the translation of several mRNAs in the Drosophila early embryo; failure to repress these targets leads to lethal developmental defects. Here we use a combination of microarray-based gene expression profiling and next-generation sequencing to identify more than 200 mRNAs that are associated with full-length PUM protein in early embryos and to define a global role for PUM in mRNA decay. Surprisingly, despite the fact that PUM is maternally supplied and thus is present from the beginning of embryogenesis, the vast majority of PUM-directed decay occurs only after zygotic genome activation. We show that the smaug mRNA, which itself encodes an RNA-binding protein that directs transcript decay, is a direct target of PUM via binding sites in the smg 3'UTR. Whereas the endogenous smaug mRNA and the transgenic reporter mRNA that carries the smaug 3'UTR undergo decay after zygotic genome activation, a reporter with an array of PUM-binding sites decays before zygotic genome activation. These data support a model in which additional cis-elements in the smg 3'UTR delay decay until after zygotic genome activation.
2017-07-01 | GSE48645 | GEO
Project description:To gain a panoramic view of Smaug function in the early embryo we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified the mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug?s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets as well as many metabolic enzymes, including several glycolytic enzymes. Embryos laid by wild-type or smaug1 homozygous mothers were collected 0-2 hours post-egglaying and lysed in an equal volume of polysome lysis buffer. The lysate was applied to polysome gradients that were fractionated into four fractions. mRNA was extracted from each fraction and converted to cDNA that was applied on custom-designed Nimblegen arrays. Three biological replicates were performed for each of the four fraction in each of the two genotypes. To demonstrate that the presence of mRNAs at the bottom of the gradient were the result of translation, we performed a puromycin experiment. smaug1 homozygous mothers were collected 0-2 hours post-egglaying and lysed in an equal volume of puromycin lysis buffer and then treated either with cycloheximide or puromycin. The lysate from each condition was applied to separate polysome gradients that were each fractionated into four fractions. mRNA was extracted from each fraction and converted to cDNA that was applied on custom-designed Nimblegen arrays. Two biological replicates were performed for each of the four fraction from each of the two conditions.
2013-12-03 | E-GEOD-50026 | biostudies-arrayexpress
Project description:To gain a panoramic view of Smaug function in the early embryo we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified the mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug?s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets as well as many metabolic enzymes, including several glycolytic enzymes.
2013-12-03 | GSE50026 | GEO
Project description:Identification of Smaug target mRNAs in the early Drosophila embryo using RIP-Chip
| PRJNA215420 | ENA
Project description:Translational control is critical for early Drosophila embryogenesis and is exerted mainly at the gene-specific level. To understand the post-transcriptional regulation during Drosophila early embryonic development, we used the sucrose polysomal gradient analyses and GeneChip analysis to illustrate the translation profile of individual mRNAs. A paper was published in Genome Biology 2007 under the tile Global analyses of mRNA translational control during early Drosophila embryogenesis". Experiment Overall Design: In this study, we have fractionated embryo extracts from a series of early stages by sedimentation on sucrose density gradients and analyzed the RNA components of these fractions. Our analysis has focused on analyzing ribosomal density, generally and for individual transcripts, global translational activity during the first 10 hours after egg laying and coordination between transcription and translation regulation.
2008-03-14 | E-GEOD-5430 | biostudies-arrayexpress
Project description:Background: Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which a subset of maternal gene products is eliminated and the zygotic genome becomes transcriptionally active. RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) – of which Argonaute 1 (AGO1) is a key component in Drosophila – target maternal mRNAs for degradation. The Drosophila Smaug, Brain tumor (BRAT) and Pumilio (PUM) RBPs direct the degradation of maternal mRNAs. Here we elucidate Smaug’s roles in regulation of miRNAs and miRISC during the MZT. Results: By global analysis of small RNAs at several stages during the MZT, we show that the vast majority of all miRNA species encoded by the Drosophila genome (85%) are expressed during the MZT. Whereas a subset of these miRNAs is loaded into oocytes by the mother and stays at constant levels during the MZT, dozens of miRNA species are either newly synthesized or re-expressed in the early embryo. Loss of Smaug has a profound effect on miRNAs but little effect on piRNAs or siRNAs. Smaug is required for production of new miRNAs during the MZT; Smaug-bound AGO1 reflects the constellation and abundance of the miRNAs present in early embryos; and Smaug is required for the increase in AGO1 protein levels that occurs during the MZT. As a consequence of low miRISC activity in smaug mutants, maternal mRNAs that are normally targeted for degradation by zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with miRISC during the MZT while the miR-309 miRNA family coregulates targets of BRAT but not PUM. Conclusions: Smaug controls the MZT through direct targeting of a subset of maternal mRNAs for degradation and, indirectly, through production and function of miRNAs and miRISC, which control clearance of a distinct subset of maternal mRNAs. BRAT and/or PUM function together with miRISC during the latter process. With respect to miRISC-dependent transcript degradation, Smaug is required (1) for the synthesis of miRNAs, (2) for synthesis and stabilization of AGO1, and (3) for action of AGO1 in association with its bound miRNAs. In smaug mutants a large number of maternal mRNAs persist and the MZT fails.
