Nonsense-mediated RNA Decay Pathway Research Articles

Primary U2AF1 S34F Mutated Hematopoietic Cells Are Sensitive to Nonsense-Mediated RNA Decay Disruption In Vivo

Expression of mutant splicing factors alters RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells, and the differential sensitivity is partially dependent on R-loop formation. However, the impact of NMD disruption on the viability of primary hematopoietic cells with splicing factor mutations has not been tested in vivo. We engineered mouse MLL-AF9 acute myeloid leukemia (AML) cells to inducibly express a wild type ( U2AF1 WT) or mutant ( U2AF1 S34F) transgene of the U2AF1 splicing factor, and tested the effects of inhibiting the NMD pathway protein kinase, SMG1, in vivo. We injected U2AF1 WT and U2AF1 S34F mouse AML cells (B6, CD45.2) into congenic, irradiated secondary recipient mice (B6, CD45.1), induced transgene expression, and then treated the mice for 2 weeks with either a SMG1 inhibitor (SMG1i) at 60 mg/kg/day or vehicle. SMG1i treatment increased the overall survival (OS) of mice bearing U2AF1S34F AML cells (median OS of 53 days compared to 42 days for mice treated with vehicle; p =0.0003), but did not impact the OS of mice with U2AF1WT AML cells (median OS of 33 days for SMG1i-treated mice compared to median OS of 32 days for vehicle-treated mice, p=0.360). We extended these findings to a second cohort of mice, where we transplanted one primary U2AF1 S34F banked mouse AML sample into 48 secondary recipient mice. To allow direct comparison of isogenic cells with or without U2AF1 S34F expression, half the mice were treated with doxycycline to induce U2AF1 S34F expression, and half of the mice did not receive doxycycline. SMG1i treatment increased the OS of mice expressing the U2AF1S34F mutant allele (median OS of 66 days, compared to median OS of 54 days for mice treated with vehicle; p<0.0001), but had no impact on survival of mice bearing AML cells where U2AF1 S34F was not expressed (median OS of 50 days, compared to median OS of 52 days for mice treated with vehicle; p=0.95). To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we first examined the effects of SMG1i treatment on cell cycle progression and cell death in primary cells. We observed that primary U2AF1 S34F-expressing AML cells treated with SMG1i ex vivo (24 hours) had an increased fraction of cells in G2/M phase compared to U2AF1 WT-treated cells (mean 48% vs. 11% at 100nM, respectively, p<0.001). In addition, the fraction of Annexin V + cells was increased in U2AF1 S34F-expressing cells treated with SMG1i ex vivo compared to U2AF1 WT treated cells (mean fold-change at 500nM relative to DMSO 4.0 vs. 2.3, respectively, p=0.027). We then isolated primary mouse AML cells after 7 days of in vivo SMG1i treatment and analyzed R-loops (S9.6 antibody) and DNA damage (gH2AX) by immunofluorescence. SMG1i-treated U2AF1 S34F cells had a marked increase in R-loop levels compared to U2AF1 WT-treated cells (mean S9.6 nuclear intensity, 306.9 vs 182.6, respectively, p<0.0001). In addition, H2AX phosphorylation was increased in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (mean gH2AX nuclear intensity, 136.7 vs 87.8, respectively p<0.0001).Thus, disrupting NMD in splicing factor mutant AML cells alters cell cycle progression and causes R-loop accumulation, DNA damage, and apoptotic cell death. The NMD pathway has been implicated in the regulation of the unfolded protein response (UPR) that controls protein homeostasis and impacts cell viability. We generated K562 cell lines expressing doxycycline-inducible U2AF1 S34F or U2AF1 WT constructs and confirmed increased sensitivity of U2AF1 S34F cells treated with SMG1i(IC50 128.1nM vs 578.5nM at 72 hours, respectively, p<0.0001). Next, we performed RNA-sequencing on U2AF1 WT and U2AF1 S34F cells that were treated with vehicle or SMG1i for 24 hours (n=3) and observed upregulation of endoplasmic reticulum and UPR pathway genes in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (GSEA; FDR<0.01). Validation of these findings using primary mouse AML cells is ongoing, including the analysis of key regulators and protein biomarkers of the UPR pathway. The vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility for therapeutic targeting of NMD to treat myeloid malignancies with aberrant splicing.

