Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Smaug target mRNAs in the early Drosophila embryo using RIP-Chip

ABSTRACT: To identify SmaugM-bM-^@M-^Ys target mRNAs on a genome-wide scale we used ribonucleoprotein (RNP) co-immunoprecipitation followed by microarray analysis of the co-purifying mRNAs (RIP-Chip). Extracts, prepared from wild-type embryos collected 0-3 hours post-egglaying, were immunoprecipitated with an anti-Smaug antibody (Smaug RIPs) while control immunoprecipitations using non-immune serum served as a negative control (control RIPs). Genome-wide transcript expression in wild-type embryos were also assessed and used as reference. There are 11 array experiments presented here: 1. gene expression profiling of total mRNA sample extracted from wild-type 0-3 hour embryos (3 technical replicates performed using a pooled input reference sample); 2. RNA co-immunoprecipitations of endogenous Smaug (Smaug RIPs) from wild-type 0-3 hour embryos (3 biological replicates and 1 technical replicates); 3. control RNA co-immunoprecipitations (control RIPs) from wild-type 0-3 hour embryos (3 biological replicates and 1 technical replicates).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Howard Lipshitz

PROVIDER: E-GEOD-49943 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which a subset of maternal gene products is eliminated and the zygotic genome becomes transcriptionally active. RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) â of which Argonaute 1 (AGO1) is a key component in Drosophila â target maternal mRNAs for degradation. The Drosophila Smaug, Brain tumor (BRAT) and Pumilio (PUM) RBPs direct the degradation of maternal mRNAs. Here we elucidate Smaugâs roles in regulation of miRNAs and miRISC during the MZT. Results: By global analysis of small RNAs at several stages during the MZT, we show that the vast majority of all miRNA species encoded by the Drosophila genome (85%) are expressed during the MZT. Whereas a subset of these miRNAs is loaded into oocytes by the mother and stays at constant levels during the MZT, dozens of miRNA species are either newly synthesized or re-expressed in the early embryo. Loss of Smaug has a profound effect on miRNAsÂ but little effect on piRNAs or siRNAs. Smaug is required for production of new miRNAs during the MZT; Smaug-bound AGO1 reflects the constellation and abundance of the miRNAs present in early embryos; and Smaug is required for the increase in AGO1 protein levels that occurs during the MZT. As a consequence of low miRISC activity in smaug mutants, maternal mRNAs that are normally targeted for degradation by zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with miRISC during the MZT while the miR-309 miRNA family coregulates targets of BRAT but not PUM. Conclusions: Smaug controls the MZT through direct targeting of a subset of maternal mRNAs for degradation and, indirectly, through production and function of miRNAs and miRISC, which control clearance of a distinct subset of maternal mRNAs. BRAT and/or PUM function together with miRISC during the latter process. With respect to miRISC-dependent transcript degradation, Smaug is required (1) for the synthesis of miRNAs, (2) for synthesis and stabilization of AGO1, and (3) for action of AGO1 in association with its bound miRNAs. In smaug mutants a large number of maternal mRNAs persist and the MZT fails. Examination of miRNA expresssion at different time points in wild type and smuag mutant early embryos .

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Scott Tenenbaum mailto:STenenbaum@uamail.albany.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). The RNA binding protein (RBP) associated mRNA sequencing track (RIP-Seq) is produced as part of the Encyclopedia of DNA Elements (ENCODE) Project (http://hgwdev.cse.ucsc.edu/ENCODE/index.html). This track displays transcriptional fragments associated with RBP in cell lines (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType) K562 and GM12878, using Ribonomic profiling via Illumina SBS. In eukaryotic organisms gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins play a major role in regulating multiple mRNAs in order to facilitate gene expression patterns. These tracks show the associated mRNAs that co-precipitate with the targeted RNA-binding proteins using RIP-Seq profiling. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf RBP-mRNA complexes were purified from cells grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). RNA samples were amplified and converted to cDNA with the Nugen (http://www.nugeninc.com/) Ovation© RNA-Seq System and prepped for sequencing with the Illumina (http://www.illumina.com/) mRNA-Seq protocol. Approximately 30 million single end sequencing reads were obtained for each K562 and GM12878. RIP samples were analyzed for signal that was at or above the 60th percentile and statistically enriched compared to the negative control. Sequences were analyzed using TopHat (http://tophat.cbcb.umd.edu/) (Trapnell et al., 2009) with Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) (Langmead et al., 2009). Peaks were called from the top 40% of TopHat normalized reads, with a max gap, min run of (24:48). Unions of overlapping peak regions from total RNA replicates (RIP-Input) are presented with p-value from a one tailed t-test for average signal from replicates versus 0 (no cut-off was used for totals). Replicate overlap for positive RIP treatment peaks (ELAVL1 and PABPC1) are presented with a p-value from one tailed t-test versus signal for same the region in negative control replicates (T7-tag). RIP peaks were from sequences longer than 120 bp and p-value < .05. For both totals (RIP-input) and RIPs, the peak scores are scaled relative p-values between treatment and control.

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Identification of Smaug target mRNAs in the early Drosophila embryo using RIP-Chip

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