Unknown,Transcriptomics,Genomics,Proteomics

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RIP-Chip of BRAT and PUM from 0-3 hour Drosophila melanogaster embryos

ABSTRACT: BRAT-associated mRNAs and PUM-associated mRNAs were identified in early Drosophila embryos by RNA co-immunoprecipitation of the endogenous proteins using synthetic antibodies, followed by microarray analysis (RIP-Chip). Nine RNA co-immunoprecipitations were performed. This includes 3 biological replicates each of 1) anti-BRAT RNA co-immunoprecipitations from wild-type 0-3 hour embryos, 2) anti-PUM RNA co-immunoprecipitations from wild-type 0-3 hour embryos, and 3) control antibody RNA co-immunoprecipitations from wild-type 0-3 hour embryos. BRAT samples and PUM samples were each normalized separately with the control samples, for a total of 12 processed samples (3 BRAT with 3 control normalized together, and 3 PUM with 3 control normalized together) from the 9 RNA co-immunoprecipitations.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Howard Lipshitz

PROVIDER: E-GEOD-60466 | 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: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) – of which Argonaute 1 (AGO1) is a key component in Drosophila – target maternal mRNAs for degradation. The Drosophila Smaug, Brain tumor (BRAT) and Pumilio (PUM) RBPs direct the degradation of maternal mRNAs. Here we elucidate Smaug’s 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 miRNAs 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.

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RIP-Chip of BRAT and PUM from 0-3 hour Drosophila melanogaster embryos

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