ABSTRACT
Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.
Subject(s)
DNA-Binding Proteins/metabolism , Proteostasis/genetics , RNA, Long Noncoding/genetics , Short Interspersed Nucleotide Elements/genetics , Apoptosis , Cell Line , Cytoplasm/metabolism , DNA Damage , Endoplasmic Reticulum Stress , Enzyme Activation , Gene Dosage , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Humans , Mitosis , Models, Biological , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Deletion/genetics , eIF-2 KinaseABSTRACT
BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.
Subject(s)
Chromosomal Instability , Colorectal Neoplasms/genetics , Neoplasms, Experimental/genetics , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aurora Kinase B/metabolism , Azoxymethane/toxicity , Carcinogenesis/genetics , Cell Line, Tumor , Colon/cytology , Colon/pathology , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/pathology , Cytogenetic Analysis , Dextrans/toxicity , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/pathology , Male , Mice , Mice, Transgenic , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/pathology , Organoids , Primary Cell Culture , Proto-Oncogene Proteins c-myc/metabolism , RNA, Long Noncoding/genetics , RNA-Binding Proteins/metabolism , Signal Transduction/geneticsABSTRACT
Diamond-Blackfan anemia (DBA) is a ribosomopathy of variable expressivity and penetrance characterized by red cell aplasia, congenital anomalies, and predisposition to certain cancers, including early-onset colorectal cancer (CRC). DBA is primarily caused by a dominant mutation of a ribosomal protein (RP) gene, although approximately 20% of patients remain genetically uncharacterized despite exome sequencing and copy number analysis. Although somatic loss-of-function mutations in RP genes have been reported in sporadic cancers, with the exceptions of 5q-myelodysplastic syndrome (RPS14) and microsatellite unstable CRC (RPL22), these cancers are not enriched in DBA. Conversely, pathogenic variants in RPS20 were previously implicated in familial CRC; however, none of the reported individuals had classical DBA features. We describe two unrelated children with DBA lacking variants in known DBA genes who were found by exome sequencing to have de novo novel missense variants in RPS20. The variants affect the same amino acid but result in different substitutions and reduce the RPS20 protein level. Yeast models with mutation of the cognate residue resulted in defects in growth, ribosome biogenesis, and polysome formation. These findings expand the phenotypic spectrum of RPS20 mutation beyond familial CRC to include DBA, which itself is associated with increased risk of CRC.
Subject(s)
Anemia, Diamond-Blackfan/genetics , Germ-Line Mutation , Ribosomal Proteins/genetics , Adolescent , Amino Acid Sequence , Child , Colorectal Neoplasms/genetics , Female , Humans , Infant, Newborn , Male , Pedigree , Penetrance , Protein Structure, Tertiary , Exome SequencingABSTRACT
Stress granules (SGs) assemble under stress-induced conditions that inhibit protein synthesis, including phosphorylation of eIF2α, inhibition of the RNA helicase eIF4a proteins or inactivation of mTORC1. Classically defined SGs are composed of translation initiation factors, 40S ribosomes, RNA-binding proteins and poly(A)+ mRNAs. As such, they represent an important compartment for storage of mRNAs and regulation of their translation. Emerging work on SGs indicates that these structures might promote cellular survival in diverse disease states. Yet, much work on SG formation and function employs acute stress conditions, which might not accurately reflect the chronic stresses that manifest in human disease. Here, we used prolonged nutrient starvation to model and investigate SG formation and function during chronic stress in a human cell line and mouse embryonic fibroblasts. Surprisingly, we found that SGs that form under chronic nutrient starvation lack 40S ribosomes, do not actively exchange their constituent components with cytoplasmic pools and promote cell death. We named these SGs starvation-induced SGs (stSGs). Our results on stSGs imply that SG assembly and function in the context of prolonged nutrient starvation stress differ significantly from what has been described for acute stress conditions.
