Nonsense-mediated mRNA decay (NMD) downmodulates mRNAs that have in-frame premature termination codons and prevents translation of potentially harmful truncated proteins from aberrant mRNAs. Two new approaches have identified physiological NMD substrates, and suggest that NMD functions as a multipurpose tool in the modulation of gene expression.

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles.

Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe-/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe-/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe-/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH.

Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model.

MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.

The NUP214-ABL1 fusion is a constitutively activated tyrosine kinase that is significantly associated with overexpression of the TLX1 and TLX3 transcription factors in T cell acute lymphoblastic leukemia (T-ALL). Here we show that NUP214-ABL1 cooperates with TLX1 in driving T-ALL development using a transgenic mouse model and human T-ALL cells. Using integrated ChIP-sequencing, ATAC-sequencing, and RNA-sequencing data, we demonstrate that TLX1 and STAT5, the downstream effector of NUP214-ABL1, co-bind poised enhancer regions, and cooperatively activate the expression of key proto-oncogenes such as MYC and BCL2. Inhibition of STAT5, downregulation of TLX1 or MYC, or interference with enhancer function through BET-inhibitor treatment leads to reduction of target gene expression and induction of leukemia cell death.

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post-termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC-bound ribosome, a central mechanistic step of RNA surveillance.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella.

Iron-loaded tumor-associated macrophages (iTAMs) show a pro-inflammatory phenotype, hallmarked by anti-tumorigenic activity and an ability to attenuate tumor growth. Here we explored the relevance of these findings in lung cancer patients by investigating the impact of the iTAM content in the tumor microenvironment (TME) on patient survival. We analyzed 102 human non-small cell lung cancer (NSCLC) paraffin-embedded archival tissue samples for iron levels and macrophage numbers. Interestingly, patients with lung adenocarcinoma accumulating iron in the TME show higher numbers of M1-like pro-inflammatory TAMs and a survival advantage compared to iron-negative patients. By contrast, in patients with lung squamous cell carcinoma iron in the TME does not affect survival, suggesting a unique influence of iron on different histological subtypes of non-small cell lung cancer (NSCLC). We conclude that in lung adenocarcinoma iron may serve as a prognostic marker for patient survival and as a potential therapeutic target for anti-cancer therapy.

Transferrin receptor 2 (Tfr2) is mainly expressed in the liver and controls iron homeostasis. Here, we identify Tfr2 as a regulator of bone homeostasis that inhibits bone formation. Mice lacking Tfr2 display increased bone mass and mineralization independent of iron homeostasis and hepatic Tfr2. Bone marrow transplantation experiments and studies of cell-specific Tfr2 knockout mice demonstrate that Tfr2 impairs BMP-p38MAPK signaling and decreases expression of the Wnt inhibitor sclerostin specifically in osteoblasts. Reactivation of MAPK or overexpression of sclerostin rescues skeletal abnormalities in Tfr2 knockout mice. We further show that the extracellular domain of Tfr2 binds BMPs and inhibits BMP-2-induced heterotopic ossification by acting as a decoy receptor. These data indicate that Tfr2 limits bone formation by modulating BMP signaling, possibly through direct interaction with BMP either as a receptor or as a co-receptor in a complex with other BMP receptors. Finally, the Tfr2 extracellular domain may be effective in the treatment of conditions associated with pathological bone formation.

Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.

The molecular pathogenesis of late complications associated with type 2 diabetes mellitus (T2DM) is not yet fully understood. While high glucose levels indicated by increased HbA1c only poorly explain disease progression and late complications, a pro-inflammatory status, oxidative stress, and reactive metabolites generated by metabolic processes were postulated to be involved. Individuals with metabolic syndrome (MetS) frequently progress to T2DM, whereby 70% of patients with T2DM show non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of MetS, and insulin resistance (IR). Epidemiological studies have shown that T2DM and steatosis are associated with alterations in iron metabolism and hepatic iron accumulation. Excess free iron triggers oxidative stress and a switch towards a macrophage pro-inflammatory status. However, so far it remains unclear whether hepatic iron accumulation plays a causative role in the generation of IR and T2DM or whether it is merely a manifestation of altered hepatic metabolism. To address this open question, we generated and characterized a mouse model of T2DM with IR, steatosis, and iron overload.

