mRNA Processing Proteins Research Articles

Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis

PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.

Reversal of tyrosine-linked ADP-ribosylation by ARH3 and PARG

ADP-ribosylation is an ancient posttranslational modification with exceptional versatility in terms of breadth of modification targets including at least seven different amino acid side chains, various moieties on nucleic acids and a variety of small chemical compounds. The spatiotemporal signalling dynamic of the different modification variations is tightly regulated and depends on the writers, erases, and readers of each type. Amongst these, tyrosine ADP-ribosylation (Tyr-ADPr) has been consistently detected as a novel modification type, but systematic analysis of its potential physiological role, modification establishment and reversal are still lacking. Here we present a reanalysis of recent ADP-ribosylome data and show that Tyr-ADPr sites are conserved and enriched amongst ribosome biogenesis and mRNA processing proteins and that these sites are affected by the status of ARH3 ADP-ribose hydrolase. To facilitate the study of Tyr-ADPr, we establish methodologies for the synthesis of well-defined Tyr-ADPr peptides and with these could show that Tyr-ADPr is reversed both by ARH3 and PARG enzymes. Together, our work lays the foundation for the future exploration of the Tyr-ADPr.

3D-bioprinted gelatin methacryloyl hydrogel culture system emulating the oviduct environment for enhanced preimplantation embryo development

Oviducts have specific biomechanical properties that support fertilization and preimplantation embryo development, both of which are essential for successful pregnancy. However, conventional plastic-based human embryo culture does not recapitulate the biomechanical environment of the oviduct. Therefore, oviduct mimic culture systems that accurately emulate biophysical conditions for reproductive cells are a significant unmet clinical need. In the present study, we designed a three-dimensional (3D)-bioprinted optimal soft hydrogel system that accurately mimics the oviduct environment and investigated signaling factors during embryo development. We developed an oviduct tube-mimic hydrogel culture dish using gelatin methacryloyl (GelMA) 3D-bioprinted hydrogel. Quantitative assessment of hydrogel mechanical properties depended on the stiffness of the GelMA 3D-bioprinted hydrogel. Embryo quality was evaluated based on cleavage speed and blastocyst ratio on the GelMA hydrogel. Whole-transcriptome next-generation sequencing (NGS) analysis of embryos was used to identify biomechanical signaling factors. Our findings revealed that 10 kPa GelMA hydrogel culture conditions performed better with respect to development speed, blastocyst ratio, and hatching ratio than the control condition. Whole transcriptome NGS revealed up-regulation of mRNA processing genes and protein transport genes by the 7 and 10 kPa hydrogels. Furthermore, the inner cell mass and the number of Oct4+ cells were significantly higher in blastocysts cultured on 10 kPa hydrogel dishes than in those cultured on conventional hard plastic dishes. These findings demonstrate that optimized oviduct-mimic hydrogel-based 3D GelMA culture dishes could improve in vitro embryo development. Hence, 3D GelMA culture dishes may be useful as human embryo culture systems for assisted reproductive techniques.

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing.

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

CMTR-1 RNA methyltransferase mutations activate widespread expression of a dopaminergic neuron-specific mitochondrial complex I gene

The mitochondrial proteome is comprised of approximately 1,100 proteins,1 all but 12 of which are encoded by the nuclear genome in C.elegans. The expression of nuclear-encoded mitochondrial proteins varies widely across cell lineages and metabolic states,2,3,4 but the factors that specify these programs are not known. Here, we identify mutations in two nuclear-localized mRNA processing proteins, CMTR1/CMTR-1 and SRRT/ARS2/SRRT-1, which we show act via the same mechanism to rescue the mitochondrial complex I mutant NDUFS2/gas-1(fc21). CMTR-1 is an FtsJ-family RNA methyltransferase that, in mammals, 2'-O-methylates the first nucleotide 3' to the mRNA CAP to promote RNA stability and translation5,6,7,8. The mutations isolated in cmtr-1 are dominant and lie exclusively in the regulatory G-patch domain. SRRT-1 is an RNA binding partner of the nuclear cap-binding complex and determines mRNA transcript fate.9 We show that cmtr-1 and srrt-1 mutations activate embryonic expression of NDUFS2/nduf-2.2, a paralog of NDUFS2/gas-1 normally expressed only in dopaminergic neurons, and that nduf-2.2 is necessary for the complex I rescue by the cmtr-1 G-patch mutant. Additionally, we find that loss of the cmtr-1 G-patch domain cause ectopic localization of CMTR-1 protein to processing bodies (P bodies), phase-separated organelles involved in mRNA storage and decay.10 P-body localization of the G-patch mutant CMTR-1 contributes to the rescue of the hyperoxia sensitivity of the NDUFS2/gas-1 mutant. This study suggests that mRNA methylation at P bodies may control nduf-2.2 gene expression, with broader implications for how the mitochondrial proteome is translationally remodeled in the face of tissue-specific metabolic requirements and stress.

Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

Transcriptional determinants of lipid mobilization in human adipocytes.

Defects in adipocyte lipolysis drive multiple aspects of cardiometabolic disease, but the transcriptional framework controlling this process has not been established. To address this, we performed a targeted perturbation screen in primary human adipocytes. Our analyses identified 37 transcriptional regulators of lipid mobilization, which we classified as (i) transcription factors, (ii) histone chaperones, and (iii) mRNA processing proteins. On the basis of its strong relationship with multiple readouts of lipolysis in patient samples, we performed mechanistic studies on one hit, ZNF189, which encodes the zinc finger protein 189. Using mass spectrometry and chromatin profiling techniques, we show that ZNF189 interacts with the tripartite motif family member TRIM28 and represses the transcription of an adipocyte-specific isoform of phosphodiesterase 1B (PDE1B2). The regulation of lipid mobilization by ZNF189 requires PDE1B2, and the overexpression of PDE1B2 is sufficient to attenuate hormone-stimulated lipolysis. Thus, our work identifies the ZNF189-PDE1B2 axis as a determinant of human adipocyte lipolysis and highlights a link between chromatin architecture and lipid mobilization.

Proteomic Analysis of Human Breast Cancer MCF-7 Cells to Identify Cellular Targets of the Anticancer Pigment OR3 from Streptomyces coelicolor JUACT03.

Search for ideal compounds with known pathways of anticancer mechanism is still a priority research focus for cancer, as it continues to be a major health challenge across the globe. Hence, in the present study, anticancer potential of a yellow pigment fraction, OR3, isolated from Streptomyces coelicolor JUACT03 was assessed on the breast cancer cell line MCF-7. TLC-fractionated OR3 pigment was subjected to HPLC and GC-MS analysis for characterization and identification of the bioactive component. MCF-7 cells were treated with IC50 concentration of OR3 and the molecular alterations were analyzed using mass spectrometry-based quantitative proteomic analysis. Bioinformatics tools such as STRING analysis and Ingenuity Pathway Analysis were performed to analyze proteomics data and to identify dysregulated signaling pathways. As per our obtained data, OR3 treatment decreased cell proliferation and induced apoptotic cell death due to significant dysregulation of protein expressions in MCF-7 cells. Altered expression included the ribosomal, mRNA processing and vesicle-mediated transport proteins as a result of OR3 treatment. Downregulation of MAPK proteins, NFkB, and estradiol signaling was identified in OR3-treated MCF-7 cells. Mainly eIF2, mTOR, and eIF4 signaling pathways were altered in OR3-treated cells. GC-MS data indicated the presence of novel compounds in OR3 fraction. It can be concluded that OR3 exhibits potent anticancer activity on the breast cancer cells mainly through altering the expression and affecting the signaling proteins which are involved in different cell proliferation/apoptotic pathways thereby causing inhibition of cancer cell proliferation, survival and metastasis.

Dynamic cytosolic foci of DPPA4 in human pluripotent stem cells

DPPA4 is essential for the pluripotent stem cell state, yet its function is poorly understood. DPPA4 is localized in the nucleus, where it is associated with active chromatin. We now report that it is also present in the cytosol, where it appears as diffused clouds, distinct foci and sometimes as spaghetti-like structures. This cytosolic localization is dynamic and DPPA4 shuttles between the cytosol and the nucleus. Its presence is almost abolished from the nucleus upon differentiation. Co-immunoprecipitation studies highlighted novel protein interactors, many of which are also found in the cytosol and are implicated in mRNA processing and RNA and protein transport between the cytosol and the nucleus. Finally, the depletion of DPPA4 resulted in cytosolic accumulation of vesicles. The cytosolic presence of DPPA4 highlights unexplored research directions that could significantly advance the understanding of DPPA4 in pluripotent stem cells and in cancer.

Serine Arginine Splicing Factor 7 (SRSF7) is a critical regulator of innate immune activation in macrophages

