ABSTRACT
Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.
Subject(s)
CCAAT-Enhancer-Binding Protein-alpha , Leukemia, Myeloid, Acute , Animals , Mice , Humans , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Transcription Factors/metabolism , Heterogeneous-Nuclear Ribonucleoproteins , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolismABSTRACT
Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.
ABSTRACT
Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.
Subject(s)
Arachidonate 15-Lipoxygenase/biosynthesis , Erythropoiesis/genetics , Gene Expression Regulation, Enzymologic , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Ribosomal Proteins/metabolism , Arachidonate 15-Lipoxygenase/genetics , Arginine/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Humans , K562 Cells , Methylation , Mitochondria/metabolism , Protein Binding , Protein BiosynthesisABSTRACT
Innate immune response is triggered by pathogen components, like lipopolysaccharides (LPS) of gram-negative bacteria. LPS initiates Toll-like receptor 4 (TLR4) signaling, which involves mitogen activated protein kinases (MAPK) and nuclear factor kappa B (NFκB) in different pathway branches and ultimately induces inflammatory cytokine and chemokine expression, macrophage migration and phagocytosis. Timely gene transcription and post-transcriptional control of gene expression confer the adequate synthesis of signaling molecules. As trans-acting factors RNA binding proteins (RBPs) contribute significantly to the surveillance of gene expression. RBPs are involved in the regulation of mRNA processing, localization, stability and translation. Thereby they enable rapid cellular responses to inflammatory mediators and facilitate a coordinated systemic immune response. Specific RBP binding to conserved sequence motifs in their target mRNAs is mediated by RNA binding domains, like Zink-finger domains, RNA recognition motifs (RRM), and hnRNP K homology domains (KH), often arranged in modular arrays. In this review, we focus on RBPs Tristetraprolin (TTP), human antigen R (HUR), T-cell intracellular antigen 1 related protein (TIAR), and heterogeneous ribonuclear protein K (hnRNP K) in LPS induced macrophages as primary responding immune cells. We discuss recent experiments employing RNA immunoprecipitation and microarray analysis (RIP-Chip) and newly developed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), photoactivatable ribonucleoside-enhanced crosslinking (PAR-iCLIP) and RNA sequencing techniques (RNA-Seq). The global mRNA interaction profile analysis of TTP, HUR, TIAR, and hnRNP K exhibited valuable information about the post-transcriptional control of inflammation related gene expression with a broad impact on intracellular signaling and temporal cytokine expression.
ABSTRACT
Acute myeloid leukemia is an aggressive disease that arises from clonal expansion of malignant hematopoietic precursor cells of the bone marrow. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of acute myeloid leukemia patients. Our deletion analysis in a cohort of 31 del(9q) acute myeloid leukemia patients further supports the importance of a minimally deleted region composed of seven genes potentially involved in leukemogenesis: GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2. Importantly, among them HNRNPK, encoding heterogeneous nuclear ribonucleoprotein K is proposed to function in leukemogenesis. We show that expression of HNRNPK and the other genes of the minimally deleted region is significantly reduced in patients with del(9q) compared with normal karyotype acute myeloid leukemia. Also, two mRNAs interacting with heterogeneous nuclear ribonucleoprotein K, namely CDKN1A and CEBPA are significantly downregulated. While the deletion size is not correlated with outcome, associated genetic aberrations are important. Patients with an additional t(8;21) show a good prognosis. RUNX1-RUNX1T1, which emerges from the t(8;21) leads to transcriptional down-regulation of CEBPA. Acute myeloid leukemia patients with mutations in CEBPA have a good prognosis as well. Interestingly, in del(9q) patients with CEBPA mutation mRNA levels of HNRNPK and the other genes located in the minimally deleted region is restored to normal karyotype level. Our data indicate that a link between CEBPA and the genes of the minimally deleted region, among them HNRNPK contributes to leukemogenesis in acute myeloid leukemia with del(9q).