Subject(s)
Apoptosis , Cytoplasmic Granules/metabolism , Animals , Cell Line, Tumor , Eukaryotic Initiation Factor-2/metabolism , Humans , Kinetics , Mice , Protein Biosynthesis , Ribosomes/metabolismABSTRACT
Stress granules (SG) are membrane-less organelles that are condensates of stalled translation initiation complexes and mRNAs. SG formation is a cytoprotective response to environmental stress and results from protein interactions involving regions of low amino acid complexity and poorly defined post-translational modifications of SG components. Many RNA-binding proteins are methylated, and we previously demonstrated that the potent SG-nucleating protein G3BP1 is methylated by protein arginine methyltransferase 1 and 5 (PRMT1 and PRMT5). G3BP1 methylation represses SG formation and is reversible. Here we functionally link JMJD6 (Jumonji C domain-containing protein 6) to G3BP1 demethylation. Our findings reveal that JMJD6 is a novel SG component that interacts with G3BP1 complexes, and its expression reduces G3BP1 monomethylation and asymmetric dimethylation at three Arg residues. Knockdown of JMJD6 repressed SG formation and G3BP1 demethylation, but SG formation and G3BP1 demethylation were rescued with catalytically active but not mutant JMJD6. These results suggest that JMJD6 functions directly or indirectly as an arginine demethylase of G3BP1 that promotes SG formation.
Subject(s)
Cytoplasmic Granules/metabolism , DNA Helicases/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Arginine/metabolism , Cell Line , Demethylation , Humans , Protein Interaction Maps , Protein Processing, Post-Translational , Stress, PhysiologicalABSTRACT
Stress granules (SGs) are cytoplasmic condensates of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins are enriched for arginine methylation and facilitate SG assembly through interactions involving regions of low amino acid complexity. How methylation of specific RNA-binding proteins regulates RNA granule assembly has not been characterized. Here, we examined the potent SG-nucleating protein Ras-GAP SH3-binding protein 1 (G3BP1), and found that G3BP1 is differentially methylated on specific arginine residues by protein arginine methyltransferase (PRMT) 1 and PRMT5 in its RGG domain. Several genetic and biochemical interventions that increased methylation repressed SG assembly, whereas interventions that decreased methylation promoted SG assembly. Arsenite stress quickly and reversibly decreased asymmetric arginine methylation on G3BP1. These data indicate that arginine methylation in the RGG domain prevents large SG assembly and rapid demethylation is a novel signal that regulates SG formation.
Subject(s)
Arsenites/pharmacology , Carrier Proteins/metabolism , Cytoplasmic Granules/metabolism , Stress, Physiological/drug effects , Arginine/genetics , Arginine/metabolism , Carrier Proteins/genetics , Cell Line, Tumor , Cytoplasmic Granules/genetics , DNA Helicases , Humans , Methylation , Poly-ADP-Ribose Binding Proteins , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , RNA Helicases , RNA Recognition Motif Proteins , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
UNLABELLED: Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-κB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2α phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity. IMPORTANCE: Stress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures and explains why G3BP1 is subverted by many viruses to promote a productive infection.
Subject(s)
Carrier Proteins/metabolism , Enterovirus/immunology , Immunity, Innate , eIF-2 Kinase/metabolism , Cell Line , DNA Helicases , Humans , Poly-ADP-Ribose Binding Proteins , Protein Interaction Mapping , RNA Helicases , RNA Recognition Motif ProteinsABSTRACT
We have shown previously that poliovirus infection disrupts cytoplasmic P-bodies in infected mammalian cells. During the infectious cycle, poliovirus causes the directed cleavage of Dcp1a and Pan3, coincident with the dispersion of P-bodies. We now show that expression of Dcp1a prior to infection, surprisingly, restricts poliovirus infection. This inhibition of infection was independent of P-body formation because expression of GFP-Dcp1a mutants that cannot enter P-bodies restricted poliovirus infection similar to wild-type GFP-Dcp1a. Expression of wild-type or mutant GFP-Dcp1a induced phosphorylation of eIF2α through the eIF2α kinase protein kinase R (PKR). Activation of PKR required the amino-terminal EVH1 domain of Dcp1a. This PKR-induced translational inhibition appears to be specific to Dcp1a because the expression of other P-body components, Pan2, Pan3, Ccr4, or Caf1, did not result in the inhibition of poliovirus gene expression or induce eIF2α phosphorylation. The translation blockade induced by Dcp1a expression suggests novel signaling linking RNA degradation/decapping and regulation of translation.
