ABSTRACT
Among the proteins predicted to be a part of the DExD box RNA helicase family, the functions of DDX49 are unknown. Here, we characterize the enzymatic activities and functions of DDX49 by comparing its properties with the well-studied RNA helicase, DDX39B. We find that DDX49 exhibits a robust ATPase and RNA helicase activity, significantly higher than that of DDX39B. DDX49 is required for the efficient export of poly (A)+ RNA from nucleus in a splicing-independent manner. Furthermore, DDX49 is a resident protein of nucleolus and regulates the steady state levels of pre-ribosomal RNA by regulating its transcription and stability. These dual functions of regulating mRNA export and pre-ribosomal RNA levels enable DDX49 to modulate global translation. Phenotypically, DDX49 promotes proliferation and colony forming potential of cells. Strikingly, DDX49 is significantly elevated in diverse cancer types suggesting that the increased abundance of DDX49 has a role in oncogenic transformation of cells. Taken together, this study shows the physiological role of DDX49 in regulating distinct steps of mRNA and pre-ribosomal RNA metabolism and hence translation and potential pathological role of its dysregulation, especially in cancers.
Subject(s)
DEAD-box RNA Helicases/metabolism , Protein Biosynthesis , RNA Helicases/metabolism , RNA Precursors/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Adenosine Triphosphate/metabolism , Carcinogenesis , Cell Line , Cell Nucleolus/enzymology , Cell Nucleolus/genetics , Cell Proliferation , DEAD-box RNA Helicases/genetics , Humans , RNA Precursors/biosynthesis , RNA Stability , RNA TransportABSTRACT
Mechanical strength and biocompatibility are considered the main prerequisites for materials in total hip replacement or joint prosthesis. Noninvasive surgical procedures are necessary to monitor the performance of a medical device in vivo after implantation. To this aim, simultaneous Gd3+ and Dy3+ additions to the ZrO2-SiO2 binary system were investigated. The results demonstrate the effective role of Gd3+ and Dy3+ to maintain the structural and mechanical stability of cubic zirconia ( c-ZrO2) up to 1400 °C, through their occupancy of ZrO2 lattice sites. A gradual tetragonal to cubic zirconia ( t-ZrO2 â c-ZrO2) phase transition is also observed that is dependent on the Gd3+ and Dy3+ content in the ZrO2-SiO2. The crystallization of either ZrSiO4 or SiO2 at elevated temperatures is delayed by the enhanced thermal energy consumed by the excess inclusion of Gd3+ and Dy3+ at c-ZrO2 lattice. The addition of Gd3+ and Dy3+ leads to an increase in the density, elastic modulus, hardness, and toughness above that of unmodified ZrO2-SiO2. The multimodal imaging contrast enhancement of the Gd3+ and Dy3+ combinations were revealed through magnetic resonance imaging and computed tomography contrast imaging tests. Biocompatibility of the Gd3+ and Dy3+ dual-doped ZrO2-SiO2 systems was verified through in vitro biological studies.
Subject(s)
Biocompatible Materials/chemistry , Contrast Media/chemistry , Dysprosium/chemistry , Gadolinium/chemistry , Silicon Dioxide/chemistry , Zirconium/chemistry , Alkaline Phosphatase/metabolism , Biocompatible Materials/chemical synthesis , Biocompatible Materials/toxicity , Cell Line, Tumor , Contrast Media/chemical synthesis , Contrast Media/toxicity , Crystallization , Dysprosium/toxicity , Elastic Modulus , Gadolinium/toxicity , Hardness , Humans , Phase Transition , Silicon Dioxide/chemical synthesis , Silicon Dioxide/toxicity , Zirconium/toxicityABSTRACT
DDX39B, a DExD RNA helicase, is known to be involved in various cellular processes such as mRNA export, splicing and translation. Previous studies showed that the overexpression of DDX39B promotes the global translation but inhibits the mRNA export in a dominant negative manner. This presents a conundrum as to how DDX39B overexpression would increase the global translation if it inhibits the nuclear export of mRNAs. We resolve this by showing that DDX39B affects the levels of pre-ribosomal RNA by regulating its stability as well as synthesis. Furthermore, DDX39B promotes proliferation and colony forming potential of cells and its levels are significantly elevated in diverse cancer types. Thus, increase in DDX39B enhances global translation and cell proliferation through upregulation of pre-ribosomal RNA. This highlights a possible mechanism by which dysregulation of DDX39B expression could lead to oncogenesis.
