Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase
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PROVIDER: PRJNA1010785 | ENA |
REPOSITORIES: ENA
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Project description:The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding site location, number, density, and context are important determinants of regulation. Moreover, the content of optimal synonymous codons exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.
2023-09-03 | GSE241940 | GEO
Project description:Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 function to negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the DCP2 mRNA decapping enzyme has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent pathway.
2021-09-09 | GSE159510 | GEO
Project description:Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome
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Project description:Control of mRNA poly(A) tail has central role to regulate mRNA metabolism: translation efficiency and mRNA stability. Gene expression in maturing oocytes largely relies on the regulation of mRNA metabolism as they are transcriptionally silent. The CCR4-NOT complex is a major deadenylase for mammals and regulates poly(A) tails of maternal mRNAs, however their function to translational regulation was not well understood. Here we show that CCR4-NOT suppresses translational activity of maternal mRNAs during oocyte maturation. Oocytes which lack entire deadenylase activity of CCR4-NOT by genetic deletion of its catalytic subunits: CNOT7 and CNOT8 showed increase of both expression of maternal mRNAs and translational activity of them during oocyte maturation.
2023-10-01 | GSE224308 | GEO
Project description:mRNA degradation critically contributes to tissue development and function as well as transcription. The CCR4-NOT complex serves as a major deadenylase that initiates mRNA degradation. We used microarrays to identify deregulated genes in the white adipose tissues lacking CNOT3, a core subunit of the CCR4-NOT complex.
2017-01-01 | GSE86930 | GEO
Project description:Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. Here, we describe a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, we detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.
2019-10-30 | GSE120412 | GEO
Project description:Combinatorial mRNA Regulation by miRNAs and Pumilio proteins
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Project description:Chlamydomonas pumilio var pumilio Cytochromome oxydase 1 (COI)
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Project description:The Ccr4-Not complex is a major regulator of stress responses that controls gene expression at multiple levels, from transcription to mRNA decay. Ccr4, a core subunit of the complex, is the main cytoplasmic deadenylase in Saccharomyces cerevisiae, however its mRNA targets have not been mapped on a genome-wide scale. Here we describe a genome-wide approach, RNA immunoprecipitation-high throughput sequencing (RIP-seq), to identify the RNAs bound to Ccr4, and two proteins that associate with it, Dhh1 and Puf5. All three proteins were preferentially bound to lowly abundant mRNAs, most often at the 3’ end of the transcript. Furthermore, Ccr4, Dhh1 and Puf5 are recruited to mRNAs that are targeted by other RNA-binding proteins that promote decay, mRNA transport and inhibit translation. Although Ccr4-Not regulates mRNA transcription and decay, Ccr4 recruitment to mRNAs correlates better with decay rates, suggesting it imparts greater control over transcript abundance through decay. Ccr4-enriched mRNAs are refractory to control by the other deadenylase complex in yeast, Pan2/3, suggesting a division of labor between these deadenylation complexes. Finally, Ccr4 and Dhh1 associate with mRNAs whose abundance increases during nutrient starvation and those that fluctuate during metabolic and oxygen consumption cycles, which explains the known genetic connections between these factors and nutrient utilization and stress pathways.
2017-12-04 | GSE72366 | GEO
Project description:The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of posttranscriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs. Two-color experimental design comparing cells with a double-knockout of the CCR4 genes to cells with a reintegrated CCR4 gene.
2010-12-10 | E-GEOD-25891 | biostudies-arrayexpress