Nonsense-mediated mRNA Decay Activity Research Articles

Abstract 6737: Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors

Abstract Introduction: Immunogenic neoantigens derived from somatic tumor mutations are essential to initiate and sustain robust anti-tumor immune responses. Frameshift insertion/deletions (fs-indels) are a key source of immunogenic neoantigens. However, fs-indels often result in the introduction of premature termination codons (PTCs), leading to mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. In human malignancies, NMD activity is increased in response to the heightened burden of somatic fs-indel mutations and effectively prevents the generation of highly immunogenic tumor-specific neoantigens, thereby promoting immune evasion. Approach: Here, we pharmacologically targeted SMG1, a core component of the NMD pathway, for the first time in patient tumor and normal samples as well as in a panel of human cancer cell lines and patient-derived organoids, to prevent the degradation of highly immunogenic frame-shifted transcripts. We analyzed the changes in transcriptome, proteome and immunopeptidome following SMG1 inhibition (SMG1i). We then made use of tumor-T cell co-cultures and Patient-Derived Tumor Fragments (PDTFs) to assess the effects of SMG1i on anti-tumor immunogenicity. Ex vivo immunological responses were measured by high-dimensional flow cytometry, cytometric bead array and single cell RNA and TCR sequencing. Results: Our data demonstrates a significant increase in T cell activation, expansion of tumor-reactive intratumoural CD8+ lymphocytes and cytokine secretion following SMG1 inhibition in ex vivo PTDFs (n=15) but not in matched normal-adjacent tissue (n=10). Mechanistically, SMG1 inhibition increased the abundance of frame-shifted mRNA transcripts, and aberrant HLA-presented peptides. Co-culture of tumor cells and patient-derived organoids with CD8+ T cells upon SMG1 inhibition induced potent MHC class I antigen-dependent T cell activation and tumor cell killing. Conclusion: Our findings, in a clinically relevant platform, highlight SMG1 inhibition as a novel cancer immunotherapy approach to promote neoantigen production and to induce anti-tumor immunogenicity irrespective of the tumor type or TMB status. Citation Format: Roberto Vendramin, Hongchang Fu, Yue Zhao, Shanila Fernandez-Patel, Danwen Qian, Lorena Ligammari, Gordon Beattie, Ronen Levy, Polina Greenberg, Osnat Bartok, Krupa Thakkar, Jun Murai, Despoina Karagianni, Chris Sng, Mansi Shah, Felipe Galvez-Cancino, Krijn Djikstra, Sergio Quezada, Yardena Samuels, TRACERx consortium, James Reading, Charles Swanton, Kevin Litchfield. Pharmacologic modulation of nonsense-mediated decay induces anti-tumor immunogenicity in ex vivo patient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6737.

Characterization of the mIF4G Domains in the RNA Surveillance Protein Upf2p.

Thirty percent of all mutations causing human disease generate mRNAs with premature termination codons (PTCs). Recognition and degradation of these PTC-containing mRNAs is carried out by the mechanism known as nonsense-mediated mRNA decay (NMD). Upf2 is a scaffold protein known to be a central component of the NMD surveillance pathway. It harbors three middle domains of eukaryotic initiation factor 4G (mIF4G-1, mIF4G-2, mIF4G-3) in its N-terminal region that are potentially important in regulating the surveillance pathway. In this study, we defined regions within the mIF4G-1 and mIF4G-2 that are required for proper function of Upf2p in NMD and translation termination in Saccharomyces cerevisiae. In addition, we narrowed down the activity of these regions to an aspartic acid (D59) in mIF4G-1 that is important for NMD activity and translation termination accuracy. Taken together, these studies suggest that inherently charged residues within mIF4G-1 of Upf2p play a role in the regulation of the NMD surveillance mechanism in S. cerevisiae.

Nonsense-mediated mRNA decay promote C2C12 cell proliferation by targeting PIK3R5.

