Ribosome profiling in Drosophila ovary enriched for late-stage oocytes (late-ovary)
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE210503 | GEO | 2022/12/31
REPOSITORIES: GEO
Similar Datasets
Project description:Ribosome profiling in Drosophila ovary enriched for late-stage oocytes (late-ovary)
| PRJNA865860 | ENA
Project description:Identification of interaction partners of the sole Drosophila hnRNP F/H homolog, Glorund, during late oogenesis.
2022-06-07 | PXD033174 | Pride
Project description:Identification of interaction partners of the sole Drosophila hnRNP F/H homolog, Glorund, during late oogenesis.
2022-06-07 | PXD033175 | Pride
Project description:Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. We investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. Our analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. We propose that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis.
2021-02-24 | PXD022142 | Pride
Project description:Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by increased ovarian androgen production, arrested follicle development, and is frequently associated with insulin resistance. These PCOS phenotypes are associated with exaggerated ovarian responsiveness to FSH and increased pregnancy loss. To examine whether the perturbations in follicle growth and the intrafollicular environment affects development of the mature PCOS oocyte, genes that are differentially expressed in PCOS compared to normal oocytes were defined using microarray analysis. This analysis detected approximately 8000 transcripts. Hierarchical clustering and principal component analysis revealed differences in global gene expression profiles between normal and PCOS oocytes. 374 genes had a statistically-significant increase or decrease in mRNA abundance in PCOS oocytes. A subset of these genes was associated with chromosome alignment and segregation during mitosis and/or meiosis, suggesting that increased mRNAs for these proteins may negatively affect oocyte maturation and/or early embryonic development. Of the 374 differentially expressed genes, 68 contained putative androgen receptor, retinoic acid receptor, and/or peroxisome proliferating receptor gamma binding sites, including 9 of the genes involved in chromosome alignment and segregation. These analyses demonstrated that normal and PCOS oocytes that are morphologically indistinguishable and of high quality exhibit different gene expression profiles. Furthermore, altered mRNA levels in the PCOS oocyte may contribute to defects in meiosis and/or mitosis which might impair oocyte competence for early development and therefore contribute to poor pregnancy outcome in PCOS. Experiment Overall Design: A single MII oocyte, defined by one polar body in the perivitelline space and no visible nuclear structure in the cytoplasm, was collected from 6 individual NL and 6 individual PCOS ovaries, placed immediately in TRIzol (Sigma, St. Louis MO), and stored at -80 C until further study. Total RNA was isolated from each oocyte and subjected to three rounds of linear amplification with the Ovation Biotin RNA Amplification and Labeling System (NuGen Technologies, San Carlos CA) per the manufacturerâs instructions. RNA from the GeneChip Eukaryotic Poly-A RNA Control Kit (Affymetrix, Santa Clara CA) was amplified and labeled under the same conditions for a positive control. Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA) were hybridized at the University of Pennsylvania Microarray Core Facility. Briefly, the linear-amplified, biotin-labled cDNA from 6 NL (N1-N6) and 6 PCOS (P1-P6) oocytes was hybridized to individual Affymetrix U133 chips. The fluorescence intensity of each chip was normalized to a trimmed mean signal of 150. Each transcript on the U133 chip was defined as present or absent in each oocyte sample using the Affymetrix Microarray Suite 5.0.
2008-06-14 | E-GEOD-5850 | biostudies-arrayexpress
Project description:RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.
| S-EPMC3020115 | biostudies-literature
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
2014-06-12 | E-GEOD-52799 | biostudies-arrayexpress
Project description:Stem cells in many systems, including Drosophila germline stem cells (GSCs), have increased ribosome biogenesis and translation during terminal differentiation. Here, we show that pseudouridylation of ribosomal RNA (rRNA) mediated by the H/ACA box is required for ribosome biogenesis and oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of mRNAs that are enriched for CAG repeats and encodes polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing TOR activity to elevate ribosome levels in H/ACA box-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.
2023-11-22 | GSE206691 | GEO
Project description:To identify the transcripts fractionated into microsome fraction in ribosome-independent manner, we isolate rough microsome fraction by sucrose density gradient ultracengrifugation, then the rough microsome fraction is centrifugated following treatment with puromycine and EDTA in high-salt buffer to remove ribosomes. The pellet and surpernatant are named naked microsome fraction (NM) and stripped ribosome fraction (SR), respectively. By calculating the ratio of the level of each mRNA in NM and SR, we identify the enriched transcripts in NM.
2016-12-25 | GSE85768 | GEO
Project description:Stem cells in many systems, including Drosophila germline stem cells (GSCs), have increased ribosome biogenesis and translation during terminal differentiation. Here, we show that pseudouridylation of ribosomal RNA (rRNA) mediated by the H/ACA box is required for ribosome biogenesis and oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of mRNAs that are enriched for CAG repeats and encodes polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing TOR activity to elevate ribosome levels in H/ACA box-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.
2023-11-22 | GSE206690 | GEO