ABSTRACT
Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.
Subject(s)
Carcinoma, Hepatocellular/pathology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Liver Neoplasms/pathology , RNA, Long Noncoding/physiology , Animals , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/geneticsABSTRACT
Cell migration requires a complicated network of structural and regulatory proteins. Changes in cellular motility can impact migration as a result of cell-type or developmental stage regulated expression of critical motility genes. Hnrnpab is a conserved RNA-binding protein found as two isoforms produced by alternative splicing. Its expression is enriched in the subventricular zone (SVZ) and the rostral migratory stream within the brain, suggesting possible support of the migration of neural progenitor cells in this region. Here we show that the migration of cells from the SVZ of developing Hnrnpab-/- mouse brains is impaired. An RNA-seq analysis to identify Hnrnpab-dependent cell motility genes led us to Eps8, and in agreement with the change in cell motility, we show that Eps8 is decreased in Hnrnpab-/- SVZ tissue. We scrutinized the motility of Hnrnpab-/- cells and confirmed that the decreases in both cell motility and Eps8 are restored by ectopically coexpressing both alternatively spliced Hnrnpab isoforms, therefore these variants are surprisingly nonredundant for cell motility. Our results support a model where both Hnrnpab isoforms work in concert to regulate Eps8 transcription in the mouse SVZ to promote the normal migration of neural cells during CNS development.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Neurons/physiology , Alternative Splicing , Animals , Cell Line , Cell Movement/genetics , Gene Expression Regulation, Developmental , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/deficiency , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Lateral Ventricles/cytology , Lateral Ventricles/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Neurological , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA Recognition Motif , Sequence Analysis, RNA , Transcription, GeneticABSTRACT
Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5'UTR of sST2. Furthermore, we found that MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation. Determination of the sST2-5'UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5'UTR to be required for optimal IRES activity. Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5'UTR, acts as an ITAF, and thus controls the activity of the sST2-IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5'UTR contains an IRES element, which is activated by a MEK/ERK-dependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2.
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Internal Ribosome Entry Sites/physiology , Protein Biosynthesis , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , 5' Untranslated Regions/drug effects , Binding Sites/drug effects , Gene Expression Regulation/drug effects , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Interleukin-1 Receptor-Like 1 Protein , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , MCF-7 Cells , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , SolubilityABSTRACT
Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Signal Transduction/physiology , Animals , Cell Line, Tumor , E2F1 Transcription Factor/physiology , Epithelial-Mesenchymal Transition , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Mice , Proto-Oncogene Proteins c-myc/physiology , Proto-Oncogene Proteins p21(ras)/genetics , Transcriptional ActivationABSTRACT
Excessive mitochondrial fission is associated with the pathogenesis of neurodegenerative diseases. Dynamin-related protein 1 (Drp1) possesses specific fission activity in the mitochondria and peroxisomes. Various post-translational modifications of Drp1 are known to modulate complex mitochondrial dynamics. However, the post-transcriptional regulation of Drp1 remains poorly understood. Here, we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates Drp1 expression at the post-transcriptional level. hnRNP A1 directly interacts with Drp1 mRNA at its 3'UTR region, and enhances translation potential without affecting mRNA stability. Down-regulation of hnRNP A1 induces mitochondrial elongation by reducing Drp1 expression. Moreover, depletion of hnRNP A1 suppresses 3-NP-mediated mitochondrial fission and dysfunction. In contrast, over-expression of hnRNP A1 promotes mitochondrial fragmentation by increasing Drp1 expression. Additionally, hnRNP A1 significantly exacerbates 3-NP-induced mitochondrial dysfunction and cell death in neuroblastoma cells. Interestingly, treatment with 3-NP induces subcellular translocation of hnRNP A1 from the nucleus to the cytoplasm, which accelerates the increase in Drp1 expression in hnRNP A1 over-expressing cells. Collectively, our findings suggest that hnRNP A1 controls mitochondrial dynamics by post-transcriptional regulation of Drp1.
