RESUMEN
Repeat-associated non-AUG (RAN) translation is a pathogenic mechanism in which repetitive sequences are translated into aggregation-prone proteins from multiple reading frames, even without a canonical AUG start codon. Since its discovery in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1), RAN translation is now known to occur in the context of 12 disease-linked repeat expansions. This review discusses recent advances in understanding the regulatory mechanisms controlling RAN translation and its contribution to the pathophysiology of repeat expansion diseases. We discuss the key findings in the context of Fragile X Tremor Ataxia Syndrome (FXTAS), a neurodegenerative disorder caused by a CGG repeat expansion in the 5' untranslated region of FMR1.
Asunto(s)
Síndrome del Cromosoma X Frágil , Enfermedades Neurodegenerativas , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Ataxia/metabolismo , Ataxia/patología , Temblor/genética , Temblor/metabolismo , Temblor/patologíaRESUMEN
BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. OBJECTIVES: We investigated whether (AGAGGG)n repeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. METHODS: XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR. RESULTS: We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells. CONCLUSIONS: In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Asunto(s)
Trastornos Distónicos , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Células HEK293 , Trastornos Distónicos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Intrones , Proteína C9orf72/genéticaRESUMEN
Specific interaction between the start codon, 5'-AUG-3', and the anticodon, 5'-CAU-3', ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.
Asunto(s)
Codón Iniciador/genética , Codón Iniciador/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Emparejamiento Base , Secuencia de Bases , Codón Iniciador/química , Biología Computacional , Células Eucariotas/metabolismo , Modelos Biológicos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , TermodinámicaRESUMEN
The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.
Asunto(s)
Motivos de Nucleótidos , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Acetiltransferasas/metabolismo , Regulación Fúngica de la Expresión Génica , Histidina/metabolismo , Sistemas de Lectura Abierta , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/genética , eIF-2 Quinasa/metabolismoRESUMEN
During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.
Asunto(s)
Factor de Transcripción Activador 4/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factor 2 Eucariótico de Iniciación/genética , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Aminoácidos/genética , Animales , Regulación Fúngica de la Expresión Génica , Humanos , Sistemas de Lectura Abierta/genética , Fosforilación , ARN Mensajero/genética , Saccharomyces cerevisiae/genéticaRESUMEN
In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.
Asunto(s)
Codón Iniciador/genética , Proteínas de Unión al ADN/genética , Factor 5 Eucariótico de Iniciación/genética , Genoma Humano , Animales , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Codón Iniciador/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 5 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Homeostasis/genética , Humanos , Unión Proteica , Biosíntesis de Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 5 Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Drosophila melanogaster/metabolismo , Factor 3 de Iniciación Eucariótica , Fibrosarcoma/patología , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Masculino , Espectrometría de Masas , Ratones DesnudosRESUMEN
Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2ß was measured as being equivalent to the published value of human eIF5 to eIF2ß, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes.
Asunto(s)
Factor de Transcripción Activador 4/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Factor de Transcripción Activador 4/biosíntesis , Animales , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/fisiología , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 5 Eucariótico de Iniciación/metabolismo , Humanos , Proteínas de Insectos/metabolismo , Sistemas de Lectura Abierta , Filogenia , Proteína Fosfatasa 1/metabolismo , Saccharomyces cerevisiae/metabolismo , Tribolium/enzimología , Tribolium/genética , Tribolium/crecimiento & desarrolloRESUMEN
The adaptive biasing force (ABF) technique allows sampling to proceed in a flat free energy surface when performing molecular dynamics (MD) simulations. Here, we present a protocol to perform MD simulations using the ABF technique and apply it to calculate the binding free energy of an RNA:RNA interaction. We describe steps for server setup, test running software, and building molecular models. We then detail procedures for running and configuring ABF-MD simulations and analyzing binding free energy and structural change. For complete details on the use and execution of this protocol, please refer to Fujita et al.1 and Kameda et al.2.
RESUMEN
Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.