Subject(s)
DEAD-box RNA Helicases/metabolism , Protein Biosynthesis , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , HEK293 Cells , HeLa Cells , Humans , Neoplasms/metabolism , Neoplasms/pathology , RNA Stability , RNA Transport , RNA, Messenger/genetics , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcription, Genetic , Tumor Stem Cell AssayABSTRACT
Traditionally, disease causal mutations were thought to disrupt gene function. However, it becomes more clear that many deleterious mutations could exhibit a "gain-of-function" (GOF) behavior. Systematic investigation of such mutations has been lacking and largely overlooked. Advances in next-generation sequencing have identified thousands of genomic variants that perturb the normal functions of proteins, further contributing to diverse phenotypic consequences in disease. Elucidating the functional pathways rewired by GOF mutations will be crucial for prioritizing disease-causing variants and their resultant therapeutic liabilities. In distinct cell types (with varying genotypes), precise signal transduction controls cell decision, including gene regulation and phenotypic output. When signal transduction goes awry due to GOF mutations, it would give rise to various disease types. Quantitative and molecular understanding of network perturbations by GOF mutations may provide explanations for 'missing heritability" in previous genome-wide association studies. We envision that it will be instrumental to push current paradigm toward a thorough functional and quantitative modeling of all GOF mutations and their mechanistic molecular events involved in disease development and progression. Many fundamental questions pertaining to genotype-phenotype relationships remain unresolved. For example, which GOF mutations are key for gene regulation and cellular decisions? What are the GOF mechanisms at various regulation levels? How do interaction networks undergo rewiring upon GOF mutations? Is it possible to leverage GOF mutations to reprogram signal transduction in cells, aiming to cure disease? To begin to address these questions, we will cover a wide range of topics regarding GOF disease mutations and their characterization by multi-omic networks. We highlight the fundamental function of GOF mutations and discuss the potential mechanistic effects in the context of signaling networks. We also discuss advances in bioinformatic and computational resources, which will dramatically help with studies on the functional and phenotypic consequences of GOF mutations.
Subject(s)
Multiomics , Precision Medicine , Genome-Wide Association Study , Mutation , Gain of Function MutationABSTRACT
Defects in homologous recombination DNA repair (HRD) both predispose to cancer development and produce therapeutic vulnerabilities, making it critical to define the spectrum of genetic events that cause HRD. However, we found that mutations in BRCA1/2 and other canonical HR genes only identified 10%-20% of tumors that display genomic evidence of HRD. Using a networks-based approach, we discovered that over half of putative genes causing HRD originated outside of canonical DNA damage response genes, with a particular enrichment for RNA-binding protein (RBP)-encoding genes. These putative drivers of HRD were experimentally validated, cross-validated in an independent cohort, and enriched in cancer-associated genome-wide association study loci. Mechanistic studies indicate that some RBPs are recruited to sites of DNA damage to facilitate repair, whereas others control the expression of canonical HR genes. Overall, this study greatly expands the repertoire of known drivers of HRD, with implications for basic biology, genetic screening, and therapy stratification.
Subject(s)
BRCA1 Protein , Neoplasms , Humans , BRCA1 Protein/genetics , Genome-Wide Association Study , BRCA2 Protein/genetics , Homologous Recombination/genetics , RNA-Binding Proteins/geneticsABSTRACT
Multiple cancers exhibit aberrant protein arginine methylation by both type I arginine methyltransferases, predominately protein arginine methyltransferase 1 (PRMT1) and to a lesser extent PRMT4, and by type II PRMTs, predominately PRMT5. Here, we perform targeted proteomics following inhibition of PRMT1, PRMT4, and PRMT5 across 12 cancer cell lines. We find that inhibition of type I and II PRMTs suppresses phosphorylated and total ATR in cancer cells. Loss of ATR from PRMT inhibition results in defective DNA replication stress response activation, including from PARP inhibitors. Inhibition of type I and II PRMTs is synergistic with PARP inhibition regardless of homologous recombination function, but type I PRMT inhibition is more toxic to non-malignant cells. Finally, we demonstrate that the combination of PARP and PRMT5 inhibition improves survival in both BRCA-mutant and wild-type patient-derived xenografts without toxicity. Taken together, these results demonstrate that PRMT5 inhibition may be a well-tolerated approach to sensitize tumors to PARP inhibition.
Subject(s)
Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Neoplasms/drug therapy , Cell Line , DNA Replication , Arginine/metabolism , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/therapeutic use , Repressor Proteins/metabolismABSTRACT
Protein arginine methyltransferase 3 (PRMT3) regulates protein functions by introducing asymmetric dimethylation marks at the arginine residues in proteins. However, very little is known about the interaction partners of PRMT3 and their functional outcomes. Using yeast-two hybrid screening, we identified Retinal dehydrogenase 1 (ALDH1A1) as a potential interaction partner of PRMT3 and confirmed this interaction using different methods. ALDH1A1 regulates variety of cellular processes by catalyzing the conversion of retinaldehyde to retinoic acid. By molecular docking and site-directed mutagenesis, we identified the specific residues in the catalytic domain of PRMT3 that facilitate interaction with the C-terminal region of ALDH1A1. PRMT3 inhibits the enzymatic activity of ALDH1A1 and negatively regulates the expression of retinoic acid responsive genes in a methyltransferase activity independent manner. Our findings show that in addition to regulating protein functions by introducing methylation modifications, PRMT3 could also regulate global gene expression through protein-protein interactions.