SARS-CoV-2 helicase might interfere with cellular nonsense-mediated RNA decay: insights from a bioinformatics study

BackgroundViruses employ diverse strategies to interfere with host defense mechanisms, including the production of proteins that mimic or resemble host proteins. This study aimed to analyze the similarities between SARS-CoV-2 and human proteins, investigate their impact on virus-host interactions, and elucidate underlying mechanisms.ResultsComparing the proteins of SARS-CoV-2 with human and mammalian proteins revealed sequence and structural similarities between viral helicase with human UPF1. The latter is a protein that is involved in nonsense-mediated RNA decay (NMD), an mRNA surveillance pathway which also acts as a cellular defense mechanism against viruses. Protein sequence similarities were also observed between viral nsp3 and human Poly ADP-ribose polymerase (PARP) family of proteins. Gene set enrichment analysis on transcriptomic data derived from SARS-CoV-2 positive samples illustrated the enrichment of genes belonging to the NMD pathway compared with control samples. Moreover, comparing transcriptomic data from SARS-CoV-2-infected samples with transcriptomic data derived from UPF1 knockdown cells demonstrated a significant overlap between datasets.ConclusionsThese findings suggest that helicase/UPF1 sequence and structural similarity might have the ability to interfere with the NMD pathway with pathogenic and immunological implications.

Nonsense-Mediated RNA Decay Disruption Induces Altered Splicing in Spliceosome Mutant Cells

Introduction: Expression of mutant splicing factors U2AF1(S34F), SF3B1(K700E), or SRSF2(P95H) alter RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells and the differential sensitivity is partially dependent on R-loop formation. The impact of NMD disruption on the viability of primary splicing factor mutant hematopoietic cells and the consequence on downstream RNA splicing are not known. Methods: Cell viability of primary mouse hematopoietic cells (untransformed and AML cells) and a human AML cell line that expresses mutant or wild-type U2AF1 after treatment with a highly-specific inhibitor of SMG1 (SMG1i), the only known protein kinase in the NMD pathway, was measured using a CellTiter-Glo assay. Gene expression and RNA splicing changes induced by mutant U2AF1 and/or SMG1 inhibition were identified by short and long-read RNA sequencing (RNA-seq). Results: We treated primary untransformed mouse U2AF1S34F or U2AF1WT expressing cKit+ hematopoietic cells ex vivo with SMG1 inhibitor and monitored cell viability. We found that SMG1i reduced the viability of primary mouse cells expressing mutant U2AF1S34F compared to U2AF1WT (IC50 52.6 nM vs 173.5 nM, respectively, p=<0.0001). Next, we tested the sensitivity of primary mouse AML cells expressing a doxycycline-inducible U2AF1S34F or U2AF1WT transgene to SMG1i that were generated using retroviral expression of the fusion oncogene MLL-AF9 to induce AML. We found that NMD disruption using SMG1i also reduced the ex vivo viability of primary U2AF1S34F compared to U2AF1WT expressing mouse AML cells (IC50 40.9 nM vs 102.2 nM, respectively, p=<0.0001), highlighting the potential utility of SMG1i treatment for spliceosome mutant myeloid malignancies. To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we generated, K562 cell lines expressing doxycycline-inducible U2AF1S34F or U2AF1WT and confirmed that NMD disruption using SMG1i reduced cell viability of mutant expressing cells in vitro (IC50 128.1nM vs 578.5nM at 72 hours, respectively, p≤0.0001). Next, we performed RNA-seq on K562 cells expressing U2AF1WT or U2AF1S34F that were treated with vehicle or 1uM SMG1i for 24 hours (n=3 replicates). We identified reduced expression of genes involved in the cell cycle, DNA replication/repair, telomere maintenance and mRNA splicing in SMG1i treated U2AF1S34F compared to U2AF1WT cells (GSEA; FDR<0.008). Next, we identified 3,393 RNA splicing alterations induced by SMG1i treatment of U2AF1S34F compared to U2AF1WT cells (rMATS; FDR<0.1, percent spliced-in >10%). Genes with altered splicing were enriched in similar pathways, including the cell cycle, DNA replication, and RNA splicing and processing pathways (Cluster Profiler; FDR≤0.02). Finally, we performed long-read RNA-seq (Nanopore) on RNA isolated from K562 cells (not expressing spliceosome mutants) that were treated with vehicle or SMG1i (0.5uM, 24hours, n=2) to directly identify full length transcripts containing premature termination codons (PTC) that are normal targets of NMD. Our preliminary data indicate that SMG1i significantly increases the number of detectable PTC containing transcripts (known and novel) compared to vehicle treatment (mean 19,291 vs 5,621 PTCs, respectively; p=0.008). In addition, PTC-containing transcripts that were overexpressed in SMG1i vs vehicle treated cells also occur in genes involved in the cell cycle, DNA repair and mRNA splicing (FDR£0.001), suggesting they are regulated by NMD under basal conditions. These gene pathways may be particularly vulnerable to further dysregulation in mutant treated cells because they are also altered by mutant U2AF1S34F expression. Conclusion: A vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility that NMD can be targeted for treating MDS with aberrant splicing. Excessive R-loop accumulation in SMG1i treated U2AF1S34F cells may contribute to the downstream gene expression and splicing alterations and reduced viability of mutant cells. Ongoing pre-clinical in vivo efficacy studies will test the potential for SMG1i to treat hematologic malignancies that have splicing factor mutations.

