Subcellular RNA localization during Drosophila embryonic neurogenesis [fractionation-Seq]
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PROVIDER: PRJNA415601 | ENA |
REPOSITORIES: ENA
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Project description:We isolated whole embryo, nuclear, and cytoplasmic lysates from Drosophila embryos during early and late embryogenesis In Drosophila melanogaster, cell-type specification during early nervous system development requires precise regulation of gene expression in time and space. To investigate the gene regulation patterns in early neuronal development, we developed a method 'DIV-SortSeq' to enrich for specific neuroglial cell types for RNA sequencing: from early columnar subdivision and specification, through neuroglial differentiation. DIV-SortSeq recapitulates many of the known protein-coding transcriptome dynamics, as well as novel transcriptional regulatory patterns. We also performed nuclear-cytoplasmic fractionation of whole embryos - Fractionation-Seq - to assess temporal changes in subcellular localization of transcripts during embryogenesis. We present these data as a resource to permit further in-depth investigation into the functional roles of the coding and noncoding transcriptome during early Drosophila neurogenesis.
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Project description:Identification of spatiotemporal RNA expression and subcellular RNA localization during Drosophila embryonic neurogenesis
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Project description:We used immunostaining and FACS to enrich for distinct neural cell types at multiple stages of Drosophila neurogenesis for RNA-Seq In Drosophila melanogaster, cell-type specification during early nervous system development requires precise regulation of gene expression in time and space. To investigate the gene regulation patterns in early neuronal development, we developed a method 'DIV-SortSeq' to enrich for specific neuroglial cell types for RNA sequencing: from early columnar subdivision and specification, through neuroglial differentiation. DIV-SortSeq recapitulates many of the known protein-coding transcriptome dynamics, as well as novel transcriptional regulatory patterns. We also performed nuclear-cytoplasmic fractionation of whole embryos - Fractionation-Seq - to assess temporal changes in subcellular localization of transcripts during embryogenesis. We present these data as a resource to permit further in-depth investigation into the functional roles of the coding and noncoding transcriptome during early Drosophila neurogenesis.
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Project description:The distribution of RNA in human embryonic stem cells (hESC) and the function of RNA localization in maintaining hESC pluripotency and differentiation are currently unknown. Here, by isolating five subcellular components of hESCs and differentiated cells, we uncovered the global subcellular RNA localization in hESC. For protein-coding mRNA, different transcripts of the same gene exhibit an “isoform switch” between subcellular components, which is regulated by localization cis-elements in their variable regions. For noncoding RNA, multiple sequence features such as polyA tail, length, and GC content jointly regulate their subcellular localization. In addition, we found that some developmental genes can be transcribed in advance and confined to chromatin in undifferentiated hESCs. Finally, we revealed significant changes in overall RNA distribution, mapped RNA dynamic localization atlas, and characterized different dynamic RNA localization patterns during hESC differentiation into mesoderm. The multiple RNA localization patterns we revealed will provide some new enlightenment for hESC stemness maintenance and differentiation.
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Project description:Analysis of subcellular location of mitochondrial gene products using two distinct cell fractions: membrane-bound (MB) and high speed (HS) obtained via biochemical fractionation from wild type individuals and mia40a zebrafish mutants, respectively.
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Project description:Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate the effect of AS on ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional Illumina short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESCs. We performed de novo transcriptome assembly from high confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Almost half of all transcripts exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 18% exhibited significant differential isoform sedimentation in ESC and NPC, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated 3’ and 5’ untranslated regions (UTR) GC-content and coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using long read sequencing-based method to study translational control.
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Project description:Atlas of Subcellular RNA Localization Revealed by APEX-seq
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Project description:We examined the subcellular localization of long noncoding RNAs (lncRNAs) in mouse embryonic stem cells.
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Project description:Objective: To assess the role of aldoketoreductases and other doxorubicin pharmacokinetic or pharmacogenomic genes in doxorubicin cytotoxicity, resistance, DNA binding activity, and subcellular localization, Methods: We conducted a whole genome microarray study to identify differences in between doxorubicin-sensitive MCF-7cc cells and doxorubicin-resistant MCF-7Dox2-12 cells in terms of their expression of genes related to doxorubicin pharmacokinetics or pharmacodynamics. Targets were then validated by pharmacologic inhibition in conjunction with drug metabolite profiling, drug localization, drug cytotoxicity, and drug DNA binding studies. Results: 2063 differentially expressed transcripts were identified, including 17% and 43% of genes or gene families associated with doxorubicin pharmacokinetics or pharmacodynamics (p values of significance of 0.05 and <0.0001, respectively). The largest changes in the expression of genes associated with doxorubicin pharmacokinetics and pharmacodynamics were chiefly among the aldo-keto reductases (AKRs) Akr1c2, Akr1c3 and Akr1b10 which convert doxorubicin to doxorubicinol. We observed that doxorubicinol exhibits dramatically reduced drug toxicity, reduced drug DNA-binding activity, and altered drug subcellular localization to lysosomes. Pharmacologic inhibition of these AKRs in MCF-7Dox2-12 cells restored drug cytotoxicity, and drug localization to the nucleus. Conclusion: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledgebases to identify highly relevant genes associated with doxorubicin resistance. The products of one or more of these genes could effectively be shown to alter the drug’s properties, while inhibiting them restored drug DNA binding, cytotoxicity, and subcellular localization. Doxorubicin resistant cell lines of breast MCF-7 cells were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with four labelling replicates of both forward and reverse labellings plus another set of 8 arrays with forward labelling. Sixteen arrays were used for this experiments. The co-cultured control cells MCF-7cc12 was generated by parallel selection process in the absence of drug.
2012-11-22 | E-GEOD-27254 | biostudies-arrayexpress