ABSTRACT
Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality. Here, we found that hnRNPU is overexpressed in HCC tissues and is correlated with the poor prognosis of HCC patients. Besides, hnRNPU is of high significance in regulating the proliferation, apoptosis, self-renewal, and tumorigenic potential of HCC cells. Mechanismly, c-Myc regulates hnRNPU expression at the transcriptional level, and meanwhile, hnRNPU stabilizes the mRNA of c-MYC. We found that the hnRNPU and c-Myc regulatory loop exerts a synergistic effect on the proliferation and self-renewal of HCC, and promotes the HCC progression. Taken together, hnRNPU functions as a novel transcriptional target of c-Myc and promotes HCC progression, which may become a promising target for the treatment of c-Myc-driven HCC.
Subject(s)
Apoptosis/physiology , Carcinoma, Hepatocellular/pathology , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-myc/metabolism , Transcription, Genetic , Animals , Cell Line, Tumor , Humans , Mice, Inbred NOD , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Telomere/metabolism , Animals , Cell Line , DNA/metabolism , G-Quadruplexes , Humans , Oligonucleotides/metabolism , Protein Binding , Replication Protein A/metabolismABSTRACT
The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.
Subject(s)
3' Untranslated Regions/genetics , Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/physiology , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Binding Sites , Cell Line, Tumor , Down-Regulation , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Humans , PC-3 Cells , Phosphoproteins/genetics , RNA Processing, Post-Transcriptional , RNA Stability , Transcription Factors , YAP-Signaling ProteinsABSTRACT
Slo2 channels are large-conductance potassium channels abundantly expressed in the nervous system. However, it is unclear how their expression level in neurons is regulated. Here we report that HRPU-2, an RNA-binding protein homologous to mammalian heterogeneous nuclear ribonucleoprotein U (hnRNP U), plays an important role in regulating the expression of SLO-2 (a homolog of mammalian Slo2) in Caenorhabditis elegans Loss-of-function (lf) mutants of hrpu-2 were isolated in a genetic screen for suppressors of a sluggish phenotype caused by a hyperactive SLO-2. In hrpu-2(lf) mutants, SLO-2-mediated delayed outward currents in neurons are greatly decreased, and neuromuscular synaptic transmission is enhanced. These mutant phenotypes can be rescued by expressing wild-type HRPU-2 in neurons. HRPU-2 binds to slo-2 mRNA, and hrpu-2(lf) mutants show decreased SLO-2 protein expression. In contrast, hrpu-2(lf) does not alter the expression of either the BK channel SLO-1 or the Shaker type potassium channel SHK-1. hrpu-2(lf) mutants are indistinguishable from wild type in gross motor neuron morphology and locomotion behavior. Together, these observations suggest that HRPU-2 plays important roles in SLO-2 function by regulating SLO-2 protein expression, and that SLO-2 is likely among a restricted set of proteins regulated by HRPU-2. Mutations of human Slo2 channel and hnRNP U are strongly linked to epileptic disorders and intellectual disability. The findings of this study suggest a potential link between these two molecules in human patients.SIGNIFICANCE STATEMENT Heterogeneous nuclear ribonucleoprotein U (hnRNP U) belongs to a family of RNA-binding proteins that play important roles in controlling gene expression. Recent studies have established a strong link between mutations of hnRNP U and human epilepsies and intellectual disability. However, it is unclear how mutations of hnRNP U may cause such disorders. This study shows that mutations of HRPU-2, a worm homolog of mammalian hnRNP U, result in dysfunction of a Slo2 potassium channel, which is critical to neuronal function. Because mutations of Slo2 channels are also strongly associated with epileptic encephalopathies and intellectual disability in humans, the findings of this study point to a potential mechanism underlying neurological disorders caused by hnRNP U mutations.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Membrane Transport Proteins/physiology , Synaptic Transmission/physiology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Epilepsy/genetics , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Heterogeneous-Nuclear Ribonucleoprotein U/genetics , Humans , Intellectual Disability/genetics , Membrane Transport Proteins/genetics , Motor Activity/physiology , Motor Neurons/physiology , Motor Neurons/ultrastructure , Mutation/geneticsABSTRACT
Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.
Subject(s)
DNA Repair , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Ku Autoantigen/physiology , Radiation Injuries/etiology , DNA Breaks, Double-Stranded , DNA Glycosylases/physiology , DNA Repair Enzymes/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/physiology , DNA-Activated Protein Kinase/physiology , DNA-Binding Proteins/physiology , HEK293 Cells , Humans , Phosphorylation , Radiation ToleranceABSTRACT
We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.
