Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MiR-309-dependent unstable maternal mRNAs are stabilized in smaug mutants

ABSTRACT: The behavior of 410 miR-309-dependent maternal mRNAs (from Bushati et al., 2008) in embryos from wild type and smg-mutant females relative to wild-type stage 14 oocyte reference RNA. All are unstable in wild-type while almost 85% are stabilized in smg mutants mRNA was extracted from staged embryos as well as stage 14 oocytes as described previously (Tadros et al., 2007a). To assay mRNA quality, known stable (rpA1) and unstable (Hsp83) transcripts were probed on Northern blots. Total RNA was then reverse transcribed with random primers (Tadros et al., 2007a) and labeled asÃ?described in the Indirect Labeling of Total RNA for Microarray Hybridization protocol at http://www.flyarrays.com. The fluorescently labeled cDNA probes were hybridized to 14Kv1 microarrays obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com; GPL3603: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL3603). Hybridization and scanning were performed using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software as previously described (Neal et al., 2003). The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer), using the adaptive quantification algorithm and analyzed using GeneTraffic Duo3.2 (Iobion Informatics/Stratagene). . The 14Kv1 arrays were normalized to a set of known stable transcripts: Rpl1, RpL32, Rps5a, Rps3, RpL22, mRpS30, mRpS22, CG6764, bonsai, RpS29, RpL11, mRpL1, CG6764, CG317, RpL37A, and RpL40.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: J Westwood

PROVIDER: E-GEOD-13288 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

Benoit Beatrice B He Chun Hua CH Zhang Fan F Votruba Sarah M SM Tadros Wael W Westwood J Timothy JT Smibert Craig A CA Lipshitz Howard D HD Theurkauf William E WE

Development (Cambridge, England) 20090301 6

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding ...[more]

PMID: 19234062

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Project description:Microarray analysis of zygotic gene expression in 2-to-3 hour wild-type (wt) and smg mutant embryos. Expression is relative to mature, stage 14 oocytes, which contain the full maternal pool of mRNA. Strictly maternal genes that are not transcribed at the MZT contribute approximately 80% of transcripts in early embryos, and are not shown. Class I zygotic genes showed high levels of expression in 2-to-3 hour embryos. 142 of the 166 Class I genes were not expressed in smg mutants. The remaining zygotically expressed genes were also present in oocytes. These genes were divided into two classes, based on analysis of 4-to-6 hour old unfertilized eggs (4-6h unf), which are transcriptionally inactive. Class-II genes produce maternal transcripts that are stable in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos. 358 of 395 Class-II genes require SMAUG for zygotic expression. Class-III genes produce maternal transcripts that are degraded in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos. 65 of 408 Class-III genes require SMAUG for expression in 2-to-3 hour embryos. mRNA was extracted from staged fertilized or unfertilized eggs, as well as stage 14 oocytes as described previously (Tadros et al., 2007a). To assay mRNA quality, known stable (rpA1) and unstable (Hsp83) transcripts were probed on Northern blots. Total RNA was then reverse transcribed with random primers (Tadros et al., 2007a) and labeled asÃ?described in the Indirect Labeling of Total RNA for Microarray Hybridization protocol at http://www.flyarrays.com. The fluorescently labeled cDNA probes were hybridized to 12Kv1 microarrays obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com; GEO platform accession number GPL1467: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1467). Hybridization and scanning were performed using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software as previously described (Neal et al., 2003). The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer), using the adaptive quantification algorithm and analyzed using GeneTraffic Duo3.2 (Iobion Informatics/Stratagene). The 12Kv1 arrays were normalized using the rank invariant LOWESS extrapolation method (Schadt et al., 2001).

Project description:In animals, egg activation triggers a cascade of posttranscriptional events that act on maternally synthesized RNAs. We show that, in Drosophila, the PAN GU (PNG) kinase sits near the top of this cascade, triggering translation of SMAUG (SMG), a multifunctional posttranscriptional regulator conserved from yeast to humans. Although PNG is required for cytoplasmic polyadenylation of smg mRNA, it regulates translation via mechanisms that are independent of its effects on the poly(A) tail. Analyses of mutants suggest that PNG relieves translational repression by PUMILIO (PUM) and one or more additional factors, which act in parallel through the smg mRNA's 3' untranslated region (UTR). Microarray-based gene expression profiling shows that SMG is a major regulator of maternal transcript destabilization. SMG-dependent mRNAs are enriched for gene ontology annotations for function in the cell cycle, suggesting a possible causal relationship between failure to eliminate these transcripts and the cell cycle defects in smg mutants. Keywords: Identification of Maternal mRNAs RNA was extracted from staged unfertilized eggs and stage 14 oocytes using a modification of the TRIzol (Invitrogen) method (Neal et al., 2003). Sample quality was evaluated by probing northern blots for known stable (rpA1) and unstable (Hsp83) transcripts. Total RNA was reverse transcribed as previously described (Neal et al., 2003), except that priming was carried out using random primers rather than oligo-dT in order not to bias the labeling toward mRNAs with long poly(A) tails. The fluorescently labeled cDNA probes were hybridized to 12Kv1 microarray slides obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com). These represent 10,500 distinct protein-coding genes, or 77% of the Drosophila protein-coding genome. Hybridization and scanning was also as previously described (Neal et al., 2003), using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software. The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer) using the adaptive quantification algorithm and analyzed using GeneTraffic Duo 3.2 (Iobion Informatics/Stratagene) after normalization to the known stable transcripts rpA1 and rp49. Of the 10,500 distinct protein-coding genes on the microarray, 9,257 were analyzed for maternal expression. The averages of all of the raw values for 21 hybridizations of wild-type stage 14 oocyte RNA (three replicates of seven experiments) were sorted from highest to lowest. Maternal expression was assessed at different levels in the list by scanning the Berkeley Drosophila Genome Project in situ database (http://www.fruitfly.org/cgi-bin/ex/insitu.pl). RNA from the genes at the top of the table is maternally loaded (90.4% of transcripts with an average raw intensity > 20,000 are maternal; n = 73), whereas RNA from genes at the bottom is absent from early embryos (only 8.2% of those with an average intensity of < 2,000 are maternal; n = 73). Hsp83 appeared near the top of the list (sixth out of 9,257). Transcripts with average values between 3,500 and 5,000 were mostly maternal (75%; n = 76). As the values decreased, there was a decreasing frequency of maternal transcripts (58.7% between 3,000 and 3,500, n = 172; 46.9% between 2,800 and 3,000, n = 160; 26.2% between 2,500 and 2,800, n = 80; 24.7% between 2,000 and 2,500, n = 81). Using a cutoff value of 3,000, we calculate that 55% of all protein-coding genes are represented in stage 14 oocytes

Dataset Information

MiR-309-dependent unstable maternal mRNAs are stabilized in smaug mutants

Publications

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

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