Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAi knock down of export factors in Drosphila Schneider cell lines

ABSTRACT: Six different endogenous Drosophila proteins (all potential export factors) have been inactivated using RNAi and leptomycin B (LMB) treatments. Five different endogenous Drosophila proteins (all potential export factors) have been targeted by RNAi. The effect on mRNA export is analyzed by monitoring the levels of mRNAs in cytoplasmic and total samples. Samples from knock-downs are compared to samples obtained from (GFP transfected) control cells processed in parallel. [The two samples compared have the same name, but the suffix _KD for knock down and _ctrl for the control]. The potential export factors targeted by RNAi are: NXF1, NXF2, NXF3, p15 and UAP56. In an additional control experiment the translation initiation factor eIF4G was targeted by RNAi (to control for the specificity of the observed effects). The samples were taken 2 (nxf1_d2), 4 (nxf1_d4, p15_d4, uap56_d4), 5 (nxf2_d5, nxf3_d5) or 6 (IF_d6) days after transfecting with dsRNA. Crm1, a transport factor responsible for protein export and the export of UsnRNAs and ribosomal subunits, has been specifically inactivated using LMB (instead of RNAi).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Luis Teixeira

PROVIDER: E-MEXP-4 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-wide analysis of nuclear mRNA export pathways in Drosophila.

Herold Andrea A Teixeira Luis L Izaurralde Elisa E

The EMBO journal 20030501 10

NXF1, p15 and UAP56 are essential nuclear mRNA export factors. The fraction of mRNAs exported by these proteins or via alternative pathways is unknown. We have analyzed the relative abundance of nearly half of the Drosophila transcriptome in the cytoplasm of cells treated with the CRM1 inhibitor leptomycin-B (LMB) or depleted of export factors by RNA interference. While the vast majority of mRNAs were unaffected by LMB, the levels of most mRNAs were significantly reduced in cells depleted of NXF ...[more]

PMID: 12743041

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Project description:Background: Despite the prevalence and biological relevance of both signalling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signalling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis of changes in alternative splicing using splicing-sensitive microarrays, induced by activation of two distinct signalling pathways, insulin and wingless, in Drosophila cells in culture. Results: Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and posttranscriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related with carbohydrate metabolism and cellular signalling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5â splice sites. Conclusion: Taken together, our data indicate that signalling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for cross-talk between different signalling pathways. To monitor transcriptional and alternative splicing changes induced by activation of the insulin and wingless pathways, a custom-designed microarray platform was employed featuring probes for all Drosophila genes for which different mRNA isoforms generated by alternative splicing have been described (see Blanchette M, Green RE, Brenner SE, Rio DC: Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 2005, 19(11):1306-1314.). Three biological replicates of total RNA isolated after pathway activation or controls (untreated cells for insulin, control dsRNA for wingless) were purified, reverse transcribed into cDNA and labelled with Cy5 or Cy3 fluorochromes and the cDNA was hybridized to the microarray,

Dataset Information

RNAi knock down of export factors in Drosphila Schneider cell lines

Publications

Genome-wide analysis of nuclear mRNA export pathways in Drosophila.

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