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The CNOT4 Subunit of the CCR4-NOT Complex is Involved in mRNA Degradation, Efficient DNA Damage Repair, and XY Chromosome Crossover during Male Germ Cell Meiosis

ABSTRACT: The CNOT4 Subunit of the CCR4-NOT Complex is Involved in mRNA Degradation, Efficient DNA Damage Repair, and XY Chromosome Crossover during Male Germ Cell Meiosis

PROVIDER: PRJNA665303 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12701004.fastq.gz	Fastqsanger.gz
	SRR12701005.fastq.gz	Fastqsanger.gz
	SRR12701006.fastq.gz	Fastqsanger.gz
	SRR12701007.fastq.gz	Fastqsanger.gz
	SRR12701008.fastq.gz	Fastqsanger.gz

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The CNOT4 Subunit of the CCR4-NOT Complex is Involved in mRNA Degradation, Efficient DNA Damage Repair, and XY Chromosome Crossover during Male Germ Cell Meiosis

Project description:The CCR4-NOT complex is a major mRNA deadenylase in eukaryotes and comprise CNOT6/6L and CNOT7/8, both of which are two types of catalytic subunits, as well as CNOT4, which is a regulatory subunit with previously undetermined functions. These subunits are previously hypothesized to play synergistic biochemical functions; however, their in vivo functions have remained poorly investigated. In this study, we constructed viable Cnot6/6l double knockout mice and the males were fertile, whereas Cnot7–/– male mice have been reported to be infertile. These results are against canonical viewpoints and indicate that CNOT7 has CNOT6/6L-independent functions in vivo. We also demonstrate that the mouse testis has an endogenous complex that contains CNOT7 and CNOT4, but not CNOT6/6L. CNOT4 is required for post-implantation embryo development and meiosis progression during spermatogenesis. Selective knockout of Cnot4 in male germ cells leads to defective DNA damage repair and homologous crossover formation between X and Y chromosomes in a cell autonomous manner. CNOT4 functions as a previously unrecognized mRNA adaptor of CCR4-NOT by targeting mRNAs to CNOT7 for deadenylation of the poly(A) tails, thereby mediating the degradation of a subset of transcripts from the zygotene to pachytene stage. Therefore, the mRNA removal promoted by CNOT4-regulated CCR4-NOT complex during the zygotene to pachytene transition is crucial for the selection of DNA repair pathways, subsequent activation of meiosis-related genes, sex chromosome paring, and ultimately, male fertility.

2021-02-19 | GSE158471 | GEO

Characterization of the CCR4-NOT complex subunits CNOT1 and CNOT4 using rapid auxin-induced degradation in cultured cells

Project description:CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation and translation. Originally discovered in yeast, this complex is highly conserved across eukaryotes, although its composition and functions differ between mammals and yeast. For example, unlike yeast Not4, human CNOT4 (E3 Ubiquitin Ligase) does not form a stable complex with CCR4-NOT, and its functions are less clear compared to its yeast counterpart. To investigate the roles of CNOT1 (the central scaffold subunit) and CNOT4, we developed a rapid auxin-induced degron cell culture system that allows for the acute depletion of these proteins. We studied the effects of their absence on complex integrity, cell growth, and mRNA expression and turnover. Our transcriptome-wide analysis revealed that depleting CNOT1 altered the expression of thousands of transcripts, with the majority showing increased abundance and a general decrease in mRNA decay. Although CNOT4 does not associate with the complex through standard biochemical methods, BioID proximity labelling confirmed its association with the complex in cells. However, depleting CNOT4 did not affect the integrity of CCR4-NOT. In contrast to the effects observed with CNOT1 depletion, reducing CNOT4 levels led to only modest changes in RNA steady-state levels and unexpectedly accelerated global mRNA decay. Finally, we show that the changes in mRNA stability in CCR4-NOT depleted cells correlated with the codon optimality of the transcripts. Our data suggest that CNOT4 exerts opposite effects on mRNA metabolism compared to CNOT1, and that CNOT4 may have functions distinct from those of complex subunits that promote mRNA degradation.

2025-10-23 | GSE277237 | GEO

The CNOT4 Subunit of the CCR4-NOT Complex is Involved in mRNA Degradation, Efficient DNA Damage Repair, and XY Chromosome Crossover during Male Germ Cell Meiosis.

Project description:The CCR4-NOT complex is a major mRNA deadenylase in eukaryotes, comprising the catalytic subunits CNOT6/6L and CNOT7/8, as well as CNOT4, a regulatory subunit with previously undetermined functions. These subunits have been hypothesized to play synergistic biochemical functions during development. Cnot7 knockout male mice have been reported to be infertile. In this study, viable Cnot6/6l double knockout mice are constructed, and the males are fertile. These results indicate that CNOT7 has CNOT6/6L-independent functions in vivo. It is also demonstrated that CNOT4 is required for post-implantation embryo development and meiosis progression during spermatogenesis. Conditional knockout of Cnot4 in male germ cells leads to defective DNA damage repair and homologous crossover between X and Y chromosomes. CNOT4 functions as a previously unrecognized mRNA adaptor of CCR4-NOT by targeting mRNAs to CNOT7 for deadenylation of poly(A) tails, thereby mediating the degradation of a subset of transcripts from the zygotene to pachytene stage. The mRNA removal promoted by the CNOT4-regulated CCR4-NOT complex during the zygotene-to-pachytene transition is crucial for the appropriate expression of genes involved in the subsequent events of spermatogenesis, normal DNA double-strand break repair during meiosis, efficient crossover between X and Y chromosomes, and ultimately, male fertility.