Progression of the pluripotent epiblast depends upon the NMD factor UPF2.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts - both canonical and alternatively processed mRNAs - including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.

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.

Single cell transcriptome sequencing on the Nanopore platform with ScNapBar.

The current ecosystem of single-cell RNA-seq platforms is rapidly expanding, but robust solutions for single-cell and single-molecule full-length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single-cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single-cell sequencing method on the 10× Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g., for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing saturation is low. ScNapBar uses unique molecular identifier (UMI) or Naïve Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore data sets. We further applied ScNapBar to pools of cells with an active or a silenced nonsense-mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

Importin β1 mediates nuclear import of the factors associated with nonsense-mediated RNA decay

In eukaryotic cells, nonsense-mediated RNA decay (NMD) is an essential physiological mechanism coupled to translation, regulating the stability of abnormal RNA containing premature termination codon (PTC) and a significant fraction of normal transcriptomes. So far, the molecular regulation mechanism of NMD pathway is far from fully elucidated. Previously, we observed the interaction between importin β1 (Impβ1) and UPF1, a core factor of NMD. Here, we demonstrated that Impβ1 knockdown stabilized NMD reporters, and Impβ1 and UPF1 interacted and co-regulated an extensive number of target transcripts. Furthermore, Impβ1 affected the interaction between UPF1 and SMG5 or MAGOH, and the nuclear distributions of UPF1, SMG1, SMG5 and MAGOH. Besides, Ran knockdown extremely promoted the dissociation of UPF1 from SMG5 or MAGOH. Our findings provide molecular insight into the potential function of Impβ1in nonsense-mediated RNA decay.

Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG.