Abstract Macrophages are immune cells that receive signals from pathogens and respond by reprogramming gene expression to activate an innate immune response. We currently have a poor understanding of how this response is fine-tuned at a post-transcriptional level. To address this, we turned to recent proteomics studies that identified mRNA processing as a pathway enriched for differentially phosphorylated proteins in macrophages infected with the important human pathogen Mycobacterium tuberculosis (Mtb). We became interested in one of these mRNA processing proteins, a splicing regulatory factor called SRSF7 (formerly known as 9G8), that was differentially phosphorylated at three serine residues during early Mtb infection. Transcriptomics analysis of SRSF7 knockdown RAW 264.7 macrophage-like cells revealed that SRSF7 is required for proper expression of a group of innate immune transcripts including the antiviral genes Mx1 and Ifit3 and the cytosolic DNA sensor Zbp1. Consistent with this phenotype, SRSF7 KD macrophages are permissive to Vesicular Stomatitis Virus (VSV) hyper replication. On the other hand, overexpression of SRSF7 in RAW 264.7 cells leads to elevated ISG abundance in resting cells and enhanced ability to restrict VSV replication. Curiously, it appears that certain ISGs are more reliant on SRSF7 than others, suggesting that SRSF7 acts at the level of the RNA itself. We have started to implicate phosphorylation of SRSF7 in these phenotypes. Together, these studies provide some of the best evidence to date that pathogen sensing cascades can functionalize RNA binding proteins during macrophage activation and highlight a previously unappreciated role for SRSF7 in controlling macrophage antiviral responses. Supported by grants from 1F31GM143893-01 NIH Predoctoral Fellowship and R35GM133720 from NIH/NIGMS.

Identification and activity of the functional complex between hnRNPL and the pseudoexfoliation syndrome-associated lncRNA, LOXL1-AS1.

Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.

The expression of SMN1, MART3, GLE1 and FUS genes in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is one of the most common genetic causes of death in infants due to a mutation of the motor neuron 1 (SMN1) gene. The SMN1 gene encodes for the multifunctional SMN protein. SMN has been shown to be implicated in pre-mRNA splicing, mRNA transport and translational control. Also other mRNA processing proteins, such as GLE1, Marten (MART3) and Fused in Sarcoma (FUS), have been linked to neurodegenerative diseases. The aim of the study was to determine the expression of SMN, GLE1, MART3 and FUS genes in cell lines of the fibroblasts derived from SMA patients and normal controls. Total RNA was extracted from purchased fibroblasts acquired from three SMA type I patients and fibroblasts of three age-matched healthy controls. The RNA was then subjected to qPCR analysis using primers specific for the GLE1, MART3, FUS and SMN1 genes vs. GAPDH as internal control gene. SMN1 mRNA levels were at least ×10 lower in fibroblasts of SMA patients compared to controls. Gle1 and MART3 gene expression was ×2 downregulated whereas FUS mRNA levels appeared to be ×3 upregulated in SMA cells when compared to controls. We found a high correlation between FUS gene expression level to the SMN1 at gene expression level of fibroblast cell lines of SMA type I patients (r = 0.994, p < 0.0001). Our preliminary data show an intriguing expression profile of Gle1, MART3 and FUS genes in SMA, and suggest a critical role of FUS protein in the SMA pathogenesis.

A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used aproteomics-based pull-down approach to map andcharacterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.

An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.

Background:Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs.Objective:We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1.Methods:We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD.Results:The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD.Conclusions:This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.

Abstract 3484: Synergism from combination of targeted therapy with tumor active phytochemicals in ovarian tumor models and changes in protein expression

Abstract Introduction Ovarian cancer is most lethal cause of gynecological types of cancer mainly because of acquired drug resistance and poor diagnosisis. In majority of cases it is detected at an advanced stage when it is likely to have spread beyond ovaries. Combination of platinum drugs with paclitaxel is the current mode of treatment. Although cancer cells respond well to the drug combination, relapse often occurs for which drugs fail to function. We have designed a number of novel platinums aimed to overcome platinum resistance in ovarian cancer. We also applied combinations of platinums and tumor active phytochemicals such as curcumin, EGCG, thymoquinone, resveratrol, ursolic acid and genistein to overcom drug resistance. This presentation will provide a brief overview of novel platinums designed in our lab with special emphasis on recent studies on combinations of platinum with phytochemicals and protein expression. Methods Activity of the compounds alone and in sequenced combination in ovarian cancer cell lines including A2780, A2780cisR and A2780ZD0473R were determined using MTT reduction assay. Drug uptake and drug-DNA binding were determined using developed protocols. Proteomic studies involving 2D gel electrophoresis and mass spectrometry were carried out to characterize key proteins associated with platinum resistance. Results Generally but not always sequenced combinations with 2 to 4 h time gap were found to be synergistic. The variation in combined drug action with the change in sequence of administration clearly indicates that the action of one drug has modulated that of the other. Proteomic studies have identified over thirty proteins believed to be associated with platinum resistance in ovarian cancer. They belong to six major groups including cytoskeletal proteins, molecular chaperone and stress related proteins, proteins involved in detoxification and drug resistance, proteins involved in metabolic processes and mRNA processing proteins. The proteins are restored to normalcy due to treatment with synergistic combinations. Conclusion If confirmed in vivo, the results of the study indicate that appropriate combinations of targeted therapy and tumor active phytochemicals may providing an effective and affordable means of overcoming drug resistance in ovarian cancer. Citation Format: Fazlul Huq. Synergism from combination of targeted therapy with tumor active phytochemicals in ovarian tumor models and changes in protein expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3484. doi:10.1158/1538-7445.AM2015-3484

Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

Abstract A10: Functional characterization of Ewing's sarcoma susceptibility loci

Abstract Background: We previously identified in a GWAS common variants near the genes TARDBP (chr1), EGR2 and ADO (both chr10), and BMF (chr15) that are associated with the susceptibility of Caucasians toward Ewing's sarcoma (ES). These variants lie in intergenic regions that appear to harbor expression quantitative trait loci (eQTLs) as they show strong modulatory effects on the expression of these “candidate genes”. While TARDBP is an mRNA and miRNA processing protein, EGR2 is a conserved transcription factor involved proliferation and differentiation of osteoprogenitors and Schwann cells. ADO, which shows a highly significant co-expression with EGR2, is the key enzyme converting cysteamine into hypotaurine. The BCL2-modifying factor BMF is involved in pro- and anti-apoptotic processes depending on the cellular context. As most SNPs identified by GWAS are usually not the causal variants but are expected to be in strong linkage disequilibrium with such variants, this project aims to comprehensively sequence and epigenetically profile the three ES susceptibility regions and to functionally characterize the role of the four candidate genes in ES. Methods and Results: Fine-selection of the genomic regions (in total 1 Mb) was performed with own and HapMap CEU data. An initial pilot experiment (48 samples) using a custom Nextera DNA enrichment system followed by paired-end 150x150bp sequencing (Illumina HiSeq2500) yielded 95% coverage with at least 20 high-quality reads/nucleotide. Comparison with matched SNP arrays revealed that both the false positive and negative discovery rates were smaller than 1%. Currently, we sequence additional 576 cases and PCA-matched controls and perform a multilayered epigenetic profiling of ES cells with/without silencing of EWS/FLI1 for our susceptibility regions. Analysis of three comprehensive paired gene expression and genotype datasets for lymphoblastoid cell lines, medulloblastoma, and neuroblastoma, proved that none of our Ewing SNPs correlated with the expression the four candidate genes, suggesting a specific effect of these eQTLs in ES, possibly via direct or indirect interaction with EWS/FLI1. In accordance, siRNA-mediated knockdown of EWS/FLI1 in ES cell lines strongly down-regulates EGR2, while EWS/FLI1 expression in human mesenchymal stem cells (MSCs) induces EGR2. In contrast, only moderate modulatory effects of EWS/FLI1 were observed for TARDBP, ADO, and BMF. However, three independent ChIP-seq studies did not find evidence of EWS/FLI1 binding to the EGR2 promoter or nearby regions, arguing for an indirect mode of regulation. On a functional level, knockdown of TARDBP, EGR2, and BMF, but not ADO reduces proliferation and clonogenicity of ES cells by blocking cell cycle progression and inducing apoptosis, which is preceded by transcriptional changes in cell cycle regulators and pro-survival genes as measured by microarrays, RNA-seq and qRT-PCR. Interestingly, all candidate genes share a highly significant overlap in modulated genes with EWS/FLI1 and their transcriptional fingerprint is similar to that conferred by EWS/FLI1 to human MSCs, the most likely cells of origin of ES. Gene-set enrichment and Ingenuity pathway analyses revealed that the candidate genes are involved in cell growth and differentiation, albeit via diverse mechanisms. Additional experiments, including the generation of inducible shRNA expression systems, are ongoing to further characterize their role and their interplay with EWS/FLI1 in ES. Conclusions: In synopsis, our data indicate that the previously identified ES susceptibility regions and candidate genes may play a prominent role in ES pathobiology. Citation Format: Thomas Grunewald, Marie-Ming Aynaud, Franck Tirode, Eleni Tomazou, Didier Surdez, Thomas Rio Frio, Virginie Bernard, Virginie Raynal, Carlo Lucchesi, Gaelle Pierron, Pascale Gilardi-Hebenstreit, Patrick Charnay, Heinrich Kovar, Olivier Delattre. Functional characterization of Ewing's sarcoma susceptibility loci. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A10.

Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.

Dynamic Association of NUP98 with the Human Genome

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors, histone-modification enzymes, and mRNA processing proteins. Recent evidence suggests that nucleoporins, well known components that control nucleo-cytoplasmic trafficking, have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation, which initially has been described in fungi and flies, also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition, we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.

Norepinephrine Deficiency Is Caused by Combined Abnormal mRNA Processing and Defective Protein Trafficking of Dopamine β-Hydroxylase

Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).