Nonsense mediated mRNA decay (NMD) is a highly conserved RNA quality control system, which can specifically clear abnormal mRNA and play an important role in tumorigenesis. Myoblast proliferation plays an important role in the repair of skeletal muscle injury and the development of myosarcoma, and is controlled by a variety of transcription factors and signals. The molecular mechanism by which NMD regulates the proliferation of myoblast cells is not completely clear. In this study, we found that the NMD activity of skeletal muscle is high in 1-week-old mice but decreases gradually with age, corresponding to a weakening capacity for muscle growth and regeneration. Here, we provide evidence that NMD plays an important role in myoblast proliferation and apoptosis. In addition, we found that PIK3R5 is an NMD substrate gene which can inhibit AKT activity and C2C12 cell proliferation. Therefore, NMD can target PIK3R5 to enhance AKT activity, which in turn promotes C2C12 cell proliferation. This study provides new insights into NMD regulatory mechanisms in muscular development and into potential novel therapeutic strategies for muscle atrophy.

IL-6/STAT3 signaling in tumor cells restricts the expression of frameshift-derived neoantigens by SMG1 induction

BackgroundThe quality and quantity of tumor neoantigens derived from tumor mutations determines the fate of the immune response in cancer. Frameshift mutations elicit better tumor neoantigens, especially when they are not targeted by nonsense-mediated mRNA decay (NMD). For tumor progression, malignant cells need to counteract the immune response including the silencing of immunodominant neoantigens (antigen immunoediting) and promoting an immunosuppressive tumor microenvironment. Although NMD inhibition has been reported to induce tumor immunity and increase the expression of cryptic neoantigens, the possibility that NMD activity could be modulated by immune forces operating in the tumor microenvironment as a new immunoediting mechanism has not been addressed.MethodsWe study the effect of SMG1 expression (main kinase that initiates NMD) in the survival and the nature of the tumor immune infiltration using TCGA RNAseq and scRNAseq datasets of breast, lung and pancreatic cancer. Different murine tumor models were used to corroborate the antitumor immune dependencies of NMD. We evaluate whether changes of SMG1 expression in malignant cells impact the immune response elicited by cancer immunotherapy. To determine how NMD fluctuates in malignant cells we generated a luciferase reporter system to track NMD activity in vivo under different immune conditions. Cytokine screening, in silico studies and functional assays were conducted to determine the regulation of SMG1 via IL-6/STAT3 signaling.ResultsIL-6/STAT3 signaling induces SMG1, which limits the expression of potent frameshift neoantigens that are under NMD control compromising the outcome of the immune response.ConclusionWe revealed a new neoantigen immunoediting mechanism regulated by immune forces (IL-6/STAT3 signaling) responsible for silencing otherwise potent frameshift mutation-derived neoantigens.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Evaluating the activity of nonsense-mediated RNA decay via Nanopore direct RNA sequencing

Nonsense-mediated mRNA decay (NMD) and its regulation play an important role in eliminating faulty transcripts and controlling gene expression. However, measuring NMD activity and characterizing its targets remain challenging. In this study, we set out to establish Nanopore direct RNA sequencing in combination with quantitative real-time PCR (qPCR) as a method for analyzing NMD activity and its targets in cultured cell lines and clinical tissue samples. Nanopore RNA sequencing could detect more isoforms than short-read sequencing, especially in identifying novel isoforms and predicting isoforms annotated with premature termination codon (PTC). Changes in transcriptional isoforms of five genes (PRS, RPL12, SRSF2, PPIA, and TMEM208) could faithfully reflect NMD activity in the three cell lines and prostate cancer (PCA) samples. NMD activity in PCA samples varied, but some patients showed an increased trend. Together, Nanopore sequencing was superior in identifying NMD targets and evaluating NMD activity compared with short-read sequencing, and the NMD markers we screened may be used for measuring NMD activity in clinical patients.

AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.

Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation.

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.

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 and nonsense-mediated mRNA decay (NMD) via various interacting proteins. The peripheral EJC-binding protein RNPS1 was reported to serve two insufficiently characterized functions: suppressing mis-splicing of cryptic splice sites and activating NMD in the cytoplasm. The analysis of transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines supports the conclusion that RNPS1 can moderately influence NMD activity, but is not a globally essential NMD factor. However, numerous aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that the RRM and C-terminal domain of RNPS1 both contribute partially to regulate RNPS1-dependent splicing events. We defined the RNPS1 core interactome using complementary immunoprecipitations and proximity labeling, which identified interactions with splicing-regulatory factors that are dependent on the C-terminus or the RRM domain 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.

Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay.

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense‐mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co‐depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome‐wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding‐deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault‐tolerant, functionally redundant NMD activators in human cells.

Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex 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, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B‐dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B‐dependent and ‐independent manner. While UPF3A is almost dispensable for NMD in wild‐type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC‐binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved “mid” domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

Elucidation of the Mechanism of Host NMD Suppression by HTLV-1 Rex: Dissection of Rex to Identify the NMD Inhibitory Domain.

The human retrovirus human T-cell leukemia virus type I (HTLV-1) infects human T cells by vertical transmission from mother to child through breast milk or horizontal transmission through blood transfusion or sexual contact. Approximately 5% of infected individuals develop adult T-cell leukemia/lymphoma (ATL) with a poor prognosis, while 95% of infected individuals remain asymptomatic for the rest of their lives, during which time the infected cells maintain a stable immortalized latent state in the body. It is not known why such a long latent state is maintained. We hypothesize that the role of functional proteins of HTLV-1 during early infection influences the phenotype of infected cells in latency. In eukaryotic cells, a mRNA quality control mechanism called nonsense-mediated mRNA decay (NMD) functions not only to eliminate abnormal mRNAs with nonsense codons but also to target virus-derived RNAs. We have reported that HTLV-1 genomic RNA is a potential target of NMD, and that Rex suppresses NMD and stabilizes viral RNA against it. In this study, we aimed to elucidate the molecular mechanism of NMD suppression by Rex using various Rex mutant proteins. We found that region X (aa20–57) of Rex, the function of which has not been clarified, is required for NMD repression. We showed that Rex binds to Upf1, which is the host key regulator to detect abnormal mRNA and initiate NMD, through this region. Rex also interacts with SMG5 and SMG7, which play essential roles for the completion of the NMD pathway. Moreover, Rex selectively binds to Upf3B, which is involved in the normal NMD complex, and replaces it with a less active form, Upf3A, to reduce NMD activity. These results revealed that Rex invades the NMD cascade from its initiation to completion and suppresses host NMD activity to protect the viral genomic mRNA.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

Metastatic colorectal cancer cells harness nonsense-mediated mRNA decay for immune evasion

Abstract Immunosuppression microenvironment allows primary tumor growth, while tumor-inherent factors that bring about immune evasion during metastasis remain elusive. Here, we observed a stronger nonsense-mediated mRNA decay (NMD) activity with a higher expression of up-frameshift protein 1 (UPF1) in colorectal cancer (CRC) metastasis than in matched primary cancer cells. In metastatic CRC SW620 cells, the higher UPF1 expression was found to arise from the increased stability of UPF1 by ubiquitin specific peptidase 10 (USP10)-mediated deubiquitination. In contrast, in primary CRC SW480 cells, autophagy promotes proteolysis of ubiquitinated UPF1 and inhibits NMD activity. Interestingly, in SW620 cells NMD selectively controlled a cluster of immune-related genes including ICAM1. Furthermore, NMD inhibition exposed SW620 cells but not SW480 cells to natural killer (NK) cell-mediated cytolysis that depended on ICAM1. Conclusion NMD activity can be recommissioned in CRC metastasis cells to escape from NK-dependent immune surveillance, and inhibiting NMD to boost anti-tumor immunity could be a novel strategy to treat CRC metastasis.

MHC‐I presentation of peptides derived from intact protein products of the pioneer round of translation

Among the earliest protein products of most cellular genes are those synthesized during the pioneer round of translation (PRT), a key step in nonsense-mediated mRNA decay (NMD) that allows scanning of new transcripts for the presence of a premature termination codon (PTC). It has been demonstrated that at least some PRT degradation products can be targeted to major histocompatibility (MHC)-I presentation. To gain new insight into this putative PRT-to-MHC-I route, we have assembled 2 pairs of reporter genes so that the 2 genes in each pair encode an identical fusion protein between a model antigenic peptide and enhanced green fluorescent protein (EGFP), one of which harbors a PTC. We expressed these genes in different mouse and human cell lines and confirmed enhanced NMD activity for the PTC(+) gene in each pair by monitoring the effect of cycloheximide on the level of the respective mRNA. We then exploited several strategies for establishing the ratio between level of peptide presentation and total amount of protein product. We consistently observed significantly higher ratios for the PTC(+) mRNAs compared with the PTC(-) ones, pointing to correlation between the turnover of otherwise identical proteins and the fate of their template mRNA. Using confocal microscopy, we showed a clear link between NMD, the presence of misfolded EGFP polypeptides, and enhanced MHC-I peptide presentation. Altogether, these findings imply that identical full-length gene products differing only in 3' noncoding sequences can be differentially degraded and targeted to the MHC-I presentation pathway, suggesting a more general role for the PRT in establishing the MHC-I peptidome.-Weinstein-Marom, H., Hendel, L., Laron, E. A., Sharabi-Nov, A., Margalit, A., Gross, G. MHC-I presentation of peptides derived from intact protein products of the pioneer round of translation.

