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1.
Nucleic Acids Res ; 47(19): 10414-10425, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665744

RESUMO

Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.


Assuntos
GTP Fosfo-Hidrolases/química , RNA Ribossômico/química , Proteínas Ribossômicas/química , Bacillus subtilis/química , Bacillus subtilis/genética , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/ultraestrutura , Hidrólise , Modelos Moleculares , Conformação Proteica , RNA Ribossômico/genética , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Ribossomos/genética , Ribossomos/ultraestrutura
2.
Nat Neurosci ; 22(10): 1696-1708, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31551601

RESUMO

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.


Assuntos
Envelhecimento/genética , Encéfalo/crescimento & desenvolvimento , Neurônios/fisiologia , Análise de Célula Única , Transcriptoma/genética , Animais , Encéfalo/citologia , Comunicação Celular/genética , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ribossomos/genética
3.
Nucleic Acids Res ; 47(19): 10400-10413, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501867

RESUMO

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.


Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Estabilidade Proteica , Ribonucleases/química , Sequência de Aminoácidos/genética , Toxinas Bacterianas/genética , Códon/química , Códon/genética , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Resposta ao Choque Térmico/genética , Estabilidade de RNA/genética , RNA Mensageiro , RNA Ribossômico 16S/genética , Ribonucleases/genética , Ribossomos/química , Ribossomos/genética
4.
Parasitol Res ; 118(10): 2789-2800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485863

RESUMO

An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric (TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon (Abbottina rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n=5m + 3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences (TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.


Assuntos
Cestoides/genética , Cromossomos/genética , DNA Ribossômico/genética , Telômero/genética , Animais , Cestoides/classificação , Cestoides/isolamento & purificação , China , Cyprinidae/genética , DNA de Helmintos/genética , Proteínas de Helminto/genética , Heterocromatina/genética , Hibridização in Situ Fluorescente , Cariótipo , Cariotipagem , Ribossomos/genética
5.
Nat Commun ; 10(1): 3920, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477696

RESUMO

Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome-mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , Ribossomos/química , Ribossomos/genética
6.
Mol Cell ; 75(6): 1117-1130.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31400849

RESUMO

Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Elongação Traducional da Cadeia Peptídica/fisiologia , Dobramento de Proteína , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP70/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Nucleic Acids Res ; 47(17): 9243-9258, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31410471

RESUMO

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.


Assuntos
Proteínas de Drosophila/genética , Fatores de Terminação de Peptídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Sequência de Aminoácidos/genética , Animais , Códon sem Sentido/genética , Códon de Terminação/genética , Drosophila/genética , Neurospora/genética
8.
Nat Neurosci ; 22(10): 1709-1717, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451803

RESUMO

Nervous system function relies on complex assemblies of distinct neuronal cell types that have unique anatomical and functional properties instructed by molecular programs. Alternative splicing is a key mechanism for the expansion of molecular repertoires, and protein splice isoforms shape neuronal cell surface recognition and function. However, the logic of how alternative splicing programs are arrayed across neuronal cells types is poorly understood. We systematically mapped ribosome-associated transcript isoforms in genetically defined neuron types of the mouse forebrain. Our dataset provides an extensive resource of transcript diversity across major neuron classes. We find that neuronal transcript isoform profiles reliably distinguish even closely related classes of pyramidal cells and inhibitory interneurons in the mouse hippocampus and neocortex. These highly specific alternative splicing programs selectively control synaptic proteins and intrinsic neuronal properties. Thus, transcript diversification via alternative splicing is a central mechanism for the functional specification of neuronal cell types and circuits.


Assuntos
Processamento Alternativo/genética , Neurônios/fisiologia , Ribossomos/genética , Transcrição Genética/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neurônios/classificação , Terminações Pré-Sinápticas/metabolismo , Prosencéfalo/citologia , Isoformas de Proteínas/genética , Células Piramidais/fisiologia
9.
Nucleic Acids Res ; 47(18): 9857-9870, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400119

RESUMO

Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.


Assuntos
Pareamento de Bases/genética , Guanosina/análogos & derivados , Conformação de Ácido Nucleico , Ribossomos/genética , Antibacterianos/farmacologia , Anticódon/química , Anticódon/genética , Dano ao DNA/genética , Escherichia coli/genética , Guanina/química , Guanosina/química , Guanosina/genética , Mutação/efeitos dos fármacos , Oxirredução , RNA Mensageiro/genética , RNA de Transferência , Aminoacil-RNA de Transferência/efeitos dos fármacos , Ribossomos/química
10.
PLoS Comput Biol ; 15(8): e1006979, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31369559

RESUMO

Regulation and maintenance of protein synthesis are vital to all organisms and are thus key targets of attack and defense at the cellular level. Here, we mathematically analyze protein synthesis for its sensitivity to the inhibition of elongation factor EF-Tu and/or ribosomes in dependence of the system's tRNA and codon compositions. We find that protein synthesis reacts ultrasensitively to a decrease in the elongation factor's concentration for systems with an imbalance between codon usages and tRNA concentrations. For well-balanced tRNA/codon compositions, protein synthesis is impeded more effectively by the inhibition of ribosomes instead of EF-Tu. Our predictions are supported by re-evaluated experimental data as well as by independent computer simulations. Not only does the described ultrasensitivity render EF-Tu a distinguished target of protein synthesis inhibiting antibiotics. It may also enable persister cell formation mediated by toxin-antitoxin systems. The strong impact of the tRNA/codon composition provides a basis for tissue-specificities of disorders caused by mutations of human mitochondrial EF-Tu as well as for the potential use of EF-Tu targeting drugs for tissue-specific treatments.


