CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway

Cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in severe lung disease. Nearly 10% of cystic fibrosis patients have at least one CFTR allele with a nonsense mutation that generates a nonsense codon in the mRNA. Nonsense mutations can result in significant reduction of gene expression partially due to rapid mRNA degradation through the nonsense-mediated decay (NMD) pathway. It has not been thoroughly investigated which branch of the NMD pathway governs the decay of CFTR mRNAs containing nonsense codons. Here we utilized antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. Interestingly, we found that CFTR mRNAs with G542X, R1162X, and W1282X nonsense codons require UPF2, UPF3, and exon junction complex proteins for NMD, whereas CFTR mRNAs with the Y122X nonsense codon do not. Furthermore, we demonstrated that all evaluated CFTR mRNAs harboring nonsense codons were degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5/SMG7-mediated exonucleolytic pathway. Finally, we found that stabilization of CFTR mRNAs by NMD inhibition alone improved functional W1282X protein production, and improved the efficiency of aminoglycoside translational readthrough of CFTR-Y122X, -G542X, and -R1162X mRNAs.

Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing, mRNA export, mRNA translation, and nonsense-mediated mRNA decay (NMD) via various interacting peripheral proteins. The EJC-binding protein RNPS1 might serve two functions: it suppresses mis-splicing of cryptic splice sites and activates NMD in the cytoplasm. When analyzing the transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines, we find no evidence for RNPS1 being a globally essential NMD factor. However, various aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that about half of these RNPS1-dependent splicing events was fully or partially rescued by the expression of the isolated RRM domain of RNPS1, whereas other splicing events are regulated by its C-terminal domain. We identified many splicing-regulatory factors, including SR proteins and U1 snRNP components, that specifically interact with the C-terminus or with the RRM of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

Eukaryotic Initiation Factor 4A-3: A Review of Its Physiological Role and Involvement in Oncogenesis

EIF4A3, a member of the DEAD-box protein family, is a nuclear matrix protein and a core component of the exon junction complex (EJC). Under physiological conditions, EIF4A3 participates in post-transcriptional gene regulation by promoting EJC control of precursor mRNA splicing, thus influencing nonsense-mediated mRNA decay. In addition, EIF4A3 maintains the expression of significant selenoproteins, including phospholipid hydroperoxide glutathione peroxidase and thioredoxin reductase 1. Several recent studies have shown that EIF4A3 promotes tumor growth in multiple human cancers such as glioblastoma, hepatocellular carcinoma, pancreatic cancer, and ovarian cancer. Molecular biology studies also showed that EIF4A3 is recruited by long non-coding RNAs to regulate the expression of certain proteins in tumors. However, its tumor-related functions and underlying mechanisms are not well understood. Here, we review the physiological role of EIF4A3 and the potential association between EIF4A3 overexpression and tumorigenesis. We also evaluate the protein’s potential utility as a diagnosis biomarker, therapeutic target, and prognosis indicator, hoping to provide new ideas for future research.

The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3’s tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint–mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3’s unexpected role in RiBi, with implications for cancer pathogenesis and treatment.

A Comprehensive Pan-Cancer Analysis of RBM8A Based on Data Mining

Background. As a member of the exon junction complex (EJC), RNA-binding motif protein 8A (RBM8A) plays a crucial role in the maintenance of mRNA and multiple activities of an organism. Immunotherapy has been proven to be a staple type of cancer treatment. However, the role of RBM8A and immunity across cancer types is unclear. Objective. This study aims to visualize the expression, prognosis, mutations, and coexpressed gene results of RBM8A across cancer types and to explore the link between RBM8A expression and immunity. Methods. In this study, data were collected from multiple online databases. We analyzed the data using the HPA, UALCAN Database, COSMIC, cBioPortal, Cancer Regulome tools, Kaplan–Meier Plotter, and TIMER website. Results. For the expression of RBM8A in normal tissues, higher expression of RBM8A was observed in immune-related cells than in nonimmune organs. The expression level of RBM8A was related to tumor type. Missense mutations in RBM8A were found in most tumors and affected the prognosis of carcinomas with coexpressed genes. RBM8A was strongly associated with immune-infiltrating cells and immune checkpoint inhibitors, especially in LIHC. Conclusions. RBM8A is a gene worth exploring and may be a unique immune target in the future.

Mammalian UPF3A and UPF3B activate NMD independently of their EJC binding

Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors - UPF1, UPF2 and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here we characterize the UPF3B-dependent and -independent NMD in human cell lines knocked-out of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, EJC-mediated NMD can operate in UPF3B-dependent and -independent manner. While UPF3A is almost completely dispensable for NMD in wild-type cells, it strongly activates EJC-mediated NMD in cells lacking UPF3B. Surprisingly, this major NMD branch can operate in UPF3-independent manner questioning the idea that UPF3 is needed to bridge UPF proteins to the EJC during NMD. Complementation studies in UPF3 knockout cells further show that EJC-binding domain of UPF3 paralogs is not essential for NMD. Instead, the conserved mid domain of UPF3B, previously shown to engage with ribosome release factors, is required for its full NMD activity. Altogether, UPF3 plays a more active role in NMD than simply being a bridge between the EJC and the UPF complex.

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

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.

The exon junction complex and intron removal prevent re-splicing of mRNA

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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

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.