Newborns with FATP4 mutations exhibit ichthyosis prematurity syndrome (IPS), and adult patients show skin hyperkeratosis, allergies, and eosinophilia. We have previously shown that the polarization of macrophages is altered by FATP4 deficiency; however, the role of myeloid FATP4 in the pathogenesis of nonalcoholic steatohepatitis (NASH) is not known. We herein phenotyped myeloid-specific Fatp4-deficient (Fatp4M-/-) mice under chow and high-fat, high-cholesterol (HFHC) diet. Bone-marrow-derived macrophages (BMDMs) from Fatp4M-/- mice showed significant reduction in cellular sphingolipids in males and females, and additionally phospholipids in females. BMDMs and Kupffer cells from Fatp4M-/- mice exhibited increased LPS-dependent activation of proinflammatory cytokines and transcription factors PPARγ, CEBPα, and p-FoxO1. Correspondingly, these mutants under chow diet displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. After HFHC feeding, Fatp4M-/- mice showed increased MCP-1 expression in livers and subcutaneous fat. Plasma MCP-1, IL4, and IL13 levels were elevated in male and female mutants, and female mutants additionally showed elevation of IL5 and IL6. After HFHC feeding, male mutants showed an increase in hepatic steatosis and inflammation, whereas female mutants showed a greater severity in hepatic fibrosis associated with immune cell infiltration. Thus, myeloid-FATP4 deficiency led to steatotic and inflammatory NASH in males and females, respectively. Our work offers some implications for patients with FATP4 mutations and also highlights considerations in the design of sex-targeted therapies for NASH treatment.NEW & NOTEWORTHY FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4M-/- mice displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. In response to HFHC feeding, male mutants were prone to hepatic steatosis, whereas female mutants showed exaggerated fibrosis. Our study provides insights into a sex-dimorphic susceptibility to NASH by myeloid-FATP4 deficiency.

Iron plays a role in many key processes in the developing brain. During pregnancy, iron supplementation is widely recommended to prevent and treat iron deficiency; however, the prevalence of iron deficiency and the risk of iron overload vary greatly between populations. Evidence on the role of high levels of maternal ferritin, a storage iron marker during pregnancy in relation to offspring neurodevelopment is lacking.

Synapsins are a family of neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. Experimental evidence suggests a role for synapsins in synaptic vesicle clustering and recycling at the presynaptic terminal, as well as in neuronal development and synaptogenesis. Synapsin knock-out (Syn1(-/-) ) mice display an epileptic phenotype and mutations in the SYN1 gene have been identified in individuals affected by epilepsy and/or autism spectrum disorder. We investigated the impact of the c.1067G>A nonsense transition, the first mutation described in a family affected by X-linked syndromic epilepsy, on the expression and functional properties of the synapsin I protein. We found that the presence of a premature termination codon in the human SYN1 transcript renders it susceptible to nonsense-mediated mRNA decay (NMD). Given that the NMD efficiency is highly variable among individuals and cell types, we investigated also the effects of expression of the mutant protein and found that it is expressed at lower levels compared to wild-type synapsin I, forms perinuclear aggregates and is unable to reach presynaptic terminals in mature hippocampal neurons grown in culture. Taken together, these data indicate that in patients carrying the W356× mutation the function of synapsin I is markedly impaired, due to both the strongly decreased translation and the altered function of the NMD-escaped protein, and support the value of Syn1(-/-) mice as an experimental model mimicking the human pathology.

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.

The exon junction complex (EJC) participates in the regulation of many post-transcriptional steps of gene expression. EJCs are deposited on messenger RNAs (mRNAs) during splicing and their core consists of eIF4A3, MLN51, Y14, and MAGOH. Here, we show that two genes encoding MAGOH paralogs (referred to as MAGOH and MAGOHB) are expressed in mammals. In macrophages, the expression of MAGOHB, but not MAGOH mRNA, increases rapidly after LPS stimulation. Both MAGOH proteins interact with other EJC components, incorporate into mRNA-bound EJCs, and activate nonsense-mediated decay. Furthermore, the simultaneous depletion of MAGOH and MAGOHB, but not individual depletions, impair nonsense-mediated decay in human cells. Hence, our results establish that the core composition of mammalian EJCs is more complex than previously recognized.