Subject(s)
GTP Phosphohydrolases/biosynthesis , Gene Expression Regulation, Neoplastic , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Microtubule-Associated Proteins/biosynthesis , Mitochondrial Dynamics/genetics , Mitochondrial Proteins/biosynthesis , Neoplasm Proteins/physiology , Neuroblastoma/genetics , RNA Processing, Post-Transcriptional , 3' Untranslated Regions/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Dynamins , GTP Phosphohydrolases/genetics , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Microtubule-Associated Proteins/genetics , Mitochondrial Proteins/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neuroblastoma/pathology , Protein Binding , Protein Transport , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/geneticsABSTRACT
AIM: Abnormalities in apoptotic signalling pathways often occur in cancer cells and limit the successful chemotherapy outcomes in cancers. Therefore, there is an urgent need to discover new anticancer agents with novel mechanisms of action to overcome the resistance effect in chemotherapy. MATERIALS AND METHODS: In the present study, the anticancer effects and the mechanisms of action of 17ßH-neriifolin (cardiac glycoside) were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and a proteomic approach in treated and non-treated SKOV-3 ovarian cancer cells. RESULTS: 17ßH-neriifolin was found to be active with IC50 values of 0.01 ± 0.001 in SKOV-3 ovarian cancer cell line, as evaluated by the sulforhodamine B (SRB) assay. RESULTS from TUNEL assay indicated that 17ßH-neriifolin caused apoptosis in SKOV-3 cells in a dose-dependent manner. Based on differential analysis of treated and non-treated SKOV-3 two-dimensional electrophoresis (2-DE) profiles, four proteins, namely vimentin (VIM), pyruvate kinase, muscle (PKM), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and transgelin (TAGLN1) were identified to be involved in apoptosis. Other proteins including piggybac transposable element derived 5 (PGBD5), DENN/MADD domain containing 2D (DENND2D) and formin-like 1(FMNL) have also been identified to be associated in SKOV-3 cell death induced by 17ßH-neriifolin. CONCLUSION: These findings may provide new insights on the potential of 17ßH-neriifolin's mechanism of action in killing ovarian cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Cardenolides/pharmacology , Ovarian Neoplasms/drug therapy , Apoptosis/drug effects , Carrier Proteins/physiology , Cell Line, Tumor , Female , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Humans , Membrane Proteins/physiology , Microfilament Proteins/physiology , Muscle Proteins/physiology , Ovarian Neoplasms/pathology , Thyroid Hormones/physiology , Thyroid Hormone-Binding ProteinsABSTRACT
Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.
Subject(s)
Cellular Senescence/genetics , DNA Repair Enzymes/biosynthesis , DNA-Binding Proteins/biosynthesis , Fibroblasts/cytology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/biosynthesis , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , MicroRNAs/physiology , Protein Disulfide-Isomerases/biosynthesis , Cell Line , Cellular Senescence/physiology , DNA Repair Enzymes/genetics , DNA Repair Enzymes/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Electrophoresis, Gel, Two-Dimensional , Gene Expression Profiling , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/physiology , Humans , Mass Spectrometry , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/physiology , Proteome , RNA Interference , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection , Up-RegulationABSTRACT
Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a large group of modular RNA-binding proteins classified according to their conserved domains. This modular nature, coupled with a large choice of alternative splice variants generates functional diversity. Here, we investigate the biological differences between 40LoVe, its splice variant Samba and its pseudoallele hnRNP AB in neural development. Loss of function experiments lead to defects in neural development with reduction of eye size, which stem primarily from increased apoptosis and reduced proliferation in neural tissues. Despite very high homology between 40LoVe/Samba and hnRNP AB, these proteins display major differences in localization, which appear to be in part responsible for functional differences. Specifically, we show that the 40Love/Samba carboxy-terminal domain (GRD) enables nucleocytoplasmic shuttling behavior. This domain is slightly different in hnRNP AB, leading to nuclear-restricted localization. Finally, we show that shuttling is required for 40LoVe/Samba function in neural development.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Heterogeneous-Nuclear Ribonucleoproteins/physiology , Nervous System/growth & development , Xenopus Proteins/physiology , Amino Acid Sequence , Animals , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , In Situ Hybridization , In Situ Nick-End Labeling , Molecular Sequence Data , Nervous System/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Xenopus , Xenopus Proteins/chemistry , Xenopus Proteins/metabolismABSTRACT
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.