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 9 , Leukemia, Myeloid, Acute/genetics , Abnormal Karyotype , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor , CCAAT-Enhancer-Binding Proteins/genetics , Female , Gene Expression , Genes, MDR , Humans , Leukemia, Myeloid, Acute/diagnosis , Male , Middle Aged , MutationABSTRACT
INTRODUCTION: Mechanical ventilation is known to activate oxidative stress and proteolytic pathways in the diaphragm. Trauma by inducing inflammation and activating proteolytic pathways may potentiate the effects of mechanical ventilation on the diaphragm. In a blunt chest trauma with concomitant injuries we tested the hypothesis that trauma via inflammation further activates the proteolytic pathways and worsens atrophy in the diaphragm. MATERIAL AND METHODS: Piglets were separated into two groups and underwent 72 h of mechanical ventilation. One group received a polytrauma (PT) by unilateral femur fracture, blunt chest trauma with lung contusion, laparotomy with standardized liver incision, and a predefined hemorrhagic shock. The second mechanically ventilated group (MV) did not receive any trauma. A non-ventilated group (Con) served as control.Diaphragmatic fiber dimensions, Western Blot analyses of proteolytic pathways, and lipid peroxidation and messenger ribonucleic acid (mRNA) levels of cytokines and nuclear factor kappa b subunit p65 were measured. RESULTS: Active Caspase-3 was significantly increased in MV (Pâ=â0.019), and in PT (Pâ=â0.02) compared with Con. Nuclear factor kappa b subunit p65, was upregulated in PT (Pâ=â0.010) compared with Con. IL-6 mRNA increased significantly in PT compared with Con (Pâ=â0.0024) but did not differ between Con and MV. CONCLUSION: Trauma and mechanical ventilation induced proteolysis and atrophy in the diaphragm, but only polytrauma induced an inflammatory response in the diaphragm. The additional traumatic inflammatory stimulus did not increase the levels of the prementioned variables. These data underline that inflammation is not a major contributor to ventilator-induced diaphragmatic dysfunction. TRIAL REGISTRY NUMBER: AZ 84-02.04.2014.A265 (Landesamt für Natur-, Umwelt- und Verbraucherschutz, LANUV NRW, Germany).
Subject(s)
Diaphragm , Multiple Trauma , Respiration, Artificial/adverse effects , Animals , Cytokines/metabolism , Diaphragm/injuries , Diaphragm/metabolism , Diaphragm/pathology , Disease Models, Animal , Lipid Peroxidation , Multiple Trauma/metabolism , Multiple Trauma/pathology , Multiple Trauma/therapy , Swine , Time Factors , Transcription Factor RelA/metabolismABSTRACT
Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Macrophages/metabolism , Prostaglandin-E Synthases/metabolism , Proteomics/methods , RNA-Binding Proteins/analysis , Animals , High-Throughput Nucleotide Sequencing/methods , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mice , Protein Interaction Maps/drug effects , RAW 264.7 Cells , RNA/metabolism , Sequence Analysis, RNA/methodsABSTRACT
Post-transcriptional regulation is crucial for structural and functional alterations in erythropoiesis. Enucleation of erythroid progenitors precedes reticulocyte release into circulation. In enucleated cells, reticulocyte 15-lipoxygenase (r15-LOX, also known as ALOX15) initiates mitochondria degradation. Regulation of r15-LOX mRNA translation by hnRNP K determines timely r15-LOX synthesis in terminal maturation. K562 cells induced for erythroid maturation recapitulate enucleation and mitochondria degradation. HnRNP K depletion from maturing K562 cells results in enhanced enucleation, which even occurs independently of maturation. We performed RIP-Chip analysis to identify hnRNP K-interacting RNAs comprehensively. Non-muscle myosin heavy chain (NMHC) IIA (also known as MYH9) mRNA co-purified with hnRNP K from non-induced K562 cells, but not from mature cells. NMHC IIA protein increase in erythroid maturation at constant NMHC IIA mRNA levels indicates post-transcriptional regulation. We demonstrate that binding of hnRNP K KH domain 3 to a specific sequence element in the NMHC IIA mRNA 3'UTR mediates translation regulation in vitro Importantly, elevated NMHC IIA expression results in erythroid-maturation-independent enucleation as shown for hnRNP K depletion. Our data provide evidence that hnRNP-K-mediated regulation of NMHC IIA mRNA translation contributes to the control of enucleation in erythropoiesis.
Subject(s)
Erythrocytes/metabolism , Erythropoiesis , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Molecular Motor Proteins/metabolism , Myosin Heavy Chains/metabolism , Protein Biosynthesis , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/metabolism , Erythrocytes/chemistry , Erythrocytes/cytology , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Humans , K562 Cells , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/genetics , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/genetics , Protein Binding , Protein Domains , RNA, MessengerABSTRACT
To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.
Subject(s)
Caspase 3/genetics , Lipopolysaccharides/pharmacology , Macrophages/drug effects , MicroRNAs/genetics , RNA, Messenger/metabolism , Animals , Apoptosis , Cell Line , Gene Expression Regulation/drug effects , Gene Library , Macrophages/cytology , Mice , MicroRNAs/metabolism , RAW 264.7 Cells , RNA, Messenger/drug effects , Sequence Analysis, RNA , Toll-Like Receptor 4/metabolismABSTRACT
Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.
