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The epitranscriptome represents the more than 140 types of chemically varying and reversable RNA modifications affecting RNA fate. Among these, the most relevant for this review are the mRNA modifications N6-methyladenosine and N6,2'-O-dimethyladenosine. Epitranscriptomic mRNA biology involves RNA methyltransferases (so-called "writers"), RNA demethylases ("erasers"), and RNA-binding proteins ("readers") that interact with methylation sites to determine the functional outcome of the modification. In this review, we discuss the role of a specific RNA demethylase encoded by the fat mass and obesity-associated gene (FTO) in cancer. FTO initially became known as the strongest genetic link for human obesity. Only in 2010, 16 years after its discovery, was its enzymatic function as a demethylase clarified, and only recently has its role in the development of cancer been revealed. FTO functions are challenging to study and interpret because of its genome-wide effects on transcript turnover and translation. We review the discovery of FTO and its enzymatic function, the tumor-promoting and suppressive roles of FTO in selected cancer types, and its potential as a therapeutic target.

Universal influenza vaccines have the potential to protect against continuously evolving and newly emerging influenza viruses. T cells may be an essential target of such vaccines as they can clear infected cells through recognition of conserved influenza virus epitopes. We evaluated a novel T cell-inducing nucleoside-modified mRNA vaccine that encodes the conserved nucleoprotein, matrix protein 1 and polymerase basic protein 1 of an H1N1 influenza virus. To mimic the human situation, we applied the mRNA vaccine as a prime-boost regimen in naïve ferrets (mimicking young children) and as a booster in influenza-experienced ferrets (mimicking adults). The vaccine induced and boosted broadly-reactive T cells in the circulation, bone marrow and respiratory tract. Booster vaccination enhanced protection against heterosubtypic infection with potential pandemic H7N9 influenza virus in influenza-experienced ferrets. Our findings show that mRNA vaccines encoding internal influenza virus proteins are a promising strategy to induce broadly-protective T-cell immunity against influenza viruses.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on β1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia.

A major goal of methodologies related to large scale gene expression analyses is to initiate comprehensive information on transcript signatures in single cells within the tissue's anatomy. Until now, this could be achieved in a stepwise experimental approach: (1) identify the majority of transcripts in a single cell (single cell transcriptome); (2) provide information on transcripts on multiple cell subtypes in a complex sample (cell heterogeneity); and (3) give information on each cell's spatial location within the tissue (zonation transcriptomics). Such genetic information will allow construction of functionally relevant gene expression maps of single cells of a given anatomically defined tissue compartment and thus pave the way for subsequent analyses, including their epigenetic modifications. Until today these aims have not been achieved in the area of cardiovascular disease research though steps toward these goals become apparent: laser capture microdissection (LCM)-based mRNA expression microarrays of atherosclerotic plaques were applied to gain information on local gene expression changes during disease progression, providing limited spatial resolution. Moreover, while LCM-derived tissue RNA extracts have been shown to be highly sensitive and covers a range of 10-16,000 genes per array/small amount of RNA, its original promise to isolate single cells from a tissue section turned out not to be practicable because of the inherent contamination of the cell's RNA of interest with RNA from neighboring cells. Many shortcomings of LCM-based analyses have been overcome using single-cell RNA sequencing (scRNA-seq) technologies though scRNA-seq also has several limitations including low numbers of transcripts/cell and the complete loss of spatial information. Here, we describe a protocol toward combining advantages of both techniques while avoiding their flaws.

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies., ZEB2 transcription factor is increased in a subset of cardiomyocytes during stress to induce cardioprotective effects after injury. Here the authors show that therapeutic delivery of ZEB2 prevents cardiac dysfunction after ischemic damage and promotes the activation of pro-angiogenic signals.