Subject(s)
Alternative Splicing/physiology , Heart/growth & development , Heart/physiology , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , RNA Precursors/metabolism , RNA, Messenger/metabolism , Animals , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Glycosylation , Heterogeneous-Nuclear Ribonucleoprotein U/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mixed Function Oxygenases/metabolism , Muscle Proteins/metabolism , Mutation , Sarcomeres/metabolismABSTRACT
Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, which are regulated by RNA trans-factors associated with exon 3 of caspase-9 pre-mRNA (C9/E3). In this study, we identified hnRNP U as an RNA trans-factor associated with C9/E3. Down-regulation of hnRNP U led to a decrease in the caspase-9a/9b mRNA ratio, demonstrating a novel enhancing function. Importantly, hnRNP U bound specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interfered with hnRNP U binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Taken together, these findings show that hnRNP U competes with hnRNP L for binding to C9/E3 to enhance the inclusion of the four-exon cassette, and this splice-enhancing function is blocked by the AKT pathway via phosphorylation of hnRNP L.
Subject(s)
Caspase 9/genetics , Heterogeneous-Nuclear Ribonucleoprotein L/metabolism , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Proto-Oncogene Proteins c-akt/metabolism , Alternative Splicing , Base Sequence , Binding Sites , Caspase 9/metabolism , Cell Line, Tumor , Exons , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal TransductionABSTRACT
The nuclear matrix-associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory noncoding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways.
Subject(s)
Alternative Splicing , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Ribonucleoprotein, U2 Small Nuclear/metabolism , Coiled Bodies/metabolism , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein U/genetics , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Humans , Mass Spectrometry , Survival of Motor Neuron 2 Protein/geneticsABSTRACT
In XX female mammals, one of the two X chromosomes is epigenetically inactivated to equalize gene expression with XY males. The formation of the inactive X chromosome (Xi) is regulated by an X-linked long noncoding RNA Xist, which accumulates on the entire length of the chromosome in cis and induces heterochromatin formation. However, the mechanism by which Xist RNA "paints" the Xi has long remained elusive. Here, we show that a matrix protein hnRNP U/SP120/SAF-A is required for the accumulation of Xist RNA on the Xi. Xist RNA and hnRNP U interact and upon depletion of hnRNP U, Xist RNA is detached from the Xi and diffusely localized into the nucleoplasm. In addition, ES cells lacking hnRNP U expression fail to form the Xi. We propose that the association with matrix proteins is an essential step in the epigenetic regulation of gene expression by Xist RNA.
Subject(s)
Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Neuroblastoma/metabolism , RNA, Untranslated/genetics , X Chromosome Inactivation , Animals , Blotting, Western , Cell Proliferation , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Heterochromatin/genetics , Histones/metabolism , Immunoenzyme Techniques , In Situ Hybridization , Male , Mice , Neuroblastoma/pathology , RNA, Long Noncoding , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , RNA, Untranslated/metabolism , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The promoter of the ornithine decarboxylase (ODC) gene contains two E-boxes, which are critical sites for transcriptional activation by the binding of c-Myc-Max heterodimers. We have identified heterogeneous nuclear ribonuclear protein U (hnRNP U) as a component of the complex formed on the E-box-containing promoter region of the ODC gene by using DNA-affinity chromatography, immunoprecipitation and chromatin immunoprecipitation assays. The N-terminal domain of hnRNP U was responsible for the association with c-Myc-Max complex. Down-regulation of hnRNP U with RNA interference blocked the induction of the ODC gene and cell growth by serum stimulation, suggesting that hnRNP U is a coactivator of the c-Myc-Max complex and essential for cell proliferation. Electrophoretic mobility-shift assays revealed that the segment between the two E-boxes in the promoter is the primary binding site of hnRNP U. The putative binding sequence was narrowed-down to a 13-nucleotide segment by comparing the sequence between the E-boxes with the binding sites of hnRNP U, which were recently identified in the promoter of Bmal1, a core component of the circadian molecular oscillator. These findings increase our knowledge of how the c-Myc-Max complex exerts its transcriptional regulatory role and suggest that hnRNP U may be a coactivator of this transcriptional activator complex.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/physiology , E-Box Elements , Heterogeneous-Nuclear Ribonucleoprotein U/physiology , Ornithine Decarboxylase/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/physiology , Amino Acid Sequence , Animals , Base Sequence , Mice , Molecular Sequence Data , NIH 3T3 Cells , Transcriptional ActivationABSTRACT
To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.