RESUMEN
Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inflamación/inmunología , Interleucina-17/metabolismo , Estabilidad del ARN/fisiología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Regulación de la Expresión Génica/inmunología , Inflamación/metabolismo , Interleucina-17/inmunología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Receptores de Interleucina-17/metabolismoRESUMEN
Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.
Asunto(s)
Células Endoteliales/metabolismo , Biosíntesis de Proteínas , Factor A de Crecimiento Endotelial Vascular/genética , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Aorta/citología , Secuencia de Bases , Bovinos , Línea Celular , Codón de Terminación , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Alineación de SecuenciaRESUMEN
Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.
Asunto(s)
Neoplasias de la Mama , Núcleo Celular , Poli(ADP-Ribosa) Polimerasa-1 , Proteínas Proto-Oncogénicas c-akt , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Núcleo Celular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Línea Celular Tumoral , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacil-ARNt Sintetasas/genética , Transporte Activo de Núcleo Celular , Señales de Localización Nuclear/metabolismoRESUMEN
Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the â¼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.
Asunto(s)
Aminoacil-ARNt Sintetasas , Aminoacil-ARNt Sintetasas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Ribosomas/metabolismoRESUMEN
Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sitios de Unión , Sistemas CRISPR-Cas , Clonación Molecular , Proteínas del Citoesqueleto/genética , ADN Complementario , Regulación de la Expresión Génica , Silenciador del Gen , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Modelos Moleculares , Fosfoproteínas/genética , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Adiponectin, an adipocyte-specific secretory protein encoded by the ADIPOQ gene has a causal role in insulin resistance. Anti-diabetic drugs increase plasma adiponectin by a poorly understood, post-transcriptional mechanism enhancing insulin sensitivity. Deletion analysis of a reporter bearing the mouse Adipoq mRNA 5'-leader identified an inhibitory cis-regulatory sequence. The 5'-leader harbours two potential upstream open reading frames (uORFs) overlapping the principal downstream ORF. Mutation of the uORF ATGs increased reporter translation ~3-fold, indicative of a functional uORF. uORFs are common in mammalian mRNAs; however, only a select group resist translational repression by the integrated stress response (ISR). Thapsigargin (TG), which induces endoplasmic reticulum (ER) stress and the ISR, enhanced expression of a reporter bearing the Adipoq 5'-leader; polysome profiling verified translation-stimulation. TG-stimulated translation was absent in cells defective in Ser51 phosphorylation of eukaryotic initiation factor 2α (eIF2α), required for the ISR. To determine its role in expression and function of endogenous adiponectin, the upstream uORF was disrupted by CRISPR-Cas9-mediated mutagenesis of differentiated mouse 3T3-L1 adipocytes. uORF disruption in adipocytes increased adiponectin expression, triacylglycerol accumulation, and glucose uptake, and inhibited paracrine muscle and liver cell expression of gluconeogenic enzymes, establishing an important role of the uORF in adiponectin-mediated responses to stress.
Asunto(s)
Adipocitos , Adiponectina , Animales , Ratones , Adiponectina/genética , Sistemas de Lectura Abierta , Células 3T3-L1 , Transporte Biológico , MamíferosRESUMEN
Metabolic pathways that contribute to adiposity and ageing are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting that there are additional downstream effectors of this pathway. Here we identify glutamyl-prolyl-tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to adiposity and ageing. Phosphorylation of EPRS at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex, which is required for execution of noncanonical functions of EPRS beyond protein synthesis. To investigate the physiological function of EPRS phosphorylation, we generated Eprs knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, similar to S6K1-deficient mice and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent. Substitution of the EprsS999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating that EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the multisynthetase complex. Interaction screening revealed that phospho-EPRS binds SLC27A1 (that is, fatty acid transport protein 1, FATP1), inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors that are critical for metabolic phenotypes.
Asunto(s)
Adiposidad , Aminoacil-ARNt Sintetasas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Adipocitos/metabolismo , Envejecimiento/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Animales , Peso Corporal , Membrana Celular/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Femenino , Insulina/metabolismo , Longevidad/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Mutación , Tamaño de los Órganos , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Transporte de Proteínas , Proteína Reguladora Asociada a mTOR , Proteínas Quinasas S6 Ribosómicas 90-kDa/deficienciaRESUMEN
LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic de novo L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.
Asunto(s)
Proteínas de Ciclo Celular/genética , Interferón gamma/genética , Elementos de Nucleótido Esparcido Largo/genética , Proteínas Nucleares/genética , Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Genoma Humano , Humanos , Factor 3 de Genes Estimulados por el Interferón/genética , Complejos Multiproteicos/genética , Unión Proteica , Inhibidores de la Síntesis de la Proteína , ARN Mensajero/genética , Retroelementos/genéticaRESUMEN
Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Caspasas/metabolismo , Aminoacil-ARNt Sintetasas/química , Dominio Catalítico , Glutamato-ARNt Ligasa/química , Glutamato-ARNt Ligasa/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , ProteolisisRESUMEN
Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population.
