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ActD timecourse in HeLa cells, HeLa cells transiently transfected with control (C2) or CPEB4-targeting siRNAs S140 and S181

ABSTRACT: ActD timecourse in HeLa cells, HeLa cells transiently transfected with control (C2) or CPEB4-targeting siRNAs S140 and S181

PROVIDER: PRJNA779950 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16929100.fastq.gz	Fastqsanger.gz
	SRR16929101.fastq.gz	Fastqsanger.gz
	SRR16929102.fastq.gz	Fastqsanger.gz
	SRR16929103.fastq.gz	Fastqsanger.gz
	SRR16929104.fastq.gz	Fastqsanger.gz

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ActD timecourse in HeLa cells, HeLa cells transiently transfected with control (C2) or CPEB4-targeting siRNAs S140 and S181

Project description:Immediate early genes (IEGs) represent a unique class of genes with rapid induction kinetics and transient expression patterns, which requires IEG mRNAs to be short-lived. Here, we establish cytoplasmic polyadenylation element-binding protein 4 (CPEB4) as a major determinant of IEG mRNA instability. We identified human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), which is known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CBEP4 in HeLa cells revealed that CPEB4 preferentially binds to the 3' untranslated region (UTR) of IEG mRNAs, at U-rich sequence motifs located in close proximity to the poly(A) site. By transcriptome-wide mRNA decay measurements, we found that the strength of CPEB4 binding correlates with short mRNA half-lives, and that loss of CBEP4 expression leads to the stabilization of IEG mRNAs. Further, we demonstrate that CPEB4 mediates mRNA degradation by recruitment of the evolutionarily conserved CCR4-NOT complex, the major eukaryotic deadenylase. While CPEB4 is primarily known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as an RBP that enhances the degradation of short-lived IEG mRNAs.

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Cpeb4-mediated Translational Regulatory Circuitry Controls Terminal Erythroid Differentiation

Project description:Erythropoiesis is essential to mammals and is regulated at multiple steps by both extracellular and intracellular factors. Many transcriptional regulatory networks in erythroid differentiation have been well characterized. However, our understanding of post-transcriptional regulatory circuitries in this developmental process is still limited. Using genomic approaches, we identified a sequence-specific RNA-binding protein, Cpeb4, which is dramatically induced in terminal erythroid differentiation (TED) by two erythroid important transcription factors, Gata1/Tal1. Cpeb4 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family that regulates translation of target mRNAs in early embryonic development, neuronal synapse, and cancer. Using primary mouse fetal liver erythroblasts, we found that Cpeb4 is required for terminal erythropoiesis by repressing the translation of a set of mRNAs highly expressed in progenitor cells. This translational repression occurs by the interaction with a general translational initiation factor, eIF3. Interestingly, Cpeb4 also binds its own mRNA and represses its translation, thus forming a negative regulatory circuitry to limit Cpeb4 protein level. This mechanism ensures that the translation repressor, Cpeb4, does not interfere with the translation of other mRNAs in differentiating erythroblasts. Our study characterized a translational regulatorycircuitry that controls TED and revealed that Cpeb4 is required for somatic cell differentiation. We used microarray to identify mRNAs associated with Cpeb4 in mouse fetal liver erythroblasts. Cpeb4 associated mRNAs were isolated from mouse fetal liver erythroblasts using anti-Cpeb4 antibody for immunoprecipitation followed by RNA extraction. Then Affymetrix microarrays were used to identify and quantify the mRNAs associated with Cpeb4.

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ActD timecourse in HeLa cells, HeLa RNF219 KO cells, HeLa RNF219 KO cells overexpressing 3xFLAG-SBP-RNF219-WT or 3xFLAG-SBP-RNF219-mut

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Project description:HeLa cells were treated with ActD and RNA sequenced to examine RNA stability

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