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome in which carriers are at an increased risk of developing a variety of tumors in multiple organ systems. A clinical diagnosis of VHL is determined by the presence of specific clinical manifestations while a molecular genetic diagnosis results from a pathogenic variant in the VHL gene. The majority of mutations occur in VHL coding exons and DNA analysis of these regions has a reported sensitivity of nearly 100%. However, rare variants in the VHL gene promoter may be detected in some cases of suspected VHL disease. We report two cases where VHL promoter variants were detected and describe the role of multi-step mRNA and protein analysis in the diagnostic evaluation of these cases.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

The first therapeutic application of messenger RNA (mRNA) was suggested more than two decades ago. However, its application was constrained by the ability of mRNA to activate the innate immune response, cytotoxicity, and poor potency. We and others recently demonstrated that these undesirable properties of mRNA may be overcome by alterating its structure. In this study, we developed a new chemically modified mRNA coding for BMP-2 with improved osteogenic features. To develop this new construct, we removed from the mRNA sequence the following undesirable elements: an upstream open reading frame in the 5-untranslated region (UTR) and a polyadenylation element together with an AU-rich tract in the 3UTR. In addition, a translation initiator of short UTRs (TISU) was introduced together with 5-iodo modified pyrimidine nucleotides. The new TISU BMP-2 chemically modified RNA (cmRNA) showed robust BMP-2 production in vitro in cell lines (HEK293 and MC3T3) and primary cells (muscle-derived mesenchymal stem cells). Stem cells additionally showed upregulation of osteogenic and angiogenic genes as a result of the TISU BMP-2 cmRNA transfection. The in vivo osteogenic properties of TISU BMP-2 cmRNA were explored in a critical-sized femoral defect in the rat. For this, the TISU BMP-2 cmRNA was loaded into collagen sponges to form transcript-activated matrices. Animals treated with TISU BMP-2 cmRNA showed superior bone formation that seemed to recapitulate endochondral ossification. The higher of the two doses examined in this model showed more robust new tissue formation. Finally, improved vascularization was detected in the healing area for animals treated with TISU BMP-2 cmRNA.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization (ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect endogenous gurken and bicoid mRNAs in Drosophila oocytes.

mRNA translation is a key step in decoding the genetic information stored in DNA. Regulation of translation efficiency contributes to gene expression control and is therefore important for cell fate and function. Here, we describe a recently developed microscopy-based method that allows for visualization of translation of single mRNAs in live cells. The ability to measure translation dynamics of single mRNAs will enable a better understanding of spatiotemporal control of translation, and will provide unique insights into translational heterogeneity of different mRNA molecules in single cells.

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.

Bone regeneration using stem cells and growth factors has disadvantages while needing to use supraphysiological growth factors concentrations. Gene therapy has been proposed as alternative, but also has limitation. Messenger RNA (mRNA)-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. Although much more efficient in delivering genes into the cell, mRNA is unfortunately unstable and immunogenic. However, recent reports indicated that chemical modifications of the mRNA molecule can improve stability and toxicity. In this study, we have combined biomaterials and chemically modified mRNA (cmRNA) encoding Metridia luciferase, eGFP, and bone morphogenetic protein (BMP)-2 to develop transcript-activated matrices (TAMs) for gene transfer to stem cells. BMP-2 cmRNA was produced to evaluate its feasibility in stimulating osteogenic differentiation. Fibrin gel and micro-macro biphasic calcium phosphate (MBCP) granules were used as biomaterials. A sustained release of hBMP-2 cmRNA from both biomaterials was observed during 7 days. This occurred significantly faster from the MBCP granules compared to fibrin gels (92% from MBCP and 43% from fibrin after 7 days). Stem cells cultured in hBMP-2 cmRNA/fibrin or on hBMP-2 cmRNA/MBCP were transfected and able to secrete significant amounts of hBMP-2. Furthermore, transfected cells expressed osteogenic markers in vitro. Interestingly, although both TAMs promoted gene expression at the same level, hBMP-2 cmRNA/MBCP granules induced significantly higher collagen I and osteocalcin gene expression. This matrix also induced more mineral deposition. Overall, our results demonstrated the feasibility of developing efficient TAMs for bone regeneration by combining biomaterials and cmRNAs. MBCP synergistically enhances the hBMP-2 cmRNA-induced osteogenic pathway.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.

UNLABELLED: Gene expression analysis in the tumor microenvironment using archived clinical samples is challenging because of formalin fixation, RNA degradation, and limiting sample volume. NanoString gene expression profiling is a RNA-DNA hybrid capture technology that does not require PCR and can accurately quantify the expression of to 800 transcripts in a single reaction. The technology requires 50-100 ng of RNA, which can be degraded ( EDITOR: is this correct?) to a 200 bp fragment size. In contrast to amplification technologies, nanoString counts the actual numbers of transcripts that are captured with transcript-specific and fluorescently-barcoded probes. This chapter describes protocols for RNA extraction, quantification, mRNA and miRNA profiling and data analysis.

