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 .

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

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