Subject(s)
Endoribonucleases/metabolism , Protein Biosynthesis/physiology , RNA Stability/physiology , Trans-Activators/metabolism , eIF-2 Kinase/metabolism , Animals , Cell Line , Endoribonucleases/genetics , Enzyme Activation/genetics , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Exoribonucleases , Mice , Mice, Knockout , Mutation , Phosphorylation/genetics , Poliomyelitis/genetics , Poliomyelitis/metabolism , Poliomyelitis/pathology , Poliovirus/genetics , Poliovirus/metabolism , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , Receptors, CCR4/genetics , Receptors, CCR4/metabolism , Repressor Proteins , Ribonucleases , Trans-Activators/genetics , eIF-2 Kinase/geneticsABSTRACT
Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.
ABSTRACT
Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.
Subject(s)
Antineoplastic Agents , Triple Negative Breast Neoplasms , Animals , Mice , Humans , Interferons , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Cell Proliferation , Tumor MicroenvironmentABSTRACT
Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation.
Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factors/metabolism , Host-Pathogen Interactions , Poliovirus/physiology , Protein Biosynthesis , Viral Proteins/biosynthesis , HeLa Cells , HumansABSTRACT
Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.
Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Homocystinuria/genetics , Host Cell Factor C1/genetics , Oxidoreductases/genetics , Repressor Proteins/genetics , Ribosomes/genetics , Vitamin B 12 Deficiency/genetics , Amino Acid Metabolism, Inborn Errors/metabolism , Amino Acid Metabolism, Inborn Errors/pathology , Animals , Disease Models, Animal , Embryo, Mammalian , Female , Gene Expression Regulation, Developmental , Homocystinuria/metabolism , Homocystinuria/pathology , Host Cell Factor C1/deficiency , Humans , Male , Mice , Mice, Knockout , Mutation , Organelle Biogenesis , Oxidoreductases/deficiency , Protein Biosynthesis , Protein Subunits/genetics , Protein Subunits/metabolism , Repressor Proteins/deficiency , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Ribosomes/pathology , Vitamin B 12/metabolism , Vitamin B 12 Deficiency/metabolism , Vitamin B 12 Deficiency/pathologyABSTRACT
Cap-independent initiation of translation is thought to promote protein synthesis on some mRNAs during times when cap-dependent initiation is down-regulated. However, the mechanism of cap-independent initiation is poorly understood. We have previously reported the secondary structure within the yeast minimal URE2 IRES element. In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using DeltaeIF2A yeast. We found that the stability of the stem-loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IRES-mediated translation can be defined to discrete regions within the URE2 coding region. Repression of internal initiation on the URE2 minimal IRES element by eIF2A is not dependent on the stability of the secondary structure within the URE2 IRES element. Our data also indicate that eIF2A-mediated repression is not specific to the URE2 IRES element, as both the GIC1 and PAB1 IRES elements are repressed by eIF2A. These data provide valuable insights into the mRNA requirements for internal initiation in yeast, and insights into the mechanism of eIF2A-mediated suppression.
Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Glutathione Peroxidase/metabolism , Nucleotides/metabolism , Prions/metabolism , Regulatory Elements, Transcriptional , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Eukaryotic Initiation Factor-2/genetics , Glutathione Peroxidase/genetics , Molecular Sequence Data , Mutation , Nucleotides/chemistry , Nucleotides/genetics , Phylogeny , Poly A/genetics , Prions/genetics , Protein Biosynthesis , RNA, Fungal/chemistry , RNA, Fungal/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence AlignmentABSTRACT
Stress granules are macromolecular aggregates of mRNA and proteins assembling in response to stresses that promote the repression of protein synthesis. Most of the work characterizing stress granules has been done under acute stress conditions or during viral infection. Comparatively less work has been done to understand stress granule assembly during chronic stress, specifically regarding the composition and function of stress granules in this alternative context. Here, we describe key aspects of stress granule biology under acute stress, and how these stress granule hallmarks differ in the context of chronic stress conditions. We will provide perspective for future work aimed at further uncovering the form and function of both acute and chronic stress granules and discuss aspects of stress granule biology that have the potential to be exploited in human disease.