Subject(s)
Alternative Splicing , Epithelial-Mesenchymal Transition/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Receptor Protein-Tyrosine Kinases/genetics , Cell Line, Tumor , Exons , HEK293 Cells , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Humans , Nonsense Mediated mRNA Decay , Receptor Protein-Tyrosine Kinases/metabolism , Regulatory Sequences, Ribonucleic AcidABSTRACT
In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.
Subject(s)
Cytokines/physiology , Hepacivirus/physiology , Virus Replication/physiology , Animals , Cell Line, Tumor , Cytokines/antagonists & inhibitors , Cytokines/genetics , Hepatocytes/metabolism , Hepatocytes/virology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Heterogeneous-Nuclear Ribonucleoprotein K , Hydroxymethylglutaryl-CoA Synthase/physiology , Isocitrate Dehydrogenase/physiology , Liver Neoplasms, Experimental/pathology , Macromolecular Substances , Mice , Mice, Inbred C57BL , Nanoparticles , Proteome , RNA Interference , RNA, Small Interfering/genetics , Ribonucleoproteins/physiology , Thioredoxins/physiology , Ubiquitins/antagonists & inhibitors , Ubiquitins/genetics , Ubiquitins/physiologyABSTRACT
To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.
Subject(s)
Gene Expression Regulation, Viral , Genes, Reporter , Genes, Viral , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Human papillomavirus 16/physiology , Catalase/biosynthesis , Catalase/genetics , Cloning, Molecular , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HeLa Cells , Host-Pathogen Interactions , Human papillomavirus 16/genetics , Humans , Nuclear Proteins/physiology , Plasmids/genetics , RNA-Binding Proteins/physiology , Regulatory Sequences, Ribonucleic Acid/genetics , Serine-Arginine Splicing Factors , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic , Viral Proteins/physiologyABSTRACT
The molecular mechanisms that govern the timing and fate of neural stem-cell differentiation toward the distinct neural lineages of the nervous system are not well defined. The contribution of post-transcriptional regulation of gene expression to neural stem-cell maintenance and differentiation, in particular, remains inadequately characterized. The RNA-binding protein Hnrpab is highly expressed in developing nervous tissue and in neurogenic regions of the adult brain, but its role in neural development and function is unknown. We raised a mouse that lacks Hnrpab expression to define what role, if any, Hnrpab plays during mouse neural development. We performed a genome-wide quantitative analysis of protein expression within the hippocampus of newborn mice to demonstrate significantly altered gene expression in mice lacking Hnrpab relative to Hnrpab-expressing littermates. The proteins affected suggested an altered pattern of neural development and also unexpectedly indicated altered glutamate signaling. We demonstrate that Hnrpab(-/-) neural stem and progenitor cells undergo altered differentiation patterns in culture, and mature Hnrpab(-/-) neurons demonstrate increased sensitivity to glutamate-induced excitotoxicity. We also demonstrate that Hnrpab nucleocytoplasmic distribution in primary neurons is regulated by developmental stage.
Subject(s)
Glutamic Acid/pharmacology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Neurons/cytology , Repressor Proteins/physiology , Animals , Cell Differentiation , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Hippocampus/cytology , Hippocampus/drug effects , Mice , Mice, Knockout , Repressor Proteins/geneticsABSTRACT
Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.
