RESUMO
The regulation of gene expression is fundamental for life. Whereas the role of transcriptional regulation of gene expression has been studied for several decades, it has been clear over the past two decades that post-transcriptional regulation of gene expression, of which translation regulation is a major part, can be equally important. Translation can be divided into four main stages: initiation, elongation, termination and ribosome recycling. Translation is controlled mainly during its initiation, a process which culminates in a ribosome positioned with an initiator tRNA over the start codon and, thus, ready to begin elongation of the protein chain. mRNA translation has emerged as a powerful tool for the development of innovative therapies, yet the detailed mechanisms underlying the complex process of initiation remain unclear. Recent studies in yeast and mammals have started to shed light on some previously unclear aspects of this process. In this Review, we discuss the current state of knowledge on eukaryotic translation initiation and its regulation in health and disease. Specifically, we focus on recent advances in understanding the processes involved in assembling the 43S pre-initiation complex and its recruitment by the cap-binding complex eukaryotic translation initiation factor 4F (eIF4F) at the 5' end of mRNA. In addition, we discuss recent insights into ribosome scanning along the 5' untranslated region of mRNA and selection of the start codon, which culminates in joining of the 60S large subunit and formation of the 80S initiation complex.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Ribossomos , Animais , Códon de Iniciação/genética , Códon de Iniciação/análise , Códon de Iniciação/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Biossíntese de Proteínas/genética , Mamíferos/genéticaRESUMO
mRNA translation relies on identifying translation initiation sites (TISs) in mRNAs. Alternative TISs are prevalent across plant transcriptomes, but the mechanisms for their recognition are unclear. Using ribosome profiling and machine learning, we developed models for predicting alternative TISs in the tomato (Solanum lycopersicum). Distinct feature sets were predictive of AUG and nonAUG TISs in 5' untranslated regions and coding sequences, including a novel CU-rich sequence that promoted plant TIS activity, a translational enhancer found across dicots and monocots, and humans and viruses. Our results elucidate the mechanistic and evolutionary basis of TIS recognition, whereby cis-regulatory RNA signatures affect start site selection. The TIS prediction model provides global estimates of TISs to discover neglected protein-coding genes across plant genomes. The prevalence of cis-regulatory signatures across plant species, humans, and viruses suggests their broad and critical roles in reprogramming the translational landscape.
Assuntos
Eucariotos , Iniciação Traducional da Cadeia Peptídica , Humanos , Iniciação Traducional da Cadeia Peptídica/genética , Eucariotos/genética , Plantas/genética , Regiões 5' não Traduzidas , RNA Mensageiro/genética , Códon de IniciaçãoRESUMO
The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo. Ribosome profiling of tma deletion strains revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3' UTR, as evidenced by peaks in the footprint data and 3' UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream open reading frames (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an 80S reinitiation process in 3' UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in recycling 40S ribosomal subunits at stop codons and translation reinitiation.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Subunidades Ribossômicas Menores de Eucariotos/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas/genética , Códon de Terminação/genética , Escherichia coli/genética , Fases de Leitura Aberta/genética , Iniciação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genéticaRESUMO
The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.
Assuntos
Regiões 5' não Traduzidas/genética , Códon de Iniciação/genética , RNA Helicases DEAD-box/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Reagentes de Ligações Cruzadas/química , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.
Assuntos
Proteínas de Ligação a DNA/genética , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Códon de Iniciação/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Células HaCaT , Células HeLa , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogênicas Virais/biossíntese , Iniciação Traducional da Cadeia Peptídica/genética , RNA Viral/genética , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.
Assuntos
COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Pandemias , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/genéticaRESUMO
The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNAcg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNAcg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria.
Assuntos
Escherichia coli , RNA de Transferência de Metionina , Escherichia coli/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Subunidades Proteicas , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.
Assuntos
Regiões 5' não Traduzidas/genética , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Fases de Leitura Aberta/genética , Iniciação Traducional da Cadeia Peptídica/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Animais , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Epiderme/embriologia , Epiderme/metabolismo , Epiderme/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Humanos , Queratinócitos , Masculino , Camundongos , Oncogenes/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Prognóstico , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.
Assuntos
Sequências Repetidas Invertidas/genética , Iniciação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Trifosfato de Adenosina/metabolismo , Arsenitos/toxicidade , Pareamento de Bases/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Ribossomos/metabolismoRESUMO
Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.
Assuntos
RNA Helicases DEAD-box/genética , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Códon de Iniciação/genética , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Fases de Leitura Aberta/genética , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Saccharomyces cerevisiae/genéticaRESUMO
Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of glycyl-tRNAGly available for translation and inhibit the first stage of elongation, the accommodation of glycyl-tRNA into the ribosomal A-site, which causes ribosomes to pause at glycine codons. Moreover, ribosome pausing activates a secondary repression mechanism at the level of translation initiation, by inducing the phosphorylation of the alpha subunit of eIF2 and the integrated stress response. Thus, CMT-GARS mutant triggers translational repression via two interconnected mechanisms, affecting both elongation and initiation of translation.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Glicina-tRNA Ligase/genética , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Mutação com Ganho de Função/genética , Expressão Gênica/genética , Glicina/genética , Células HEK293 , Humanos , Fosforilação , Biossíntese de Proteínas/genética , RNA de Transferência de Glicina/genéticaRESUMO
Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377-448) whereas linker corresponds to the long α-helix (aa 460-512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90-166) that constitutes a previously unassigned section of the main density and resides between DHX29's C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29's function.