AIMS/HYPOTHESIS: The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. METHODS: Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. RESULTS: At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p

Erythropoiesis is regulated at many levels, including control of mRNA translation. Changing environmental conditions, such as hypoxia or the availability of nutrients and growth factors, require a rapid response enacted by the enhanced or repressed translation of existing transcripts. Cold shock domain protein e1 (Csde1/Unr) is an RNA-binding protein required for erythropoiesis and strongly upregulated in erythroblasts relative to other hematopoietic progenitors. The aim of this study is to identify the Csde1-containing protein complexes and investigate their role in post-transcriptional expression control of Csde1-bound transcripts. We show that Serine/Threonine kinase receptor-associated protein (Strap/Unrip), was the protein most strongly associated with Csde1 in erythroblasts. Strap is a WD40 protein involved in signaling and RNA splicing, but its role when associated with Csde1 is unknown. Reduced expression of Strap did not alter the pool of transcripts bound by Csde1. Instead, it altered the mRNA and/or protein expression of several Csde1-bound transcripts that encode for proteins essential for translational regulation during hypoxia, such as Hmbs, eIF4g3 and Pabpc4. Also affected by Strap knockdown were Vim, a Gata-1 target crucial for erythrocyte enucleation, and Elavl1, which stabilizes Gata-1 mRNA. The major cellular processes affected by both Csde1 and Strap were ribosome function and cell cycle control.

To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 C. elegans presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence in situ hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed C. elegans protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes. SET-17 drives the transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid development. SET-17 is concentrated in stable chromatin-associated nuclear foci at actively transcribed msp (major sperm protein) gene clusters, which we term msp locus bodies. Our results reveal the function of a PRDM9/7-family SET-domain protein in spermatocyte transcription. We propose that the spatial intranuclear organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription., National Institutes of Health (U.S.) (grant GM24663), NIH/National Cancer Institute Physical Sciences Oncology Center (U54CA1438 74), National Institutes of Health (U.S.) Pioneer Award (8 DP1 CA174420- 05)

G-protein coupled receptor (GPCR) mediated activation of the MAPK signalling cascade is a key pathway in the induction of hypertrophic remodelling of the heart – a response to pathological cues including hypertension and myocardial infarction. While levels of pro-hypertrophic hormone agonists of GPCRs increase during periods of greater workload to enhance cardiac output, hypertrophy does not necessarily result. Here we investigated the relationship between the duration of exposure to the pro-hypertrophic GPCR agonist endothelin-1 (ET-1) and the induction of hypertrophic remodelling in neonatal rat ventricular myocytes (NRVM) and in the adult rat heart in vivo. Notably, a 15 min pulse of ET-1 was sufficient to induce markers of hypertrophy that were present when measured at 24 h in vivo and 48 h in vitro. The persistence of ET-1 action was insensitive to ET type A receptor (ETA receptor) antagonism with BQ123. The extended effects of ET-1 were dependent upon sustained MAPK signalling and involved persistent transcription. Inhibitors of endocytosis however conferred sensitivity upon the hypertrophic response to BQ123, suggesting that endocytosis of ETA receptors following ligand binding preserves their active state by protection against antagonist. Contrastingly, α1 adrenergic-induced hypertrophic responses required the continued presence of agonist and were sensitive to antagonist. These studies shed new light on strategies to pharmacologically intervene in the action of different pro-hypertrophic mediators., Highlights • Acute ET-1 exposure elicits a long-lasting cardiac myocyte hypertrophic response. • ET-1 effects depend on persistent MAPK signalling and active transcription. • ET-1 elicited hypertrophy is insensitive to subsequent ETA receptor antagonism. • Endocytosis inhibition potentiates ET-1-induction of hypertrophy markers. • Endocytosis inhibition sensitises effects of ET-1 to ETA receptor antagonist.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Contains fulltext : 171962.PDF (Publisher’s version ) (Open Access) The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p