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Genome-wide distribution analysis of RNA Helicase Maleless (MLE) in Drosophila Melanogaster early male embryo, before (0-2 Hr) and after (2-4 Hr) maternal to zygotic transition (MZT) and in absence of CLAMP

ABSTRACT: Purpose: Study how maternal Pioneer Transcription factor CLAMP affects sex-specifc splicing during initial embryonic development by regulating occupancy of spliceosome component Maleless (MLE), a RNA helicase on chromatin Method: Cut and Run (CUT&RUN) DNA libraries (Uyehara and McKay 2019) using anti-MLE antibody were generated from male embryos at 0-2 Hr pre-MZT and 2-4 hr post-MZT stages in presence and absence of maternal CLAMP. Maternal CLAMP was depleted by driving UAS-CLAMPRNAi in the ovaries using MTD-GAL4 maternal triple driver. As a control UAS-GFPRNAI was driven using the MTD-GAL4. Embryos were sexed using the meiotic drive system that produces sperm with only Y chromosomes (Reider et al 2017), resulting in progeny of only male genotypes. Results: We identified regions on chromatin where MLE binds during 0-2 Hr pre-MZTand 2-4 Hr post-MZT embryonic stages in males. In 0-2 Hr and 2-4 Hr old control embryos 28,279 and 24,910 MLE peaks were detected. After depletion of maternal CLAMP, 41.97% of 0-2 Hr peaks (22183 remaining, 6096 lost) and 59.1% of 2-4 Hr peaks (15735 remaining, 9175 lost) are lost. We also found loss of maternal CLAMP resulted in 26% and 35.25% new MLE peaks at 0-2 hr pre-MZT and 2-4 Hr post-MZT embryonic stages. Our motif search shows presence of putative motifs for MLE binding on chromatin which needs further validation. Conclusion: This is the first study showing genome-wide MLE occupancy in sexed embryos during 0-2 hr pre-MZT and 2-4 hr post-MZT stages. We show that the maternally deposited transcription factor CLAMP affects MLE occupancy on chromatin in early embryos. Thus, we conclude that maternal transcription factors can affect spliceosome function by regulating occupancy of their components on chromatin.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE174781 | GEO | 2022/05/01

REPOSITORIES: GEO

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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 .

Dataset Information

Genome-wide distribution analysis of RNA Helicase Maleless (MLE) in Drosophila Melanogaster early male embryo, before (0-2 Hr) and after (2-4 Hr) maternal to zygotic transition (MZT) and in absence of CLAMP

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