Subject(s)
Aldehyde Dehydrogenase 1 Family/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Retinal Dehydrogenase/metabolism , Tretinoin/metabolism , Down-Regulation/genetics , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Protein Binding , Protein-Arginine N-Methyltransferases/physiology , Signal Transduction/drug effects , Tretinoin/pharmacologyABSTRACT
Protein arginine methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues in various proteins affecting diverse cellular processes such as transcriptional regulation, splicing, DNA repair, differentiation, and cell cycle. Elevated levels of PRMT5 are observed in several types of cancers and are associated with poor clinical outcomes, making PRMT5 an important diagnostic marker and/or therapeutic target for cancers. Here, using yeast two-hybrid screening, followed by immunoprecipitation and pull-down assays, we identify a previously uncharacterized protein, FAM47E, as an interaction partner of PRMT5. We report that FAM47E regulates steady-state levels of PRMT5 by affecting its stability through inhibition of its proteasomal degradation. Importantly, FAM47E enhances the chromatin association and histone methylation activity of PRMT5. The PRMT5-FAM47E interaction affects the regulation of PRMT5 target genes expression and colony-forming capacity of the cells. Taken together, we identify FAM47E as a protein regulator of PRMT5, which promotes the functions of this versatile enzyme. These findings imply that disruption of PRMT5-FAM47E interaction by small molecules might be an alternative strategy to attenuate the oncogenic function(s) of PRMT5.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Signal Transduction/genetics , Two-Hybrid System Techniques , Arginine/metabolism , Cell Proliferation/genetics , Chromatin/metabolism , Gene Expression , Gene Expression Regulation , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Methylation , Protein Binding , Protein Stability , Protein-Arginine N-Methyltransferases/genetics , RNA, Messenger/genetics , TransfectionABSTRACT
Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several proteins involved in diverse biological processes. SET7/9-mediated methylation often regulates the stability, sub-cellular localization and protein-protein interactions of its substrate proteins. Here, we aimed to identify novel biological processes regulated by SET7/9 by identifying new interaction partners. For this we used yeast two-hybrid screening and identified the large subunit ribosomal protein, eL42 as a potential interactor of SET7/9. We confirmed the SET7/9-eL42 interaction by co-immunoprecipitation and GST pulldown studies. The N-terminal MORN domain of SET7/9 is essential for its interaction with eL42. Importantly, we identified that SET7/9 methylates eL42 at three different lysines - Lys53, Lys80 and Lys100 through site-directed mutagenesis. By puromycin incorporation assay, we find that SET7/9-mediated methylation of eL42 affects global translation. This study identifies a new role of the functionally versatile SET7/9 lysine methyltransferase in the regulation of global protein synthesis.
Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Ribosomal Proteins/metabolism , Amino Acid Sequence , HEK293 Cells , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/genetics , Humans , Lysine/chemistry , Methylation , Protein Biosynthesis , Protein Domains , Protein Interaction Domains and Motifs , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Two-Hybrid System TechniquesABSTRACT
The ability of ß-Tricalcium phosphate [ß-TCP, ß-Ca3(PO4)2] to host iron at its structural lattice and its associated magnetic susceptibility, hyperthermia effect, biocompatibility and mechanical characteristics is investigated. The studies revealed the ability of ß-Ca3(PO4)2 to host 5.02mol% of Fe3+ at its Ca2+(5) site. Excess Fe3+ additions led to the formation of trigonal Ca9Fe(PO4)7 and moreover a minor amount of CaFe3(PO4)3O crystallization was also observed. A gradual increment in the iron content at ß-Ca3(PO4)2 results in the simultaneous effect of pronounced hyperthermia effect and mechanical stability. However, the presence of CaFe3(PO4)3O contributes for the reduced hyperthermia effect and mechanical stability of iron substituted ß-Ca3(PO4)2. Haemolytic tests, cytotoxicity tests and ALP gene expression analysis confirmed the biocompatibility of the investigated systems.
Subject(s)
Calcium Phosphates/chemical synthesis , Crystallization , IronABSTRACT
Pure and five silver-doped (0-5Ag) ß-tricalcium phosphate [ß-TCP, ß-Ca3(PO4)2]/chitosan composite coatings were deposited on Titanium (Ti) substrates and their properties that are relevant for applications in hard tissue replacements were assessed. Silver, ß-TCP and chitosan were combined to profit from their salient and complementary antibacterial and biocompatible features.The ß-Ca3(PO4)2 powders were synthesized by co-precipitation. The characterization results confirmed the Ag(+) occupancy at the crystal lattice of ß-Ca3(PO4)2. The Ag-dopedß-Ca3(PO4)2/chitosan composite coatings deposited by electrophoresis showed good antibacterial activity and exhibited negative cytotoxic effects towards the human osteosarcoma cell line MG-63. The morphology of the coatings was observed by SEM and their efficiency against corrosion of metallic substrates was determined through potentiodynamic polarization tests.