Subject(s)
Cell Hypoxia/genetics , Cell Hypoxia/physiology , Endoplasmic Reticulum/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Cell Line , Codon, Initiator , Cytoplasm/metabolism , Gene Expression , Genetic Markers , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , Protein Biosynthesis , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Ribosomes/metabolism , TranscriptomeABSTRACT
Abstract Analysis of arginine methylation, which affects specific protein interactions in eukaryotic cells, requires access to methylated protein for biophysical and biochemical studies. Methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon co-expression with protein arginine methyltransferase 1 in E. coli was monitored by mass spectrometry and found to be identical to the modification of hnRNP K purified from mammalian cells. Recombinant non-methylated and arginine-methylated hnRNP K (MethnRNP K) were used to characterize self-aggregation and nucleic acid binding. Analytical ultracentrifugation and static light scattering experiments revealed that hnRNP K methylation does not impact reversible self-aggregation, which can be prevented by high ionic strength and organic additives. Filter binding assays were used to compare the binding of non-methylated and MethnRNP K to the pyrimidine repeat-containing differentiation control element (DICE) of reticulocyte 15-lipoxygenase mRNA 3' UTR. No affinity differences were detected for both hnRNP K variants. A series of oligonucleotides carrying various numbers of C4 motifs at different positions was used in steady state competition assays with fluorescently-labeled functional differentiation control element (2R). Quantitative evaluation indicated that all hnRNP K homology domains of hnRNP K contribute differentially to RNA binding, with KH1-KH2 acting as a tandem domain and KH3 as an individual binding domain.
Subject(s)
Arginine/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Nucleic Acids/metabolism , Animals , Arginine/chemistry , Binding Sites , Cells, Cultured , Escherichia coli/enzymology , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Mass Spectrometry , Methylation , Mice , Protein-Arginine N-Methyltransferases/chemistry , Protein-Arginine N-Methyltransferases/metabolismABSTRACT
DDX6 (Rck/p54), a member of the DEAD-box family of helicases, is highly conserved from unicellular eukaryotes to vertebrates. Functions of DDX6 and its orthologs in dynamic ribonucleoproteins contribute to global and transcript-specific messenger RNA (mRNA) storage, translational repression, and decay during development and differentiation in the germline and somatic cells. Its role in pathways that promote mRNA-specific alternative translation initiation has been shown to be linked to cellular homeostasis, deregulated tissue development, and the control of gene expression in RNA viruses. Recently, DDX6 was found to participate in mRNA regulation mediated by miRNA-mediated silencing. DDX6 and its orthologs have versatile functions in mRNA metabolism, which characterize them as important post-transcriptional regulators of gene expression.
Subject(s)
Cell Differentiation/genetics , DEAD-box RNA Helicases/genetics , Gene Expression Regulation, Viral/genetics , Protein Biosynthesis/genetics , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Gene Silencing , Humans , MicroRNAs/genetics , Molecular Sequence Data , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Sequence AlignmentABSTRACT
Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signaling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous nuclear ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating hematopoietic cells, was studied in noninduced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed several mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor-ß-activated kinase 1 (TAK1) a central player in TLR4 signaling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis or stability but enhances TAK1 mRNA translation, resulting in elevated TNF-α, IL-1ß, and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in noninduced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesized TAK1 boosts macrophage inflammatory response.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Lipopolysaccharides/immunology , MAP Kinase Kinase Kinases/genetics , Macrophage Activation/immunology , Macrophages/immunology , Protein Biosynthesis/genetics , RNA, Messenger/genetics , 3' Untranslated Regions/genetics , 3' Untranslated Regions/immunology , Animals , Cell Line , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Heterogeneous-Nuclear Ribonucleoprotein K/immunology , Inflammation/genetics , Inflammation/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , MAP Kinase Kinase Kinases/immunology , Macrophage Activation/genetics , Mice , Protein Biosynthesis/immunology , RNA Processing, Post-Transcriptional/genetics , RNA Processing, Post-Transcriptional/immunology , RNA, Messenger/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5'-untranslated region (5'-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5'-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5'-UTR and 3'-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5'-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.