The up-frameshift suppressor 1 homolog (UPF1) RNA surveillance gene is a core element in the nonsense-mediated RNA decay (NMD) pathway, which impacts a broad spectrum of biological processes in a cell-specific manner. In the present study, the contribution of the NMD pathway to psoriasis lesions and its moderating effects on the biological processes of keratinocytes was reported. Sanger sequencing for skin scales from two patients with psoriasis identified two mRNA mutations (c.2935_2936insA and c.2030-2081del) in the UPF1 gene. The somatic mutants produced truncated UPF1 proteins and perturbed the NMD pathway in cells, leading to the upregulation of NMD substrates. As the most abundant epidermal growth factor receptor ligand in keratinocytes, it was concluded that amphiregulin (AREG) mRNA is a natural NMD substrate, that is dependent on its 3′ untranslated region sequence. Perturbed NMD modulated keratinocyte homeostasis in an AREG-dependent but nonidentical manner, which highlighted the unique characteristics of NMD in keratinocytes. By targeting AREG mRNA post-transcriptionally, the UPF1-NMD pathway contributed to an imbalance between proliferation on the one hand, and apoptosis and abnormal differentiation, migration and inflammatory response on the other, in keratinocytes, which indicated a role of the NMD pathway in the full development of keratinocyte-related morbidity and skin diseases.

Nonsense-mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR.

The recessive genetic disease cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR (CF transmembrane conductance regulator) gene. Approximately 10% of patients with CF have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense-mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies that specifically target nonsense mutations in CFTR. Here, we identified antisense oligonucleotides (ASOs) that target the NMD factor SMG1 to inhibit the NMD pathway, and determined their effects on the W1282X CFTR mutation. First, we developed and validated two in vitro models of the W1282X CFTR mutation. Next, we treated these cells with antisense oligonucleotides to inhibit NMD and measured the effects of these treatments on W1282X expression and function. SMG1-ASO-mediated NMD inhibition upregulated the RNA, protein, and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increased the CFTR chloride channel function in cells homozygous for the W1282X mutation. Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in patients with CF and the W1282X mutation.

PF213 THE GENETIC LANDSCAPE OF FAMILIAL MDS/AML; RECURRING MUTATIONS IN THE RNA HELICASE DHX34 LEADING TO DEFECTS IN NONSENSE‐MEDIATED RNA DECAY

Background:The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve both the recognition and the management of this group of at risk individuals. Inherited forms account for &lt;5% of new diagnoses and their genetic basis can be separated into 3 groups: where approved testing exists (group 1); genes associated with other inherited haematological syndromes or emerging from basic research and requiring validation (group 2); and cases in which the specific gene mutations have not been established (group 3).Aims:Characterisation of novel genes mutated in familial AML/MDS.Methods:Familial AML/MDS references families where two or more family members were diagnosed with a haematological malignancy (predominantly AML, MDS, aplastic anaemia) or a prodromal cytopenia (e.g. thrombocytopenia) but at least one case was MDS or AML. Bone marrow or peripheral blood samples were collected from at least one affected individual from 86 families together with the corresponding laboratory and clinical information.Results:A primary genetic lesion associated with groups 1 &amp; 2 was detected in 48 families (56%). WES analysis in the remaining 38 families rationalised 147 germline variants in 60 candidate genes. These genes were divisible into 3 discrete groups based on association with rare haematological syndromes, loci with relevant functions mutated in &gt;3 families, or loci overlapping with other series of potential familial MDS/AML or IBMF syndromes. Germline variants in 22 of the selected 60 genes (37%) have been implicated across a broad range of inherited disorders including T cell‐negative, B cell‐negative, natural killer cell‐negative severe combined immunodeficiency (ADA, p.Gln3∗ and p.Glu88Argfs∗34), Platelet‐type bleeding disorder‐11 (GP6, p.Arg159∗ and p.Met430Lys) and Congenital Dyserythropoietic Anemia Type II (SEC23B, p.Pro542Leu and p.Glu361Asp). Among the non‐syndromic genes, we noted 4 families with variants in TET2, an epigenetic regulator and additional families harboured variants in DHX34, an RNA helicase that has been shown to function in the Nonsense‐mediated RNA decay (NMD) pathway and to co‐purify with the spliceosome, suggesting its potential involvement in pre‐mRNA splicing. All 4 DHX34 variants (p.Tyr515Cys, p.Arg897Cys, p.Asp638Asn and p.Glu444Asp), compromised NMD activity, as shown by their inability to promote phosphorylation of UPF1, which is essential for NMD activation. Cross referencing our WES data with the familial MDS/AML candidate loci documented by other groups highlighted the candidacy of 15 additional loci, including variants in DNAH9 in 3 families, a gene recurrently mutated in sporadic AML (2% of cases), one family with a variant in SH2B3 (p.Ala49Thr) and a germline duplication detected in one additional family NAPRT1 (p.Val216_Phe219dup).Summary/Conclusion:Familial AML/MDS represent a challenge where our genetic capability has raced ahead of the capacity to bring to bear much of our new knowledge into the clinical arena. Herein we propose several new recurrent predisposing genes in familial MDS/AML, validate the significant overlap with the genetics of inherited BMF syndromes and rare hematological syndromes, and demonstrate that heterozygous mutations in DHX34, detected in 4 families, individually impact activity of the NMD pathway. Overall these new findings are not only important for the respective families but offer novel insights into the aetiology of acute leukaemia.