2016-09-06 | GSE82194 | GEO
Project description:Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-Sequencing (TL-Seq), a technique combining enzymatic capture of m7G-capped mRNA 5'-ends with high-throughput sequencing. TL-Seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Transcription initiation sites occur in at least 5% of protein-coding regions and are concentrated near the 5'ends of ORFs. These truncated mRNAs are translated, based on ribosome density analysis. Translation Associated TL-Seq (TATL-Seq), which combines TL-Seq with polysome fractionation, revealed substantial differences in translation of alternative TL isoforms. Globally, while some TL features are associated with poor translation and nonsense-mediated mRNA decay (uAUGs, very short length), others (secondary structure, long length) have much less impact than predicted from analyses of individual genes. TL-Seq and TATL-Seq can be applied to any eukaryote to investigate TL-mediated regulation of gene expression. TL-Seq (+/- pyrophosphatase), TATL-Seq (TL-Seq libraries from polysome gradient)
2013-04-03 | E-GEOD-39074 | biostudies-arrayexpress
Project description:During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug plays an essential role in progression through the MZT by regulating the translational repression and degradation of a large number of maternal mRNA species. The Smaug protein itself is rapidly cleared at the end of the MZT by the Skp/Cullin/F-box (SCF) E3-ligase complex; clearance of Smaug requires zygotic transcription. Here, we show that an F-box protein, which we name Bard (encoded by CG14317), is required for degradation of Smaug. Bard is expressed zygotically and physically interacts with Smaug at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of Smaug. We show that shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of Smaug protein, a phenotype that is rescued by transgenes expressing Bard .
2021-10-06 | MSV000088199 | MassIVE
Project description:The maternal mRNA nanos is required for anterior-posterior patterning in the early Drosophila melanogaster development. The nanos-mRNA is translationally repressed and deadenylated during the first two hours of embryogenesis. A stem-loop structure in the 3'-UTR of this RNA (SRE - Smaug recogniiton element) is bound by the Smaug protein which recruits a protein complex to the RNA to sequester its translation and intiate transcript degradation via the ccr4/not-complex. To analyse the composition of this repressor complex, biotinylated RNAs carrying two SRE stem-loops were used for an affinity purification of bound proteins. As a negative control an RNA was used that contained two point mutations in the SRE loop, that abolish binding of Smaug. Seven subunits were identified as components of the repressor complex. The main deadenylase in Drosophila, the ccr4/not-complex was also significantly enriched.
2017-07-18 | PXD006596 | Pride
Project description:Background: Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which a subset of maternal gene products is eliminated and the zygotic genome becomes transcriptionally active. RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) â of which Argonaute 1 (AGO1) is a key component in Drosophila â target maternal mRNAs for degradation. The Drosophila Smaug, Brain tumor (BRAT) and Pumilio (PUM) RBPs direct the degradation of maternal mRNAs. Here we elucidate Smaugâs roles in regulation of miRNAs and miRISC during the MZT. Results: By global analysis of small RNAs at several stages during the MZT, we show that the vast majority of all miRNA species encoded by the Drosophila genome (85%) are expressed during the MZT. Whereas a subset of these miRNAs is loaded into oocytes by the mother and stays at constant levels during the MZT, dozens of miRNA species are either newly synthesized or re-expressed in the early embryo. Loss of Smaug has a profound effect on miRNAs but little effect on piRNAs or siRNAs. Smaug is required for production of new miRNAs during the MZT; Smaug-bound AGO1 reflects the constellation and abundance of the miRNAs present in early embryos; and Smaug is required for the increase in AGO1 protein levels that occurs during the MZT. As a consequence of low miRISC activity in smaug mutants, maternal mRNAs that are normally targeted for degradation by zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with miRISC during the MZT while the miR-309 miRNA family coregulates targets of BRAT but not PUM. Conclusions: Smaug controls the MZT through direct targeting of a subset of maternal mRNAs for degradation and, indirectly, through production and function of miRNAs and miRISC, which control clearance of a distinct subset of maternal mRNAs. BRAT and/or PUM function together with miRISC during the latter process. With respect to miRISC-dependent transcript degradation, Smaug is required (1) for the synthesis of miRNAs, (2) for synthesis and stabilization of AGO1, and (3) for action of AGO1 in association with its bound miRNAs. In smaug mutants a large number of maternal mRNAs persist and the MZT fails. Examination of miRNA expresssion at different time points in wild type and smuag mutant early embryos .
2016-09-06 | E-GEOD-82194 | biostudies-arrayexpress