The F-box protein Bard (CG14317) targets the Smaug RNA-binding protein for destruction during the Drosophila maternal-to-zygotic transition.
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ABSTRACT:
SUBMITTER: Cao WX
PROVIDER: S-EPMC8733446 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug plays an essential role in progression through the MZT by regulating the translational repression and degradation of a large number of maternal mRNA species. The Smaug protein itself is rapidly cleared at the end of the MZT by the Skp/Cullin/F-box (SCF) E3-ligase complex; clearance of Smaug requires zygotic transcription. Here, we show that an F-box protein, which we name Bard (encoded by CG14317), is required for degradation of Smaug. Bard is expressed zygotically and physically interacts with Smaug at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of Smaug. We show that shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of Smaug protein, a phenotype that is rescued by transgenes expressing Bard .
2021-10-06 | MSV000088199 | MassIVE
Project description: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 protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.
| S-EPMC2727558 | biostudies-literature
Project description:Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process, RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT), and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. Here, we have carried out global analysis of small RNAs both in wild-type and in smg mutants. Our results show that 85% of all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. We hypothesize that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs.
| S-EPMC5100853 | biostudies-literature
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 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. Examination of miRNA expresssion at different time points in wild type and smuag mutant early embryos .
2016-09-06 | E-GEOD-82194 | biostudies-arrayexpress
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.
2016-09-06 | GSE82194 | GEO
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).
2010-06-19 | E-GEOD-13287 | biostudies-arrayexpress
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.
2009-04-23 | GSE13287 | GEO
Project description:During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA-protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of these RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3' UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA-protein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.
| S-EPMC5495070 | biostudies-literature
Project description:BACKGROUND:Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. RESULTS:Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. CONCLUSIONS:Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.
| S-EPMC4460960 | biostudies-literature
Project description:The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.
| S-EPMC4935972 | biostudies-literature