Asunto(s)
Factor 1 Eucariótico de Iniciación/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Factor 1 Eucariótico de Iniciación/química , Factor 1 Eucariótico de Iniciación/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/genética , Unión Proteica , Biosíntesis de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Línea Celular , Factor 2 Eucariótico de Iniciación/análisis , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Ratones , Imitación Molecular , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated proteins in cultured human and fly cells. We detail one-step anti-FLAG affinity purification and whole-lane mass spectrometry analysis of samples resolved by SDS-PAGE. This protocol allows for quantitative evaluation of the effect of 5MP mutations on its molecular interactions, to elucidate translational control by 5MP. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Humanos , Ribosomas/metabolismo , Proteínas/metabolismo , Citosol/metabolismo , Espectrometría de MasasRESUMEN
eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
Asunto(s)
Factor 5 Eucariótico de Iniciación , Enfermedades Neurodegenerativas , Calorimetría , Factor 2 Eucariótico de Iniciación , Factor 3 de Iniciación Eucariótica , Humanos , InterferometríaRESUMEN
In contrast to prokaryotes wherein GUG and UUG are permissive start codons, initiation frequencies from non-AUG codons are generally low in eukaryotes, with CUG being considered as strongest. Here, we report that combined 5-cytosine methylation (5mC) and pseudouridylation (Ψ) of near-cognate non-AUG start codons convert GUG and UUG initiation strongly favored over CUG initiation in eukaryotic translation under a certain context. This prokaryotic-like preference is attributed to enhanced NUG initiation by Ψ in the second base and reduced CUG initiation by 5mC in the first base. Molecular dynamics simulation analysis of tRNAiMet anticodon base pairing to the modified codons demonstrates that Ψ universally raises the affinity of codon:anticodon pairing within the ribosomal preinitiation complex through partially mitigating discrimination against non-AUG codons imposed by eukaryotic initiation factor 1. We propose that translational control by chemical modifications of start codon bases can offer a new layer of proteome diversity regulation and therapeutic mRNA technology.
RESUMEN
Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.
Asunto(s)
Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Saccharomyces cerevisiae/enzimología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Codón Iniciador/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Mutación/genética , Sistemas de Lectura Abierta/genética , Péptidos , Unión Proteica , Estructura Terciaria de Proteína , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA(i)(Met), show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.
Asunto(s)
Codón Iniciador/metabolismo , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , ARN de Hongos , ARN Ribosómico 18S , Subunidades Ribosómicas Pequeñas de Eucariotas , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , ARN Ribosómico 18S/química , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.
Asunto(s)
Codón Iniciador/genética , Proteínas de Unión al ADN/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Biosíntesis de Proteínas/genética , Expansión de Repetición de Trinucleótido/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Proteínas de Unión al ADN/química , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/química , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Iniciación de la Cadena Peptídica Traduccional , Unión Proteica , Dominios Proteicos , Receptores Inmunológicos/metabolismoRESUMEN
BACKGROUND: Translational reprogramming through controlled initiation from non-AUG start codons is considered a crucial driving force in tumorigenesis and tumor progression. However, its clinical impact and underlying mechanism are not fully understood. METHODS: Using a bioinformatics approach, we identified translation initiation regulator 5MP1/BZW2 on chromosome 7p as a potential oncogenic driver gene in colorectal cancer (CRC), and explored the biological effect of 5MP1 in CRC in vitro or in vivo. Pathway analysis was performed to identify the downstream target of 5MP1, which was verified with transcriptomic and biochemical analyses. Finally, we assessed the clinical significance of 5MP1 expression in CRC patients. FINDINGS: 5MP1 was ubiquitously amplified and overexpressed in CRC. 5MP1 promoted tumor growth and induced cell cycle progression of CRC. c-Myc was identified as its potential downstream effector. c-Myc has two in-frame start codons, AUG and CUG (non-AUG) located upstream of the AUG. 5MP1 expression increased the AUG-initiated c-Myc isoform relative to the CUG-initiated isoform. The AUG-initiated c-Myc isoform displayed higher protein stability and a stronger transactivation activity for oncogenic pathways than the CUG-initiated isoform, accounting for 5MP1-driven cell cycle progression and tumor growth. Clinically, high 5MP1 expression predicts poor survival of CRC patients. INTERPRETATION: 5MP1 is a novel oncogene that reprograms c-Myc translation in CRC. 5MP1 could be a potential therapeutic target to overcome therapeutic resistance conferred by tumor heterogeneity of CRC. FUND: Japan Society for the Promotion of Science; Priority Issue on Post-K computer; National Institutes of Health; National Science Foundation; KSU Johnson Cancer Center.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Cromosomas Humanos Par 7 , Codón Iniciador , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Amplificación de Genes , Técnicas de Inactivación de Genes , Humanos , Ratones , Persona de Mediana Edad , Oncogenes , Pronóstico , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).