RIP-CHIP data from CNOT6L WT and KO hepatocytes
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE62365 | GEO | 2019/03/29
REPOSITORIES: GEO
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Project description:The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of arious biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. We compared the expression profiles of the cellular genes between control and CCR4b/CNOT6L-depleted cells by microarray analysis
2011-08-03 | GSE31148 | GEO
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 stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of arious biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. We compared the expression profiles of the cellular genes between control and CCR4b/CNOT6L-depleted cells by microarray analysis Total RNAs were extracted from control or CCR4b-depleted cells. Following the isolation of RNA, all subsequent technical procedures, including quality control of RNA, labeling, hybridization, and scanning of the arrays, were performed in BIO MATRIX Research, Inc., cDNA, and biotinlabeled cRNA was synthesized according to protocols for Affymetrix array analysis. Biotin-labeled cRNAs (15 g) were hybridized to the GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA). To determine the average differences for each probe set, changes were calculated by a global normalization method using Affymetrix GCOS (GeneChipOperating Software) software.
2011-08-02 | E-GEOD-31148 | biostudies-arrayexpress
Project description:Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3’-untranslated regions (3’-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.
2020-03-30 | E-MTAB-6941 | biostudies-arrayexpress
Project description:Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
2020-04-16 | GSE144250 | GEO
Project description:Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
2020-04-16 | GSE131408 | GEO
Project description:Roquin2, and its paralog Roquin1, are key regulator of mRNA decay. Roquin proteins recognize mRNAs containing a 3’-UTR stem-loop motif, called constitutive decay element (CDE), through the ROQ domain. Upon target engagement, Roquin proteins recruit the CCR4-CAF1-NOT complex, mediating mRNA deadenylation and destabilization. To study the effect of Roquin2 stabilization, we sought out to identify the relevant Roquin2 mRNA targets in human DLBCL. To this purpose, we measured differential gene expression via RNA-seq analysis in U-2932 cells expressing Roquin2 or non-degradable Roquin2 upon BCR crosslinking.
2018-04-27 | GSE93675 | GEO
Project description:miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. AGO1 is a key protein required for miRNA-mediated gene silencing. In animal cells, mRNA degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1 or NOT1. We show that approximately 45% of AGO1-targets are regulated by both CAF1 and NOT1, indicating deadenylation is a widespread effect of miRNA regulation.<br><br>We employed RNA interference using long double-stranded RNAs to deplete cultured S2 cells of AGO1 (CG6671, 2 independent samples), CAF1 (CG5684, 2 independent samples), or NOT1 (CG1884, 3 independent samples). Further, we used Affymetrix oligonucleotide microarrays to analyze expression profiles in these samples. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (3 and 2 independent samples, respectively).<br>
2008-12-03 | E-MEXP-1713 | biostudies-arrayexpress
Project description:The evolutionarily conserved CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon histone deacetylase (HDAC) inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified ring finger protein 219 (RNF219) as an acetylation-regulated cofactor. We provide evidence that RNF219 is an active RING-type E3 ubiquitin ligase which stably associates with the CCR4-NOT complex via the NOT9 module through a short linear motif located in the C-terminal low-complexity region of RNF219. Using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 acts as an inhibitor of deadenylation. Accordingly, our transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, independently of its E3 ligase activity. Our results establish RNF219 as a negative regulator of CCR4-NOT-mediated mRNA deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.
2021-12-15 | PXD027237 | Pride
Project description:The control of mRNA stability plays a central role in regulating gene expression. In metazoans, the earliest stages of development are driven by maternally supplied mRNAs. The degradation of these maternal mRNAs is critical for promoting the maternal-to-zygotic transition of developmental programs, although the underlying mechanisms are poorly understood in vertebrates. Here, we characterized maternal mRNA degradation pathways in zebrafish using a transcriptome analysis and systematic reporter assays. Our data demonstrate that ORFs enriched with uncommon codons promote deadenylation by the CCR4-NOT complex in a translation-dependent manner. This codon-mediated mRNA decay is conditional on the context of the 3â² UTR, with long 3â² UTRs conferring resistance to deadenylation. These results indicate that the combined effect of codon usage and 3â² UTR length determines the stability of maternal mRNAs in zebrafish embryos. Our study thus highlights the codon-mediated mRNA decay as a conserved regulatory mechanism in eukaryotes. zebrafish embryonic mRNA profile at 2 different stages (2 hpf and 6 hpf) in wildtype and 3 additional conditions (miR-430 inhibition, RNApol II inhibition and CNOT7 inhibition) at 6 hpf. All experiments are performed as triplicates
2016-03-17 | E-GEOD-71609 | biostudies-arrayexpress