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An RNAi Screen of RNA Helicases Identifies eIF4A3 as a Regulator of Embryonic Stem Cell Identity [CLIP-seq]

ABSTRACT: RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

ORGANISM(S): Mus musculus

PROVIDER: GSE194010 | GEO | 2022/12/09

REPOSITORIES: GEO

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eIF4A3 interactome by IP-MS in mouse ESCs

Project description:RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). We mapped the eIF4A3 interactomes in mouse ESCs, revealing that eIF4A3 is widely involved in the post-transcriptional regulation of gene expression. Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

2023-03-11 | PXD031362 | Pride

An RNAi Screen of RNA Helicases Identifies eIF4A3 as a Regulator of Embryonic Stem Cell Identity [Ribo-seq]

Project description:RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

2022-12-09 | GSE193940 | GEO

An RNAi Screen of RNA Helicases Identifies eIF4A3 as a Regulator of Embryonic Stem Cell Identity [RNA-Seq]

Project description:RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

2022-12-09 | GSE193938 | GEO

RNA-Seq of control and EJC factor (EIF4A3, RBM8A, MAGOH) knockdowns in part with EIF4A3 rescue in HeLa Tet-Off cells

Project description:The exon junction complex is involved in gene expression regulation on multiple co- and post-transcriptional levels. We aimed to investigate in HeLa Tet-Off cells which gene expression alterations can be observed upon knockdown of core EJC factors (EIF4A3, RBM8A and MAGOH) or upon EIF4A3 knockdown and rescue with EIF4A3 wild type construct by using RNA-Seq analyses. The rescue construct was stably integrated into the genome using the PiggyBac transposon system. As control Luciferase (Luc) siRNA was used.

2022-05-06 | E-MTAB-10771 | biostudies-arrayexpress

PRPF8 iCLIP on HeLa cells in response to eIF4A3 Knockdown

Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.

2018-10-14 | E-MTAB-7269 | biostudies-arrayexpress

Genome-wide identification of circRNAs translated by eIF4A3

Project description:Exon junction complexes (EJCs) deposited on spliced mRNAs play multifunctional roles in the regulation of gene expression. Whereas the formation and components of EJCs are well characterized, the underlying molecular mechanisms for gene regulation remain poorly understood. Here we find that a eukaryotic translation initiation factor (eIF) 4A3, a core component of EJC directly interacts with eIF3g, a subunit of eIF3 complex. This interaction serves as a linker between the EJC and eIF3 complex, consequently driving an internal ribosomal recruitment. Accordingly, artificially tethered EJC component or cellular EJC deposited on mRNA after splicing promotes internal initiation of translation in a way that is resistant to cellular stress induced by serum starvation. We also demonstrate that translatable endogenous or reporter circular RNAs depend on EJC for their association with polysomes. Our results uncover an internal initiation driven by EJC, expanding the protein-coding potential of human transcriptome including circular RNAs.

2023-08-31 | GSE131563 | GEO

eIF4A3 - Modified iCLIP

Project description:eIF4A3 - Modified iCLIP

| PRJEB11647 | ENA

The Exon Junction Complex Core Represses Cancer-specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing

Project description:Using the TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that the mRNA re-splicing is controlled by specific repressors and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

2021-06-15 | GSE178102 | GEO

DEAD box RNA helicases are pervasive protein kinase interactors and activators

Project description:DEAD box (DDX) RNA helicases are a large family of ATPases, many of which have unknown functions. There is emerging evidence that besides their role in RNA biology, DDX proteins may stimulate protein kinases. To investigate if protein kinase-DDX interaction is a more widespread phenomenon, we conducted three orthogonal large-scale screens, including proteomics analysis with 32 RNA helicases, protein array profiling, and kinome-wide in vitro kinase assays. We retrieved Ser/Thr protein kinases as prominent interactors of RNA helicases and report hundreds of binary interactions. We extracted members of eleven protein kinase families, which bind to - and are stimulated by - DDX proteins, including CAMK, CDK, CK1, CK2, DYRK, MARK, NEK, PRKC, SPRK, STE7/MAP2K, and STE20/PAK family members. We identified MARK1 in all screens and validated that DDX proteins accelerate MARK1 catalytic rate. The findings indicate pervasive interactions between protein kinases and DEAD box RNA helicases and provide a rich resource to explore their regulatory relationships.

2024-03-10 | PXD043289 | Pride

The CWC27/CWC22 heterodimer: a spliceosome building block linked to the inflammation response in retinal pigment epithelium

Project description:Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the Exon Junction Complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.

2020-04-06 | GSE145872 | GEO

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