RESUMO
Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.
Assuntos
Proteínas Reguladoras de Apoptose , Microscopia Crioeletrônica , Ligação Proteica , RNA Mensageiro , Proteínas de Ligação a RNA , Subunidades Ribossômicas Menores de Eucariotos , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Sítios de Ligação , Biossíntese de Proteínas , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Modelos MolecularesRESUMO
During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ribossomos/química , Ribossomos/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Modelos Moleculares , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Dobramento de RNA , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , AnimaisRESUMO
Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.
Assuntos
Fator de Iniciação 4E em Eucariotos , Biossíntese de Proteínas , Subunidades Ribossômicas Menores de Eucariotos , Humanos , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fator de Iniciação 4E em Eucariotos/metabolismoRESUMO
The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.
Assuntos
Precursores de RNA , RNA Ribossômico 18S , RNA Nucleolar Pequeno , Subunidades Ribossômicas Menores de Eucariotos , Precursores de RNA/genética , RNA Ribossômico 18S/genética , RNA Nucleolar Pequeno/genética , Saccharomyces cerevisiae/genética , Conformação de Ácido Nucleico , Subunidades Ribossômicas Menores de Eucariotos/genéticaRESUMO
The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.
Assuntos
Proteínas Ribossômicas , Proteínas de Saccharomyces cerevisiae , Humanos , Biossíntese de Proteínas , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Engenharia Genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HCT116 , Células HEK293 , Humanos , Proteólise , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) enzymes have a dual genetic origin. Mechanisms regulating the expression of nucleus-encoded OXPHOS subunits in response to metabolic cues (glucose versus glycerol) are well understood, while the regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3, a DEAD/H box helicase gene, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunits in cells consistent with its role in regulating translation elongation based on the Arg8m reporter system. IRC3-deleted cells retained mitochondrial DNA despite a growth defect on glycerol plates. Glucose-grown Δirc3ρ+ and irc3 temperature-sensitive cells at 37°C have reduced translation rates from the majority of mRNAs. In contrast, when galactose was the carbon source, a reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ+ cells but no defect was observed in irc3 temperature-sensitive cells, at 37°C. In support of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, Δirc3 suppressor strains isolated for restoration of growth on glycerol medium restore mitochondrial protein synthesis differentially in the presence of glucose versus glycerol.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Mitocôndrias/genética , Fosforilação Oxidativa , Elongação Traducional da Cadeia Peptídica/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Mitocondrial/genética , Galactose/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Elongação Traducional da Cadeia Peptídica/fisiologia , RNA Helicases/metabolismo , RNA Mensageiro/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genéticaRESUMO
Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/genéticaRESUMO
Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-ß-cyclodextrin (MßCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Células Endoteliais/microbiologia , Fosfopiruvato Hidratase/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Streptococcus suis/patogenicidade , Animais , Linhagem Celular , Imunofluorescência , Células HEK293 , Humanos , Fosfopiruvato Hidratase/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Sorogrupo , Streptococcus suis/classificação , Streptococcus suis/enzimologia , Fatores de VirulênciaRESUMO
During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.
Assuntos
Adenilato Quinase/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Proteínas Nucleares/genética , Nucleosídeo-Trifosfatase/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Sítios de Ligação , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Biogênese de Organelas , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469â000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.
Assuntos
Interações Hospedeiro-Patógeno/genética , Lisina/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Biologia Computacional/métodos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Lisina/metabolismo , Mutação , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismoRESUMO
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.
Assuntos
COVID-19/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/virologia , SARS-CoV-2/genética , SARS-CoV-2/ultraestrutura , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
Translation in eukaryotes is dependent on the activity of translation initiation factor (eIF) 4G family of proteins, a scaffold protein that, during the initiation step, coordinates the activity of other eIFs to recruit the 40S ribosomal subunit to the mRNA. Three decades of research on protein synthesis and its regulation has provided a wealth of evidence supporting the crucial role of cap-dependent translation initiation, which involves eIF4G. However, the recent discovery of a surprising variety of alternative mechanisms to initiate translation in the absence of eIF4G has stirred the orthodox view of how protein synthesis is performed. These mechanisms involve novel interactions among known eIFs, or between known eIFs and other proteins not previously linked to translation. Thus, a new picture is emerging in which the unorthodox translation initiation complexes contribute to the diversity of mechanisms that regulate gene expression in eukaryotes.