Subject(s)
Gene Expression , HIV-1/genetics , Heterogeneous-Nuclear Ribonucleoprotein D/physiology , HIV-1/metabolism , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein D0 , Heterogeneous-Nuclear Ribonucleoprotein D/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein D/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Humans , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA Interference , RNA, Viral/analysis , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/metabolism , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Ovarian cancer is a gynecological malignancy with the highest mortality. Chemoresistance is an important subject for the treatment of ovarian cancer, because obtaining significant drug resistance to the first line chemotherapy, paclitaxel, causes major therapeutic obstacles. It is essential to improve the survival rate of ovarian cancer patients by mining the biomarkers indicating the drug resistance and prognosis, and by further understanding underlying mechanisms of drug resistance. In the present study, we established paclitaxel-resistant subline (SKpac) from human epithelial ovarian cancer cell line, SKOV3, and performed comparative analysis of whole proteomes between paclitaxel-resistant SKpac sublines and paclitaxel-sensitive parental SKOV3 cells to identify differentially expressed proteins and useful biomarkers indicating chemoresistance. Proteins related to chemoresistant process were identified by two-dimensional gel electrophoresis (2DE) with mass spectrometry (MALDI-TOF and LC-MS/MS). Eighteen spots were differentially expressed and were identified in SKpac chemoresistant cells compared to SKOV3. The expressions of ALDH 1A1, annexin A1, hnRNP A2, and GDI 2 proteins were validated by Western blot, which was consistent with proteomic analysis. Among the selected proteins, downregulation of hnRNP A2 and GDI 2 was found to be the most significant finding in SKpac cells and chemoresistant ovarian cancer tissues. Our results suggest that hnRNP A2 and GDI 2 may represent potential biomarkers of the paclitaxel-resistant ovarian cancers for tailored cancer therapy.
Subject(s)
Drug Resistance, Neoplasm , Guanine Nucleotide Dissociation Inhibitors/analysis , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/analysis , Ovarian Neoplasms/physiopathology , Paclitaxel/pharmacology , Cell Line, Tumor , Female , Guanine Nucleotide Dissociation Inhibitors/physiology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Humans , Ovarian Neoplasms/drug therapy , Proteomics/methodsABSTRACT
Annexin-A7 (ANXA7) tumor suppressor role has been shown in various tumors, and ANXA7 expression has been particularly lost in androgen-resistant prostate cancers. In this study, we studied ANXA7 regulation in normal prostate versus androgen-sensitive and -resistant prostate cancer cells. Deletion mapping analysis showed lowest ANXA7-promoter activities in androgen-sensitive LNCaP prostate cancer cells. Genomatix analysis of ANXA7 promoter identified a cluster of steroid nuclear hormone receptor elements, including V$GREF (V$GRE.02/ARE.02). Gelshift analysis clearly indicated distinct nuclear protein occupancy at this ANXA7-promoter site (-1086/-890) in prostate cancer (LNCaP, DU145, and PC3) versus normal prostate (PrEC) cells. In matrix-assisted laser desorption time-of-flight mass spectrometry-based search for ANXA7 nuclear regulators, we identified several heterogeneous nuclear ribonucleoproteins (hnRNPs) (A1, A2/B1 and K) attached to the steroid-associated ANXA7-promoter site in the androgen-resistant PC3 prostate cancer cells with high ANXA7 gene copy number, but not in PrEC. The hnPNP role in ANXA7 regulation (that was validated by hnRNPA2/B1 antibody interference) resulted in multiple ANXA7 cDNA and protein products in PC3, but not in PrEC. Ingenuity pathways analysis showed plausible molecular paths between ANXA7 and the hnRNP-associated network in prostate cancer progression. Thus, a multi-hnRNP complex can be responsible for aberrant ANXA7 transcription and splicing, thereby affecting ANXA7 expression pattern and tumor suppressor function in prostate cancer.
