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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178102 | GEO | 2021/06/15

REPOSITORIES: GEO

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Transcriptome-wide modulation of splicing by the exon junction complex

Project description:We report that knockdown of EJC core proteins, eIF4A3, Y14, Magoh, causes a transcript-wide changes in alternative splicing, as well as some transcriptional changes. These changes are specific to EJC core proteins, and KD of UPF1 protein caused different sets of alterantive splicing changes. These changes are linked to the rate of transcription. Examination of 4 different knockdown, as well as GFP knockdown in HeLa cells, 2 replicates each condition.

2014-11-14 | E-GEOD-63091 | biostudies-arrayexpress

Transcriptome-wide modulation of splicing by the exon junction complex

2014-11-14 | GSE63091 | GEO

Exon junction complex shapes m6A epitranscriptome during splicing

Project description:N6-methyladenosine (m6A) is the most abundant post-transcriptional methylation of mature mRNA and plays a crucial regulatory role in various biological functions, involving development and cancer progression. m6A is deposited by methyltransferase complex composed of METTL3 and METTL14 cotranscriptionally, and removed by demethylase FTO or ALKBH5. m6A is highly enriched within the 3’ UTRs and in the vicinity of the stop codon of mature mRNA. However, the mechanism that causes this distribution pattern is still enigmatic. Here, we show that EJC packages mRNAs to shape mRNA m6A landscape. We first tested the possibility that demethylase removes m6A during spicing and found that ALKBH5 depletion had a minor effect on m6A levels. Thus, we ruled out the demethylation model. We then hypothesized that splicing factors inhibit the methylation process of internal exons, but not the 3’ UTR. Knockdown of EIF4A3, a core component of the exon junction complex, significantly increased m6A levels in mature mRNAs. The hypermethylated sites are enriched in short internal exons. Furthermore, EIF4A3 depletion upregulates METTL3 binding affinity to mRNA to deposit m6As. In conclusion, Our results demonstrate that EIF4A3 blocks METTL3 binding and methylating internal short exons of mRNA. These findings shed light on the fact that RNA packaging formed during splicing acts as a regulator that shapes mRNA modifications.

2022-12-06 | GSE207663 | GEO

Magoh, Y14, eIF4AIII interactomes by TurboID proximity labelling provide no evidence for a canonical EJC in (almost) intron-less trypanosomatids

Project description:In metazoans, mRNA quality is tightly monitored from transcription to translation. A key role lies with the exon junction complex (EJC) that is placed upstream of the exon-exon junction after splicing. The EJC inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and non-sense mediated decay (NMD, UPF1/2/3). The protozoan parasite Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of the three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested as an adaptation of the EJC function to mark trans-spliced mRNAs. Here we explore the interactome of Magoh, Y14, eIF4AIII in T. brucei by TurboID proximity labelling.

2024-03-25 | PXD050946 | Pride

mRNAseq of Drosophila S2 cells after knockdown of EJC components

Project description:This study used Drosophila melanogaster S2 cells treated with siRNA against the EJC components mago or eIF4A3, or a non-targeting control siRNA (three samples per condition). mRNA-seq libraries were prepared from the samples and the data was used to examine 'RS-exons' - exons which reconstitute a 5' splice site when they are spliced to the upstream exon. In contrast to mammalian cells, RS-exons are not recursively spliced out from Drosophila transcripts after perturbation of the EJC.

2018-10-15 | E-MTAB-7271 | biostudies-arrayexpress

The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

Project description:In metazoans, the exon junction complex (EJC) is a central component of spliced messenger ribonucleoprotein particles (mRNPs). CASC3 has been reported to be a core component of the EJC and to be crucial for assembly, the splicing regulating function of the EJC and nonsense-mediated mRNA decay (NMD). However, recent evidence suggests that CASC3 functions differently from other EJC core components. To elucidate the cellular role of CASC3, we have established human cell lines in which CASC3 was inactivated by means of CRISPR-Cas9 genome editing. We show that in these cells CASC3 is dispensable for the splicing regulatory role of the EJC. However, we find that CASC3 depletion results in the upregulation of many known and novel NMD substrates, suggesting that CASC3 is required for the efficient execution of EJC-dependent NMD. Taken together, our results challenge the model of CASC3 as an assembly factor and core component of the EJC. Our data rather show that CASC3 is involved in the degradation of NMD substrates and therefore uncover the primary molecular function of CASC3 in human cells.

2021-09-09 | PXD019161 | Pride

The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

2021-02-25 | PXD015754 | Pride

The protein complex of kinesin motor KIF1C and its hereditary spastic paraplegia associated mutant G102A

Project description:Regulated local translation, RNA transport and protein localization are critical for spatio-temporal gene expression in neurons. The localization of mRNA and subsequent local protein synthesis contribute to neuronal functions like synaptogenesis, synapse pruning, axon guidance, axonal regeneration and synaptic plasticity. Thus, mutations in motor proteins and subsequent cargo transport failure lead to motoneuron diseases. The kinesin family member 1 C has been shown to play a role in different cargo transport alongside the microtubule network, but the pathomechanisms behind KIF1C deficiency mediated motoneuron diseases has not been deciphered yet. Additionally, the exon junction complex has been suggested as a molecular link between splicing and cytoplasmic mRNA localization and local translation. Here we identified KIF1C as a EJC transporter in neuronal cells. We investigated the KIF1C proteome in differentiated SH-SY5Y cells and found an interaction of KIF1C with EJC components and other RNA-binding proteins in KIF1C overexpressing SH-SY5Y cells. The interaction could be validated via immunoprecipitation and an endogenous eIF4A3 co-immunoprecipitation. The co-localization of eIF4A3 and RBM8A with wildtpye KIF1C at protrusions of SH-SY5Ys further confirms the interaction. The mislocalization of eIF4A3 and RMB8A due to KIF1C mutation suggests a transport of the EJC by KIF1C. Furthermore, the interaction of KIF1C with PABPC1 and EJC components is RNA mediated. We additionally demonstrate via complex capture with KIF1C overexpressing HEK293 cells that KIF1C interacts with RNA itself. We therefore suggest the interaction of KIF1C not only with the EJC, but with a complex containing RNA and the EJC. Hence KIF1C is a transporter of mRNA and the EJC.

2023-12-28 | PXD037387 | 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

RNAseq of HEK293 cells after Chtop knockdown

Project description:During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. While early studies suggested that the Exon Junction Complex (EJC) may provide a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex (CBC) to the 5’ end of RNAs, as part of the TREX complex. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3’-end processing. Consequently, Alyref can alter splicing decisions and Chtop regulates alternative polyadenylation. Surprisingly, we observe that eIF4A3 stimulates Alyref deposition on single exon or spliced RNAs close to EJC sites. Additional experiments suggest that Alyref is recruited to the 5’-end of RNAs by CBC before binding RNAs near EJCs. Our study reveals mechanical insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

2019-05-01 | GSE113952 | GEO

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