MiR-222 stimulates M2b macrophage polarization in severely burned mice through the degradation of long noncoding RNA GAS5

Abstract M2bMφ have been characterized as cells responsible for the increased susceptibility of severely burned mice to various opportunistic infections. We have previously demonstrated that a long noncoding RNA GAS5 is not expressed by M2bMφ, significantly. This RNA is involved in CCL1 gene silencing, and CCL1 is essential for the prolongation of M2bMφ. In this study, the mechanism involved in the reduction of a GAS5 level in M2bMφ was investigated. Mφ were isolated from the mesenteric lymph nodes of mice 1–15 days after severe burn injury (3rd degree, 25% TBSA burn). The level of phosphorylated UPF1 (pUPF1), a marker of the nonsense-mediated RNA decay (NMD) pathway, was determined in various Mφ preparations by Western blotting. In some experiments, these Mφ were transfected with miR-222 mimic, and tested for the GAS5 RNA and CCL1 mRNA levels. miR-222 has been shown to directly binds to GAS5. In the results, the levels of pUPF1 and miR-222 were increased in Mφ from mice 10–15 days after burn injury. These Mφ did not express GAS5 RNA and expressed CCL1 mRNA. The GAS5 reduction was also demonstrated in normal mouse Mφ transfected with miR-222, but not in the same Mφ treated with an inhibitor of the NMD pathway. M2bMφ were not demonstrated in severely burned mice injected with an miR-222 inhibitor. These results suggest that, through the reduction of long noncoding RNA GAS5, miR-222 appeared in Mφ of severely burned mice stimulates the M2bMφ polarization.

A Novel Heterozygous Deletion Variant in KLOTHO Gene Leading to Haploinsufficiency and Impairment of Fibroblast Growth Factor 23 Signaling Pathway.

Hyperphosphatemia is commonly present in end-stage renal disease. Klotho (KL) is implicated in phosphate homeostasis since it acts as obligate co-receptor for the fibroblast growth factor 23 (FGF23), a major phosphaturic hormone. We hypothesized that genetic variation in the KL gene might be associated with alterations in phosphate homeostasis resulting in hyperphosphatemia. We performed sequencing for determining KL gene variants in a group of resistant hyperphosphatemic dialysis patients. In a 67-year-old female, blood DNA sequencing revealed a heterozygous deletion of a T at position 1041 (c.1041delT) in exon 2. This variation caused a frameshift with substitution of isoleucine for phenylalanine and introduction of a premature termination codon (p.Ile348Phefs*28). cDNA sequencing showed absence of deletion-carrier transcripts in peripheral blood mononuclear cells suggesting degradation of these through a nonsense-mediated RNA decay pathway. Experiments in vitro showed that p.Ile348Phefs*28 variant impaired FGF23 signaling pathway, indicating a functional inactivation of the gene. In the patient, serum levels of KL were 2.9-fold lower than the mean level of a group of matched dialysis subjects, suggesting a compromise in the circulating protein concentration due to haploinsufficiency. These findings provide a new loss-of-function variant in the human KL gene, suggesting that genetic determinants might be associated to clinical resistant hyperphosphatemia.