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Effect of depletion of CNOT4 on gene transcription in human cells.

Project description:Codon usage bias of an organism has been reported to affect the level of gene expression by influencing this process at multiple steps. Here, we have identified a role of a subunit of CCR4-NOT complex, CNOT4, in the transcriptional regulation of genes in a codon usage dependent manner.

2025-10-17 | GSE294397 | GEO

Effect of depletion of CNOT4 on gene expression in human cells

Project description:Codon usage bias of an organism plays an important role in determining its gene expression levels. Here, we have identified that a subunit of CCR4-NOT complex, CNOT4, plays a significant role in mediating codon usage effect on gene expression in human cells by affecting mRNA levels in a codon usage dependent manner.

2025-10-17 | GSE261504 | GEO

RPA directly stimulates Mer3/HFM1 helicase processivity to ensure normal crossover formation in meiosis

Project description:Meiotic crossover formation is critical for generating viable gametes and enhancing genetic diversity. The helicase Mer3 (HFM1 in humans) is a highly conserved factor essential for promoting crossovers and ensuring their proper distribution. Here, we identify replication protein A (RPA) as a direct interactor of budding yeast Mer3. We demonstrate that this interaction is conserved between human HFM1 and RPA. Cross-linking mass spectrometry and structural modelling with AlphaFold2 reveal a conserved and specific Mer3-RPA interface. Single-molecule magnetic tweezers assays demonstrate that direct RPA interaction is required for Mer3 helicase processivity under conditions of low DNA tension. Consistently, mer3 mutant deficient in RPA binding exhibit reduced crossover frequencies and accumulate unresolved recombination intermediates during budding yeast meiosis. Via genome-wide localisation experiments, we link this effect to a weakened recruitment to double-strand break sites of the mer3 mutant. Our findings provide mechanistic insights into coordination of meiotic recombination by the Mer3 helicase through interactions with the canonical DNA repair machinery, highlighting a conserved mechanism underlying crossover control during sexual reproduction.

2025-11-02 | GSE305755 | GEO

TOPBP1 Shapes XY Silencing Dynamics during Meiosis to Ensure Male Fertility

Project description:TOPBP1 Shapes XY Silencing Dynamics during Meiosis to Ensure Male Fertility

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Global Analysis of the Meiotic Crossover Landscape

Project description:Using microarrays to genotype the parental origin of progeny resulting from a cross between S96 and YJM789 yeast strains, we mapped the distribution of crossovers that occurred during meiosis. Knowledge of the crossover distribution allowed us to assess changes in crossover control in wild type and mutant strains. The S96 strain is a S288 derivative and thus its DNA sequence has high homology to the oligo sequences used to create the S98 Affymetrix Gene chip. The YJM789 strain is ~ 0.6% divergent from S288. Keywords: wild type and mutant analysis

2008-09-16 | GSE12170 | GEO

TOPBP1 Shapes XY Silencing Dynamics Independently of Sex Body Formation to Ensure Male Fertility

Project description:In mammals and several other taxa, the ability of males to cope with the limited synapsis of the X and Y chromosomes during prophase I of meiosis relies on the process of meiotic sex chromosome inactivation (MSCI). Components of the somatic DNA damage response machinery, including ATR, TOPBP1, MDC1 and BRCA1 play key roles in MSCI, although how they establish XY silencing remains incompletely understood. In particular, it remains unclear how DDR factors coordinate XY silencing with DNA repair, chromosome synapsis and the formation of the sex body, a distinct phase-separated sub-nuclear structure formed during prophase I to house the unsynapsed XY bivalent. Here we report a mutant mouse (Topbp1B5/B5), harboring mutations in the BRCT5 domain of Topbp1, that shows impaired XY silencing but grossly normal sex body formation. While Topbp1B5/B5 mice are viable, without detectable somatic defects, males are completely infertile. Distinct from mice lacking ATR or TOPBP1 specifically during meiosis, Topbp1B5/B5 males exhibit normal chromosome synapsis and canonical markers of DNA repair in early prophase I. ATR signaling is mostly intact in Topbp1B5/B5 spermatocytes, although specific ATR-dependent events are disrupted, including localization of the RNA:DNA helicase Senataxin to chromatin loops of the XY. Strikingly, while Topbp1B5/B5 spermatocytes are able to initiate MSCI the completion of gene silencing is defective, with a subset of X chromosome genes displaying distinct patterns of transcriptional deregulation. These findings suggest a non-canonical role for the ATR-TOPBP1 signaling axis in XY silencing dynamics at advanced stages in pachynema. This is the first DDR mutant that separates XY silencing from sex body formation, as well as TOPBP1’s role in spermatogenesis from its roles in organismal viability.

2024-02-09 | PXD042199 | Pride

Characterization of the CCR4-NOT complex subunits CNOT1 and CNOT4 using rapid auxin-induced degradation in cultured cells

Project description:Characterization of the CCR4-NOT complex subunits CNOT1 and CNOT4 using rapid auxin-induced degradation in cultured cells

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