CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

Abstract The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

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CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

10.1101/811018 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer V. Gerbracht ◽

Volker Boehm ◽

Thiago Britto-Borges ◽

Sebastian Kallabis ◽

Janica L. Wiederstein ◽

...

Keyword(s):

Exon Junction Complex ◽

Core Component ◽

Mrna Isoforms ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Endonucleolytic Cleavage ◽

Core Proteins ◽

Messenger Ribonucleoprotein ◽

Cytoplasmic Processes

AbstractThe exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

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Role of the Nonsense-Mediated Decay Factor hUpf3 in the Splicing-Dependent Exon-Exon Junction Complex

Science ◽

10.1126/science.1062829 ◽

2001 ◽

Vol 293 (5536) ◽

pp. 1832-1836 ◽

Cited By ~ 175

Author(s):

V. N. Kim

Keyword(s):

Exon Junction Complex ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Decay Factor

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Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster

eLife ◽

10.7554/elife.19881 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 13

Author(s):

Subhendu Roy Choudhury ◽

Anand K Singh ◽

Tina McLeod ◽

Marco Blanchette ◽

Boyun Jang ◽

...

Keyword(s):

Specific Interaction ◽

Polytene Chromosomes ◽

Exon Junction Complex ◽

Exon Junction ◽

Cell Extracts ◽

Intronless Genes ◽

Core Proteins ◽

Central Protein ◽

Nascent Transcripts

Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.

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Faculty Opinions recommendation of Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001848.14206 ◽

2001 ◽

Author(s):

Matthias Hentze

Keyword(s):

Exon Junction Complex ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Decay Factor

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Faculty Opinions recommendation of Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001848.15206 ◽

2001 ◽

Author(s):

Andrea Barta

Keyword(s):

Exon Junction Complex ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Decay Factor

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Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing nonsense-mediated decay pathways

Genome Biology ◽

10.1186/s13059-021-02309-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Carrie Kovalak ◽

Scott Donovan ◽

Alicia A. Bicknell ◽

Mihir Metkar ◽

Melissa J. Moore

Keyword(s):

Alternative Splicing ◽

Deep Sequencing ◽

Mrna Decay ◽

Hek293 Cells ◽

Exon Junction Complex ◽

Rna Seq ◽

Mrna Isoforms ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Evolutionarily Conserved

Abstract Background Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.

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Faculty Opinions recommendation of Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001848.12176 ◽

2001 ◽

Author(s):

Elsebet Lund

Keyword(s):

Exon Junction Complex ◽

Nonsense Mediated Decay ◽

Exon Junction ◽

Decay Factor

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

International Journal of Molecular Sciences ◽

10.3390/ijms22126519 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6519

Author(s):

Yuta Otani ◽

Ken-ichi Fujita ◽

Toshiki Kameyama ◽

Akila Mayeda

Keyword(s):

Cancer Cells ◽

Control Factor ◽

Exon Junction Complex ◽

Core Component ◽

Normal Cells ◽

Knock Down ◽

Exon Junction ◽

Mature Mrna ◽

Complex Core ◽

Specific Mrna

Using 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 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.

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