Compound C inhibits nonsense-mediated RNA decay independently of AMPK.

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development.

The No-Go and Nonsense-Mediated RNA Decay Pathways Are Regulated by Inflammatory Cytokines in Insulin-Producing Cells and Human Islets and Determine β-Cell Insulin Biosynthesis and Survival.

Stress-related changes in β-cell mRNA levels result from a balance between gene transcription and mRNA decay. The regulation of RNA decay pathways has not been investigated in pancreatic β-cells. We found that no-go and nonsense-mediated RNA decay pathway components (RDPCs) and exoribonuclease complexes were expressed in INS-1 cells and human islets. Pelo, Dcp2, Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by inflammatory cytokines in INS-1 cells under conditions where central β-cell mRNAs were downregulated. These changes in RDPC mRNA or corresponding protein levels were largely confirmed in INS-1 cells and rat/human islets. Cytokine-induced upregulation of Pelo, Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by inducible nitric oxide synthase inhibition, as were endoplasmic reticulum (ER) stress, inhibition of Ins1/2 mRNA, and accumulated insulin secretion. Reactive oxygen species inhibition or iron chelation did not affect RDPC expression. Pelo or Xrn1 knockdown (KD) aggravated, whereas Smg6 KD ameliorated, cytokine-induced INS-1 cell death without affecting ER stress; both increased insulin biosynthesis and medium accumulation but not glucose-stimulated insulin secretion in cytokine-exposed INS-1 cells. In conclusion, RDPCs are regulated by inflammatory stress in β-cells. RDPC KD improved insulin biosynthesis, likely by preventing Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but Smg6 KD ameliorated, cytokine-mediated β-cell death, possibly through prevention of proapoptotic and antiapoptotic mRNA degradation, respectively.

Erratum to: Stress and the nonsense-mediated RNA decay pathway.

The original version of this article unfortunately contained errors in the section entitled "NMD in stress responses in plants".

M2b macrophage polarization accompanied with reduction of long noncoding RNA GAS5

Macrophages (Mϕ) are highly plastic and change their functional phenotypes depending on microenvironmental signals. Recent studies have shown that microRNAs are involved in the polarization of Mϕ. In this study, we demonstrated that the phenotype of M2bMϕ [CCL1(+) IL-10(+) LIGHT(+)] switches to other phenotypes with interchangeability attained through the increased expression of growth arrest-specific 5 RNA (GAS5 RNA), a long noncoding RNA. GAS5 RNA has been described as a silencer of the CCL1 gene. Various phenotypes of Mϕ were prepared from bone marrow-derived Mϕ (BMDMϕ) after stimulation with IFNγ [M(IFNγ)/M1Mϕ], IL-4 [M(IL-4)/M2aMϕ], LPS and immobilized IgG [M(LPS + IC)/M2bMϕ], and IL-10 [M(IL-10)/M2cMϕ]. BMDMϕ cultured with medium [M(no)/quiescent Mϕ] were used as a control. As compared to Μ(no), M(IFNγ), M(IL-4) and M(IL-10), the reduced level of GAS5 RNA was shown in M(LPS + IC). CCL1 and LIGHT mRNAs (typical biomarkers of M2bMϕ) were not expressed by M(LPS + IC) transduced with a GAS5 gene using lentiviral vector. The reduction of GAS5 RNA in M(LPS + IC) was mediated by the activation of nonsense-mediated RNA decay (NMD) pathway. BMDMϕ overexpressed with GAS5 RNA after GAS5 gene transduction did not polarize to M2bMϕ even though they were stimulated with LPS and IC in combination. These results indicate that the reduction of GAS5 RNA influenced by the NMD pathway activation leads to the Mϕ polarization stimulated with LPS and IC in combination.

Stress and the nonsense-mediated RNA decay pathway.