MicroRNA 4651 regulates nonsense-mediated mRNA decay by targeting SMG9 mRNA

Nonsense-mediated mRNA decay (NMD) is originally identified as a conserved RNA surveillance mechanism that rapidly degrades aberrant mRNA containing premature termination codons (PTCs). However, the molecular regulation mechanisms by which microRNAs inhibit NMD has not been well understood. Here we identified that miR-4651 participated in the NMD pathway by downregulating expression levels of SMG9. We provided evidences that (1) Overexpression of miR-4651 mimic significantly inhibited the expression of SMG9 (P < 0.05); (2) NMD substrates genes, TBL2 and GADD45B were both increased at mRNA and protein expression levels when SMG9 was suppressed by siRNA, whereas decreased by SMG9 overexpression; (3) Expression of SMG9 was significantly increased but TBL2, GADD45B were significantly decreased when cells transfected with miR-4651 inhibitor (P < 0.05). These results indicated that miR-4651 regulated NMD by targeting SMG9 mRNA. Our study highlights that miR-4651 represses NMD. miR-4651 targets SMG9 and represses the expression levels of SMG9. NMD activity is decreased additionally when SMG9 is inhibited. The present study provides evidence for microRNA/NMD regulatory mechanism.

Drosophila CrebB is a Substrate of the Nonsense-Mediated mRNA Decay Pathway that Sustains Circadian Behaviors.

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.

Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

Nonsense-mediated mRNA decay (NMD) is a cellular mechanism that eliminates mRNAs that harbor premature translation termination codons (PTCs). Here, we investigated the effects of environmental stresses (oxidative stress and endoplasmic reticulum (ER) stress) on NMD activity. Methylmercury (MeHg) was used to cause oxidative stress and thapsigargin to stress the ER. NMD suppression, evidenced by upregulation of NMD-sensitive mRNAs and a decrease in UPF1 phosphorylation, was observed in MeHg-treated myogenic cells, cerebral cortical neuronal cells, and astroglial cells. Mild ER stress amplified NMD suppression caused by MeHg. To elucidate the cause of stress-induced NMD suppression, the role of the phospho-eIF2α/ATF4 pathway was investigated. Knockdown and non-phosphorylatable eIF2α-transfection studies demonstrated the critical role of phospho-eIF2α-mediated repression of translation in mild ER stress-induced NMD suppression. However, NMD suppression was also observed in phospho-eIF2α-deficient cells under mild ER stress. Mechanistic target of rapamycin suppression-induced inhibition of cap-dependent translation, and downregulation of the NMD components UPF1, SMG7, and eIF4A3, were probably involved in stress-induced NMD suppression. Our results indicate that stress-induced NMD suppression has the potential to affect the condition of cells and phenotypes of PTC-related diseases under environmental stresses by stabilizing NMD-targeted gene expression.

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs fromhuman cells via peripheral proteins RNPS1 and CASC3. We show that the EJC originates as anSR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. The switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependentNMD.

Targeting nonsense-mediated mRNA decay in colorectal cancers with microsatellite instability

Nonsense-mediated mRNA decay (NMD) is responsible for the degradation of mRNAs with a premature termination codon (PTC). The role of this system in cancer is still quite poorly understood. In the present study, we evaluated the functional consequences of NMD activity in a subgroup of colorectal cancers (CRC) characterized by high levels of mRNAs with a PTC due to widespread instability in microsatellite sequences (MSI). In comparison to microsatellite stable (MSS) CRC, MSI CRC expressed increased levels of two critical activators of the NMD system, UPF1/2 and SMG1/6/7. Suppression of NMD activity led to the re-expression of dozens of PTC mRNAs. Amongst these, several encoded mutant proteins with putative deleterious activity against MSI tumorigenesis (e.g., HSP110DE9 chaperone mutant). Inhibition of NMD in vivo using amlexanox reduced MSI tumor growth, but not that of MSS tumors. These results suggest that inhibition of the oncogenic activity of NMD may be an effective strategy for the personalized treatment of MSI CRC.