Subject(s)
Annexin A7/genetics , Gene Expression Regulation, Neoplastic , Heterogeneous-Nuclear Ribonucleoproteins/physiology , Prostatic Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Heterogeneous-Nuclear Ribonucleoprotein K/physiology , Humans , Male , Phosphatidylinositol 3-Kinases/physiology , Promoter Regions, Genetic , Prostate/metabolism , Prostatic Neoplasms/prevention & control , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The mammalian proteins hnRNP A1 and hnRNP H control many splicing decisions in viral and cellular primary transcripts. To explain some of these activities, we have proposed that self-interactions between bound proteins create an RNA loop that represses internal splice sites while simultaneously activating the external sites that are brought in closer proximity. Here we show that a variety of hnRNP H binding sites can affect 5' splice site selection. The addition of two sets of hnRNP H sites in a model pre-mRNA modulates 5' splice site selection cooperatively, consistent with the looping model. Notably, binding sites for hnRNP A1 and H on the same pre-mRNA can similarly collaborate to modulate 5' splice site selection. The C-terminal portion of hnRNP H that contains the glycine-rich domains (GRD) is essential for splicing activity, and it can be functionally replaced by the GRD of hnRNP A1. Finally, we used the bioluminescence resonance energy transfer (BRET) technology to document the existence of homotypic and heterotypic interactions between hnRNP H and hnRNP A1 in live cells. Overall, our study suggests that interactions between different hnRNP proteins bound to distinct locations on a pre-mRNA can change its conformation to affect splicing decisions.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/physiology , RNA Splice Sites , Base Sequence/physiology , Binding Sites/genetics , Cells, Cultured , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Humans , Nucleic Acid Conformation , Protein Binding/physiology , RNA Splice Sites/genetics , RNA Splice Sites/physiology , Sequence Homology, Nucleic Acid , Substrate Specificity/genetics , TransfectionABSTRACT
Recent evidence suggests that the canonical miRNA processing pathway can b regulated by a number of positive and negative trans-acting factors. This chapter provides an overview of hnRNP A1-mediated regulation of miR-18a biogenesis. Our laboratory has recently established that the multifunctional RNA-binding protein hnRNP A1 is required for the processing of miR-18a at the nuclear of Drosha-mediated processing. By combining structural and functional analysis of RNA, we showed that hnRNP A1 regulates the processing of pri-miR-18a by binding to its terminal loop and reshaping its stem-loop structure, thus allowing for a more effective Drosha cleavage. Furthermore, we linked the event of hnRNP A1-binding to the pri-miR-18a with an unusual phylogenetic sequence conservation of its terminal loop. Bioinformatic and mutational analysis revealed that a number of pri-miRNAs have highly conserved terminal loops, which are predicted to act as landing pads for trans-acting factors influencing miRNA processing. These results underscore a previously uncharacterized role for general RNA-binding proteins as factors that facilitate the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA production and function.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , MicroRNAs/physiology , Animals , Base Sequence , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , MicroRNAs/chemistry , Molecular Sequence Data , Ribonuclease III/physiologyABSTRACT
Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Gene Expression Regulation, Viral , HIV Infections/metabolism , HIV-1/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Active Transport, Cell Nucleus , Cell Nucleus/genetics , Cytoplasm/genetics , HIV Infections/virology , HIV-1/physiology , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Virus InternalizationABSTRACT
Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.
Subject(s)
Alternative Splicing , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/physiology , Animals , Base Sequence , Caenorhabditis elegans , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Models, Genetic , Molecular Sequence Data , RNA/chemistry , RNA Interference , RNA Splicing , Regulatory Elements, Transcriptional , Sepharose/chemistryABSTRACT
Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early marker for several cancers. This protein is associated with proto-oncogenes and tumor suppressor genes and has itself been described as a proto-oncogene. Our earlier experiments drew a connection between hnRNP A2/B1 levels and cell proliferation and raised the possibility that this protein contributes to the uncontrolled cell division that characterizes cancer. Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22 283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. These results reinforce the conclusion that hnRNP A2/B1 is associated with cellular processes that affect the cell cycle and proliferation.