Subject(s)
Oxidative Stress/physiology , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , Stress, Physiological/physiology , Animals , Humans , RNA, Messenger/geneticsABSTRACT
Down syndrome (DS) is the most common genetic cause of intellectual disability. Protein homeostasis is essential for normal brain function, but little is known about its role in DS pathophysiology. In this study, we found that the integrated stress response (ISR)-a signaling network that maintains proteostasis-was activated in the brains of DS mice and individuals with DS, reprogramming translation. Genetic and pharmacological suppression of the ISR, by inhibiting the ISR-inducing double-stranded RNA-activated protein kinase or boosting the function of the eukaryotic translation initiation factor eIF2-eIF2B complex, reversed the changes in translation and inhibitory synaptic transmission and rescued the synaptic plasticity and long-term memory deficits in DS mice. Thus, the ISR plays a crucial role in DS, which suggests that tuning of the ISR may provide a promising therapeutic intervention.
Subject(s)
Down Syndrome/physiopathology , Down Syndrome/psychology , Neuronal Plasticity , Proteostasis/physiology , Stress, Physiological/physiology , Synaptic Transmission , Animals , Brain/physiopathology , Eukaryotic Initiation Factor-2/metabolism , Memory, Long-Term , Mice , Mice, Mutant Strains , Protein Biosynthesis , Stress, Physiological/drug effects , Stress, Physiological/genetics , eIF-2 Kinase/antagonists & inhibitors , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolismABSTRACT
The antidiabetic drug metformin has been associated with reduced colorectal cancer (CRC) risk and improved prognosis of CRC patients. However, the detailed mechanisms underlying such beneficial effects remain unknown. In this study, we aimed to evaluate metformin activity in CRC models and unveil the underlying molecular mechanisms. We showed that metformin inhibits CRC cell proliferation by arresting cells in the G1 phase of the cell cycle and dramatically reduces colony formation of CRC cells. We discovered that metformin causes a robust reduction of MYC protein level. Through the use of luciferase assay and coincubation with either protein synthesis or proteasome inhibitors, we demonstrated that regulation of MYC by metformin is independent of the proteasome and 3' UTR-mediated regulation, but depends on protein synthesis. Data from polysome profiling and ribopuromycylation assays showed that metformin induced widespread inhibition of protein synthesis. Repression of protein synthesis by metformin preferentially affects cell cycle-associated proteins, by altering signaling through the mTOR-4EBP-eIF4E and MNK1-eIF4G-eIF4E axes. The inhibition of MYC protein synthesis may underlie metformin's beneficial effects on CRC risk and prognosis.
Subject(s)
Colorectal Neoplasms , Eukaryotic Initiation Factor-4G , Intracellular Signaling Peptides and Proteins , Metformin/pharmacology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-myc , Signal Transduction , TOR Serine-Threonine Kinases , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , HCT116 Cells , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5' UTRs with a high frequency of polypurine sequences, several of which harbored (CGG)4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT-driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A.Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229-40. ©2018 AACR.
Subject(s)
5' Untranslated Regions/genetics , Eukaryotic Initiation Factor-4A/genetics , Protein Biosynthesis/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Wnt Signaling Pathway/genetics , 5' Untranslated Regions/drug effects , Animals , Mice , Protein Biosynthesis/drug effects , RNA Helicases/genetics , RNA, Messenger/genetics , Ribosomes/drug effects , Ribosomes/genetics , Triterpenes/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.
Subject(s)
Carrier Proteins/metabolism , Casein Kinase II/metabolism , Cytoplasmic Granules/metabolism , Stress, Physiological , Arsenites/toxicity , Casein Kinase II/antagonists & inhibitors , Cell Line, Tumor , Cytoplasmic Granules/drug effects , DNA Helicases , Genes, Dominant , Humans , Phosphorylation/drug effects , Poly-ADP-Ribose Binding Proteins , Protein Kinase Inhibitors/pharmacology , Protein Subunits/metabolism , RNA Helicases , RNA Recognition Motif Proteins , Stress, Physiological/drug effectsABSTRACT
UNLABELLED: Stress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition. IMPORTANCE: Our previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity.
Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cytoplasmic Granules/metabolism , Mengovirus/immunology , RNA, Double-Stranded/metabolism , eIF-2 Kinase/metabolism , Animals , Cell Line , DNA Helicases , Host-Pathogen Interactions , Humans , Mengovirus/growth & development , Mice, Knockout , Poly-ADP-Ribose Binding Proteins , Protein Binding , Protein Interaction Mapping , RNA Helicases , RNA Recognition Motif ProteinsABSTRACT
Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1:CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circadian clock.