Assuntos
Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genética , RNA Helicases/genética , RNA Mensageiro/genética , Ribossomos/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Microscopia Crioeletrônica , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestruturaRESUMO
Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Ferro/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas/metabolismoRESUMO
The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas Ribossômicas/genética , Escherichia coli/metabolismo , Humanos , Conformação Proteica , RNA Ribossômico 16S/genética , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.
Assuntos
Vírus da Febre Aftosa/genética , Regulação Viral da Expressão Gênica/genética , Sítios Internos de Entrada Ribossomal/genética , Iniciação Traducional da Cadeia Peptídica/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Proliferação de Células/genética , Chlorocebus aethiops , Vírus da Febre Suína Clássica/genética , Cricetinae , Vírus da Febre Aftosa/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Picornaviridae/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Suínos , Células Vero , Replicação Viral/genética , NucleolinaRESUMO
Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.
Assuntos
Adenosina/análogos & derivados , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas/genética , Adenosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Códon de Iniciação/genética , Sequência Conservada , Epigênese Genética , Evolução Molecular , Sequência Rica em GC/genética , Humanos , Metilação , Camundongos , Especificidade de Órgãos , Iniciação Traducional da Cadeia Peptídica/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae , Transcriptoma/genéticaRESUMO
High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.
Assuntos
Códon/metabolismo , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas/genética , Códon/genética , Haloferax volcanii/efeitos dos fármacos , Harringtoninas/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/efeitos dos fármacos , Pegadas de Proteínas , Fases de Leitura/genética , Ribossomos/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
Ribosome profiling and mass spectroscopy have identified canonical and noncanonical translation initiation codons (TICs) that are upstream of the main translation initiation site and used to translate oncogenic proteins. There have previously been conflicting reports about the patterns of nucleotides that surround noncanonical TICs. Here, we use a Kozak Similarity Score algorithm to find that nearly all of these TICs have flanking nucleotides closely matching the Kozak sequence. Remarkably, the nucleotides flanking alternative noncanonical TICs are frequently closer to the Kozak sequence than the nucleotides flanking TICs used to translate the gene's main protein. Of note, the 5' untranslated region (5'UTR) of cancer-associated genes with an upstream TIC tend to be significantly longer than the same region in genes not associated with cancer. The presence of a longer-than-typical 5'UTR increases the likelihood of ribosome binding to upstream noncanonical TICs, and may be a distinguishing feature of a number of genes overexpressed in cancer. Noncanonical TICs that are located in the 5'UTR, although thought by some to be disadvantageous and suppressed by evolution, may translate oncogenic proteins because of their flanking nucleotides.
Assuntos
Neoplasias , Regiões 5' não Traduzidas/genética , Algoritmos , Códon/genética , Códon de Iniciação/genética , Humanos , Neoplasias/genética , Nucleotídeos , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genéticaRESUMO
CD4 expression identifies a subset of mature T cells primarily assisting the germinal center reaction and contributing to CD8+ T-cell and B-cell activation, functions, and longevity. Herein, we present a family in which a novel variant disrupting the translation-initiation codon of the CD4 gene resulted in complete loss of membrane and plasma soluble CD4 in peripheral blood, lymph node, bone marrow, skin, and ileum of a homozygous proband. This inherited CD4 knockout disease illustrates the clinical and immunological features of a complete deficiency of any functional component of CD4 and its similarities and differences with other clinical models of primary or acquired loss of CD4+ T cells. The first inherited loss of any functional component of CD4, including soluble CD4, is clinically distinct from any other congenital or acquired CD4 T-cell defect and characterized by compensatory changes in T-cell subsets and functional impairment of B cells, monocytes, and natural killer cells.
Assuntos
Antígenos CD4/deficiência , Antígenos CD4/genética , Síndromes de Imunodeficiência/genética , Iniciação Traducional da Cadeia Peptídica/genética , Doenças da Imunodeficiência Primária/genética , Medula Óssea/imunologia , Medula Óssea/metabolismo , Antígenos CD4/análise , Antígenos CD4/sangue , Linfócitos T CD4-Positivos/imunologia , Códon de Iniciação , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Humanos , Íleo/imunologia , Íleo/metabolismo , Imunidade Inata , Síndromes de Imunodeficiência/imunologia , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Masculino , Monócitos/imunologia , Mutação de Sentido Incorreto , Linhagem , Doenças da Imunodeficiência Primária/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.