E-GEOD-50026 - Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein- Polysome gradient/microarray study
Released on 3 December 2013, last updated on 9 December 2013
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.
transcription profiling by array
Jason Gardiner Dumelie <email@example.com>, Craig A Smibert, Howard D Lipshitz, J T Westwood, Jason G Dumelie, John D Laver, Linan Chen, Matthew H Cheng, Najeeb U Siddiqui, Quaid Morris, Xiao Li, Zhiyong Yang