Subject(s)
DEAD-box RNA Helicases/physiology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Hypoxia , Proto-Oncogene Proteins/physiology , Vascular Endothelial Growth Factor A/biosynthesis , 3' Untranslated Regions , 5' Untranslated Regions , Cytoplasm/metabolism , DEAD-box RNA Helicases/chemistry , Human Umbilical Vein Endothelial Cells , Humans , In Situ Hybridization, Fluorescence , MCF-7 Cells , Mass Spectrometry/methods , Micrococcal Nuclease/metabolism , Neovascularization, Pathologic , Proto-Oncogene Proteins/chemistry , RNA Interference , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Recombinant Proteins/metabolismABSTRACT
Studies on the post-transcriptional control of gene expression in hematopoietic cells have uncovered that a subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs) is involved in cytoplasmic gene regulation. Among them hnRNP K and hnRNPs E1/E2 share common structural motifs, the hnRNP K homology (KH) domains that provide a functional basis for RNA binding. Specific sub-cellular localization and differentiation dependent post-translational modifications modulate the interaction of these proteins with mRNA and proteins in messenger ribonucleoprotein complexes (mRNPs), the latter generating connectivity to cell signaling events. As components of different mRNPs, hnRNP K and hnRNPs E1/E2 function as crucial modulators of mRNA stability and translation in hematopoietic cell differentiation.
Subject(s)
Hematopoiesis/genetics , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , RNA Processing, Post-Transcriptional/genetics , Animals , Cell Differentiation/genetics , Humans , RNA Stability/geneticsABSTRACT
Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , DEAD-box RNA Helicases/metabolism , Protein Biosynthesis , Proto-Oncogene Proteins/metabolism , RNA Interference , RNA Transport , Reticulocytes/metabolism , Ribonucleoproteins/metabolism , Arachidonate 15-Lipoxygenase/genetics , Humans , K562 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.
Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Proteomics/methods , Ubiquitin-Protein Ligases/metabolism , Cell Line , Chaperonin 60/metabolism , Electrophoresis, Gel, Two-Dimensional , Heterogeneous-Nuclear Ribonucleoprotein K , Humans , Mass Spectrometry , Protein Interaction Mapping , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Ribonucleoproteins/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/geneticsABSTRACT
The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) and structured sequence elements within the 3'UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5'cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3'UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5'UTR and 3'UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5'cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5'UTR and HCV 3'UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5'UTR and/or HCV 3'UTR, recruits eIF3 and enhances HCV IRES-mediated translation.
Subject(s)
3' Untranslated Regions , Hepacivirus/genetics , Peptide Chain Initiation, Translational , RNA-Binding Proteins/metabolism , 5' Untranslated Regions , Animals , Binding Sites/genetics , Cell Line , Cells, Cultured , Eukaryotic Initiation Factor-3/metabolism , Genome, Viral , Hepacivirus/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , Open Reading Frames , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Rats , Replicon , Ribosomes/metabolismABSTRACT
Erythroid precursor cells undergo nuclear extrusion and degradation of mitochondria when they mature to erythrocytes. It has been suggested before that the reticulocyte 15-lipoxygenase (r15-LOX) plays an important role in initiating the breakdown of mitochondria in rabbit reticulocytes. The expression of rabbit r15-LOX is regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) K and E1 at the translational level. However, this mechanism has never been confirmed in human erythropoiesis. Based on K562 cells we have set up an inducible human erythroid cell system. We show that, during induction, K562 cells exhibit changes in morphology and protein expression that are characteristic for terminal erythroid maturation: nuclear exclusion, expression of endogenous human r15-LOX regulated by hnRNP K and hnRNP E1, and loss of mitochondria. Importantly, induction of terminal erythroid maturation in primary human CD34(+) cells recapitulated the results obtained in K562 cells. Employing the physiologically relevant K562 cell system we uncovered a new mechanism of interdependent post-transcriptional regulation of gene expression. The timely expression of the tyrosine kinase c-Src, which phosphorylates hnRNP K in later stages, is controlled by hnRNP K in early stages of erythroid maturation. hnRNP K binds to the 3'-untranslated region of the c-Src mRNA and inhibits its translation by blocking 80 S ribosome formation. In premature erythroid cells, small interfering RNA-mediated knockdown of hnRNP K, but not of hnRNP E1, leads to the de-repression of c-Src synthesis.
Subject(s)
Erythroid Cells/metabolism , Gene Expression Regulation , Gene Silencing , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/metabolism , 3' Untranslated Regions , Animals , Antigens, CD34/biosynthesis , CSK Tyrosine-Protein Kinase , Humans , K562 Cells , Mitochondria/metabolism , Rabbits , Reticulocytes/metabolism , Transcription, Genetic , src-Family KinasesABSTRACT
Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 5' and 3' termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein. HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 5'-3' interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.