Assuntos
Fator de Iniciação Eucariótico 4G/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Sequência de Aminoácidos/genética , Fator de Iniciação 4E em Eucariotos/genética , Regulação da Expressão Gênica/genética , Iniciação Traducional da Cadeia Peptídica/genéticaRESUMO
PDCD2 is an evolutionarily conserved protein with previously characterized homologs in Drosophila (zfrp8) and budding yeast (Tsr4). Although mammalian PDCD2 is essential for cell proliferation and embryonic development, the function of PDCD2 that underlies its fundamental cellular role has remained unclear. Here, we used quantitative proteomics approaches to define the protein-protein interaction network of human PDCD2. Our data revealed that PDCD2 specifically interacts with the 40S ribosomal protein uS5 (RPS2) and that the PDCD2-uS5 complex is assembled co-translationally. Loss of PDCD2 expression leads to defects in the synthesis of the small ribosomal subunit that phenocopy a uS5 deficiency. Notably, we show that PDCD2 is important for the accumulation of soluble uS5 protein as well as its incorporation into 40S ribosomal subunit. Our findings support that the essential molecular function of PDCD2 is to act as a dedicated ribosomal protein chaperone that recognizes uS5 co-translationally in the cytoplasm and accompanies uS5 to ribosome assembly sites in the nucleus. As most dedicated ribosomal protein chaperones have been identified in yeast, our study reveals that similar mechanisms exist in human cells to assist ribosomal proteins coordinate their folding, nuclear import and assembly in pre-ribosomal particles.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Sequência Conservada/genética , Células HeLa , Humanos , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genéticaRESUMO
Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENRâ¢MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENRâ¢MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENRâ¢MCTS1 dependent manner.
Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genéticaRESUMO
In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.
Assuntos
Arginina/análogos & derivados , Fator de Iniciação 2 em Eucariotos/genética , RNA Mensageiro/genética , Substituição de Aminoácidos/genética , Arginina/genética , Códon de Iniciação/genética , Humanos , Complexos Multiproteicos/genética , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genéticaRESUMO
The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. We observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5' untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.
Assuntos
Betacoronavirus/química , Betacoronavirus/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Regiões 5' não Traduzidas , Betacoronavirus/genética , Microscopia Crioeletrônica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/genéticaRESUMO
rRNA-modifying enzymes participate in ribosome assembly. However, whether the catalytic activities of these enzymes are important for the ribosome assembly and other cellular processes is not fully understood. Here, we report the crystal structure of WT human dimethyladenosine transferase 1 (DIMT1), an 18S rRNA N6,6-dimethyladenosine (m26,6A) methyltransferase, and results obtained with a catalytically inactive DIMT1 variant. We found that DIMT1+/- heterozygous HEK 293T cells have a significantly decreased 40S fraction and reduced protein synthesis but no major changes in m26,6A levels in 18S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WT and DIMT1+/- cells significantly decreased m26,6A levels in 18S rRNA, indicating a dominant-negative effect of this variant on m26,6A levels. However, expression of the DIMT1-E85A variant restored the defects in 40S levels. Of note, unlike WT DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the WT enzyme extended to translation fidelity and gene expression patterns in DNA damage response pathways. These results suggest that the catalytic activity of DIMT1 is involved in protein translation and that the overall protein scaffold of DIMT1, regardless of the catalytic activity on m26,6A in 18S rRNA, is essential for 40S assembly.
Assuntos
Metiltransferases/química , Biossíntese de Proteínas , RNA Ribossômico 18S/química , Substituição de Aminoácidos , Catálise , Cristalografia por Raios X , Células HEK293 , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação de Sentido Incorreto , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S-IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S-IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S-IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S-IRES complex does not occur prematurely.