Asunto(s)
Apoptosis , Proteínas Bacterianas/metabolismo , Enzimas de Restricción del ADN/metabolismo , Escherichia coli/enzimología , Adaptación Fisiológica , Proteínas Bacterianas/genética , Western Blotting , Muerte Celular , Enzimas de Restricción del ADN/genética , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Viabilidad Microbiana , Microscopía Confocal , Densidad de Población , Crecimiento Demográfico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Mycobacterium tuberculosis (Mtb) is a formidable pathogen which has the ability to survive the hostile environment of the host by evading the host defense system. The re-configuration of its transcriptional and metabolic process allows the pathogen to confront the adverse environment within the host macrophages. The factors that assist the transcription and modulate the DNA topology would have to play a key role in the regulation of global gene expression of the organism. How transcription of these essential housekeeping genes alters in response to growth conditions and environmental stress has not been addressed together in a set of experimental conditions in Mtb. Now, we have mapped the transcription start sites (TSS) and promoters of several genes that play a central role in the regulation of DNA topology and transcription in Mtb. Using in vivo reporter assays, we validated the activity of the identified promoter elements in different growth conditions. The variation in transcript abundance of these essential genes was also analyzed in growth phase-dependent manner. These data provide the first glimpse into the specific adaptive changes in the expression of genes involved in transcription and DNA topology modulation in Mtb.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Humanos , Mycobacterium tuberculosis/crecimiento & desarrollo , Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg(2+) ion higher than 500 µM mediates promiscuous activity, Ca(2+) suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca(2+)-mediated cleavage but imparting high cleavage fidelity with Mg(2+). High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca(2+)-mediated cleavage activity by altering the geometry of the Ca(2+)-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.
Asunto(s)
División del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/química , Calcio/química , Dominio Catalítico , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Magnesio/química , Simulación de Dinámica Molecular , Mutación , Especificidad por SustratoRESUMEN
Most bacterial genomes harbor restriction-modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host-parasite coevolutionary "arms race."
Asunto(s)
Bacteriófagos/metabolismo , Evolución Biológica , Enzimas de Restricción-Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Aptitud Genética/genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/virología , Bacteriófagos/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Interacciones Huésped-Patógeno , Oligonucleótidos/genéticaRESUMEN
Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.
Asunto(s)
Adenosina , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Homocigoto , Metiltransferasas , Mutación Missense , ARN Mensajero , Femenino , Humanos , Masculino , Adenosina/análogos & derivados , Adenosina/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas del Tejido Nervioso , Factores de Empalme de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3'-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3'-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3'-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality.
Asunto(s)
ARN Viral , Regulón , SARS-CoV-2 , ARN Subgenómico , Humanos , COVID-19/genética , Regulón/genética , ARN Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Virales/metabolismo , ARN Subgenómico/genéticaRESUMEN
Most of the restriction endonucleases (REases) are dependent on Mg(2+) for DNA cleavage, and in general, Ca(2+) inhibits their activity. R.KpnI, an HNH active site containing ßßα-Me finger nuclease, is an exception. In presence of Ca(2+), the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg(2+)-induced promiscuous activity. To elucidate the mechanism of unusual Ca(2+)-mediated activity, we generated alanine variants in the putative Ca(2+) binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca(2+). We demonstrate that ExDxD residues are involved in Ca(2+) coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca(2+)-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca(2+) binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg(2+), the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca(2+) binding motif that confers site specific cleavage upon Ca(2+) binding is also critical for the promiscuous activity of the Mg(2+)-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.
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Calcio/farmacología , División del ADN/efectos de los fármacos , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencias de Aminoácidos/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/efectos de los fármacos , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Cinética , Klebsiella pneumoniae/enzimología , Magnesio/farmacología , Especificidad por SustratoRESUMEN
Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, 'alt-proteins' lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.
RESUMEN
Increasing evidence suggests that intratumoral inflammation has an outsized influence on antitumor immunity. Here, we report that IL-17, a proinflammatory cytokine widely associated with poor prognosis in solid tumors, drives the therapeutic failure of anti-PD-L1. By timing the deletion of IL-17 signaling specifically in cancer-associated fibroblasts (CAFs) in late-stage tumors, we show that IL-17 signaling drives immune exclusion by activating a collagen deposition program in murine models of cutaneous squamous cell carcinoma (cSCC). Ablation of IL-17 signaling in CAFs increased the infiltration of cytotoxic T cells into the tumor mass and sensitized otherwise resistant cSCC to anti-PD-L1 treatment. Mechanistically, the collagen deposition program in CAFs was driven by IL-17-induced translation of HIF1α, which was mediated by direct binding of Act1, the adaptor protein of IL-17 receptor, to a stem-loop structure in the 3' untranslated region (UTR) in Hif1α mRNA. Disruption of Act1's binding to Hif1α mRNA abolished IL-17-induced collagen deposition and enhanced anti-PD-L1-mediated tumor regression.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma de Células Escamosas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Interleucina-17 , Neoplasias Cutáneas , Animales , Antígeno B7-H1/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-17/metabolismo , Ratones , ARN Mensajero , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
Restriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation.
Asunto(s)
Enzimas de Restricción del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Mutación Puntual , Sitios de Unión , ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Iones , Cinética , Magnesio/química , Metales/química , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Recombinación GenéticaRESUMEN
CRISPR-Cas9-mediated, site-directed mutagenesis in mice generates mosaic founder mice with varied efficiency of desired point mutation and other non-homologous end-joined variants. Here, we present a protocol for design, sample preparation, and analysis for identification of mice with the desired mutation. Deep sequencing provides the proportion of reads of a particular allele for each mouse line. Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021).