Cells respond to internal and external cellular stressors by activating stress-response pathways that re-establish homeostasis. If homeostasis is not achieved in a timely manner, stress pathways trigger programmed cell death (apoptosis) to preserve organism integrity. A highly conserved stress pathway is the unfolded protein response (UPR), which senses excessive amounts of unfolded proteins in the ER. While a physiologically beneficial pathway, the UPR requires tight regulation to provide a beneficial outcome and avoid deleterious consequences. Recent work has demonstrated that a conserved and highly selective RNA degradation pathway-nonsense-mediated RNA decay (NMD)-serves as a major regulator of the UPR pathway. NMD degrades mRNAs encoding UPR components to prevent UPR activation in response to innocuous ER stress. In response to strong ER stress, NMD is inhibited by the UPR to allow for a full-magnitude UPR response. Recent studies have indicated that NMD also has other stress-related functions, including promoting the timely termination of the UPR to avoid apoptosis; NMD also regulates responses to non-ER stressors, including hypoxia, amino-acid deprivation, and pathogen infection. NMD regulates stress responses in species across the phylogenetic scale, suggesting that it has conserved roles in shaping stress responses. Stress pathways are frequently constitutively activated or dysregulated in human disease, raising the possibility that "NMD therapy" may provide clinical benefit by downmodulating stress responses.

The nonsense-mediated RNA decay pathway is disrupted in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumors (IMTs) are characterized by myofibroblast proliferation and an inflammatory cell infiltrate. Little is known about the molecular pathways that precipitate IMT formation. Here, we report the identification of somatic mutations in UPF1, a gene that encodes an essential component of the nonsense-mediated RNA decay (NMD) pathway, in 13 of 15 pulmonary IMT samples. The majority of mutations occurred in a specific region of UPF1 and triggered UPF1 alternative splicing. Several mRNA targets of the NMD pathway were upregulated in IMT samples, indicating that the UPF1 mutations led to reduced NMD magnitude. These upregulated NMD targets included NIK mRNA, which encodes a potent activator of NF-κB. In human lung cells, UPF1 depletion increased expression of chemokine-encoding genes in a NIK-dependent manner. Elevated chemokines and IgE class switching events were observed in IMT samples, consistent with NIK upregulation in these tumors. Together, these results support a model in which UPF1 mutations downregulate NMD, leading to NIK-dependent NF-κB induction, which contributes to the immune infiltration that is characteristic of IMTs. The molecular link between the NMD pathway and IMTs has implications for the diagnosis and treatment of these tumors.

A36: The TP53 mutations in the Russian patients with de novo DLBCL

Background TP53 dysfunction is implicated in lymphomagenesis and disease progression. Information about the frequency and spectrum of TP53 mutations in the Russian pathients with diffuse large B-cell lymphoma (DLBCL) in the current version of the IARC TP53 Mutation Database R17 is not represented. The goal of this work was to study the frequency, spectrum and functional significance of TP53 mutations in Russian patients with DLBCL. Material and methods At the present time the pilot group of 14 patients were included in the study. Diagnosis was assessed according to the criteria of the WHO classification system. Genomic DNA was isolated from formalin-fixed, paraffin embedded tissue blocks. Direct sequence analysis of gene TP53 was performed according to the IARC protocol, 2010 update. Results In two patients were identified single nucleotide substitutions that are not described in the current version of the PubMed database. All of mutations occurred in the DNA-binding domain of p53. The nonsense mutation Arg196Ter was detected in one patient. Previously it was shown that formation of this premature stop codon might activate the nonsense-mediated RNA decay pathway. The second patient had two missense mutations – Leu130Phe and Arg156Cys. The first of them leads to p53 inactivation according to the analysis of the functional importance of amino acid substitutions using service PolyPhen-2. Conclusion We detected TP53 mutation in 14% cases. The mutational rate in our study is in good agreement with other studies where the frequency of the TP53 mutations in patients with DLBCL ranged mostly from 13% to 23%.

The unfolded protein response is shaped by the NMD pathway.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.