RESUMO
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.
Assuntos
Araquidonato 15-Lipoxigenase/biossíntese , Eritropoese/genética , Regulação Enzimológica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Proteínas Ribossômicas/metabolismo , Araquidonato 15-Lipoxigenase/genética , Arginina/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Humanos , Células K562 , Metilação , Mitocôndrias/metabolismo , Ligação Proteica , Biossíntese de ProteínasRESUMO
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.
Assuntos
Eritrócitos/metabolismo , Eritropoese , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Biossíntese de Proteínas , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Eritrócitos/química , Eritrócitos/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Células K562 , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Ligação Proteica , Domínios Proteicos , RNA MensageiroRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Macrófagos/metabolismo , Prostaglandina-E Sintases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/análise , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Mapas de Interação de Proteínas/efeitos dos fármacos , Células RAW 264.7 , RNA/metabolismo , Análise de Sequência de RNA/métodosRESUMO
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.
Assuntos
Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Retículo Endoplasmático/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linhagem Celular , Códon de Iniciação , Citoplasma/metabolismo , Expressão Gênica , Marcadores Genéticos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Ribossomos/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Lipopolissacarídeos/imunologia , MAP Quinase Quinase Quinases/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/imunologia , Inflamação/genética , Inflamação/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , MAP Quinase Quinase Quinases/imunologia , Ativação de Macrófagos/genética , Camundongos , Biossíntese de Proteínas/imunologia , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/imunologia , RNA Mensageiro/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
Assuntos
Caspase 3/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , MicroRNAs/genética , RNA Mensageiro/metabolismo , Animais , Apoptose , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Macrófagos/citologia , Camundongos , MicroRNAs/metabolismo , Células RAW 264.7 , RNA Mensageiro/efeitos dos fármacos , Análise de Sequência de RNA , Receptor 4 Toll-Like/metabolismoRESUMO
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.
Assuntos
RNA Helicases DEAD-box/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hipóxia , Proteínas Proto-Oncogênicas/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Citoplasma/metabolismo , RNA Helicases DEAD-box/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Hibridização in Situ Fluorescente , Células MCF-7 , Espectrometria de Massas/métodos , Nuclease do Micrococo/metabolismo , Neovascularização Patológica , Proteínas Proto-Oncogênicas/química , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Arginina/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Ácidos Nucleicos/metabolismo , Animais , Arginina/química , Sítios de Ligação , Células Cultivadas , Escherichia coli/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Espectrometria de Massas , Metilação , Camundongos , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
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.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT , Leucemia Mieloide Aguda , Animais , Camundongos , Humanos , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismoRESUMO
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.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , RNA Helicases DEAD-box/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Transporte de RNA , Reticulócitos/metabolismo , Ribonucleoproteínas/metabolismo , Araquidonato 15-Lipoxigenase/genética , Humanos , Células K562 , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Regiões 3' não Traduzidas , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas , Animais , Sítios de Ligação/genética , Linhagem Celular , Células Cultivadas , Fator de Iniciação 3 em Eucariotos/metabolismo , Genoma Viral , Hepacivirus/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fases de Leitura Aberta , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ratos , Replicon , Ribossomos/metabolismoRESUMO
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.
RESUMO
The protein tyrosine kinase c-Src is regulated by two intramolecular interactions. The repressed state is achieved through the interaction of the Src homology 2 (SH2) domain with the phosphorylated C-terminal tail and the association of the SH3 domain with a polyproline type II helix formed by the linker region between SH2 and the kinase domain. hnRNP K, the founding member of the KH domain protein family, is involved in chromatin remodeling, regulation of transcription, and translation of specific mRNAs and is a target in different signal transduction pathways. In particular, it functions as a specific activator and a substrate of the tyrosine kinase c-Src. Here we address the question how hnRNP K interacts with and activates c-Src. We define the proline residues in hnRNP K in the proline-rich motifs P2 (amino acids [aa] 285 to 297) and P3 (aa 303 to 318), which are necessary and sufficient for the specific activation of c-Src, and we dissect the amino acid sequence (aa 216 to 226) of hnRNP K that mediates a second interaction with c-Src. Our findings indicate that the interaction with c-Src and the activation of the kinase are separable functions of hnRNP K. hnRNP K acts as a scaffold protein that integrates signaling cascades by facilitating the cross talk between kinases and factors that mediate nucleic acid-directed processes.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Domínios de Homologia de src , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Glutationa Transferase/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Dados de Sequência Molecular , Fosforilação , Plasmídeos , Prolina/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Chaperonina 60/metabolismo , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
RESUMO
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).
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 9 , Leucemia Mieloide Aguda/genética , Cariótipo Anormal , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Feminino , Expressão Gênica , Genes MDR , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
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).
Assuntos
Diafragma , Traumatismo Múltiplo , Respiração Artificial/efeitos adversos , Animais , Citocinas/metabolismo , Diafragma/lesões , Diafragma/metabolismo , Diafragma/patologia , Modelos Animais de Doenças , Peroxidação de Lipídeos , Traumatismo Múltiplo/metabolismo , Traumatismo Múltiplo/patologia , Traumatismo Múltiplo/terapia , Suínos , Fatores de Tempo , Fator de Transcrição RelA/metabolismoRESUMO
hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/química , Proteínas Tirosina Quinases/química , Tirosina/química , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Araquidonato 15-Lipoxigenase/química , Proteína Tirosina Quinase CSK , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Quinases da Família srcRESUMO
hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.
Assuntos
Inativação Gênica , Biossíntese de Proteínas , Proteínas Tirosina Quinases/metabolismo , Ribonucleoproteínas/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Proteína Tirosina Quinase CSK , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Dados de Sequência Molecular , Mutação , Fosfopiruvato Hidratase/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , RNA Mensageiro , Ribonucleoproteínas/metabolismo , Tirosina/genética , Tirosina/metabolismo , Domínios de Homologia de src , Quinases da Família srcRESUMO
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.