Assuntos
Códon/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA de Transferência/genética , Códon/metabolismo , Biologia Computacional , Simulação por Computador , Humanos , Modelos Biológicos , Mutação , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Fator Tu de Elongação de Peptídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Ribossomos/metabolismo
11.
Mol Cell ; 75(5): 996-1006.e8, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31377116

RESUMO

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.


Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae
12.
Mol Cell ; 75(6): 1256-1269.e7, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31378463

RESUMO

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.


Assuntos
Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Conformação de Ácido Nucleico , RNA Fúngico/metabolismo , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Chaetomium/genética , Proteínas Fúngicas/genética , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Ribossomos/genética
13.
Nucleic Acids Res ; 47(17): 9358-9367, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31392980

RESUMO

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5' mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.


Assuntos
Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/genética , Ribossomos/genética , Regiões 5' não Traduzidas/genética , Códon/genética , Regulação da Expressão Gênica/genética , Fases de Leitura Aberta/genética , Sequências Reguladoras de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética
14.
Nucleic Acids Res ; 47(16): 8649-8661, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392997

RESUMO

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.


Assuntos
Lycopersicon esculentum/genética , Doenças das Plantas/genética , Biossíntese de Proteínas , RNA de Plantas/genética , RNA Ribossômico 18S/genética , Ribossomos/metabolismo , Viroides/genética , Citrus/virologia , Interações Hospedeiro-Patógeno/genética , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/virologia , Biogênese de Organelas , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Interferência de RNA , RNA de Plantas/antagonistas & inibidores , RNA de Plantas/metabolismo , RNA Ribossômico 18S/antagonistas & inibidores , RNA Ribossômico 18S/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Estresse Fisiológico/genética , Viroides/metabolismo , Viroides/patogenicidade
15.
Mol Cell ; 75(6): 1270-1285.e14, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31351877

RESUMO

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduces DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi.


Assuntos
Neoplasias da Mama/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Neoplásico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribossomos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , RNA Helicases DEAD-box/genética , Reparo do DNA , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , RNA Neoplásico/genética , RNA Nucleolar Pequeno/genética , Ribossomos/genética
16.
Mol Cell ; 75(3): 427-441.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353208

RESUMO

The translation machinery and the genes it decodes co-evolved to achieve production throughput and accuracy. Nonetheless, translation errors are frequent, and they affect physiology and protein evolution. Mapping translation errors in proteomes and understanding their causes is hindered by lack of a proteome-wide experimental methodology. We present the first methodology for systematic detection and quantification of errors in entire proteomes. Following proteome mass spectrometry, we identify, in E. coli and yeast, peptides whose mass indicates specific amino acid substitutions. Most substitutions result from codon-anticodon mispairing. Errors occur at sites that evolve rapidly and that minimally affect energetic stability, indicating selection for high translation fidelity. Ribosome density data show that errors occur at sites where ribosome velocity is higher, demonstrating a trade-off between speed and accuracy. Treating bacteria with an aminoglycoside antibiotic or deprivation of specific amino acids resulted in particular patterns of errors. These results reveal a mechanistic and evolutionary basis for translation fidelity.


Assuntos
Substituição de Aminoácidos/genética , Biossíntese de Proteínas , Proteoma/genética , Seleção Genética , Aminoácidos/genética , Anticódon/genética , Códon/genética , Escherichia coli/genética , RNA de Transferência/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética
17.
Nucleic Acids Res ; 47(15): 8111-8125, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31340039

RESUMO

It has been a long debate whether the 98% 'non-coding' fraction of human genome can encode functional proteins besides short peptides. With full-length translating mRNA sequencing and ribosome profiling, we found that up to 3330 long non-coding RNAs (lncRNAs) were bound to ribosomes with active translation elongation. With shotgun proteomics, 308 lncRNA-encoded new proteins were detected. A total of 207 unique peptides of these new proteins were verified by multiple reaction monitoring (MRM) and/or parallel reaction monitoring (PRM); and 10 new proteins were verified by immunoblotting. We found that these new proteins deviated from the canonical proteins with various physical and chemical properties, and emerged mostly in primates during evolution. We further deduced the protein functions by the assays of translation efficiency, RNA folding and intracellular localizations. As the new protein UBAP1-AST6 is localized in the nucleoli and is preferentially expressed by lung cancer cell lines, we biologically verified that it has a function associated with cell proliferation. In sum, we experimentally evidenced a hidden human functional proteome encoded by purported lncRNAs, suggesting a resource for annotating new human proteins.


Assuntos
Biossíntese de Proteínas , Proteoma/genética , Proteômica/métodos , RNA Longo não Codificante/genética , Células A549 , Animais , Linhagem Celular Tumoral , Evolução Molecular , Perfilação da Expressão Gênica/métodos , Código Genético , Humanos , Fases de Leitura Aberta/genética , Peptídeos/genética , Primatas/genética , Proteoma/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Ribossomos/genética
18.
Nucleic Acids Res ; 47(16): 8807-8820, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31299085

RESUMO

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação Fúngica da Expressão Gênica , Terminação Traducional da Cadeia Peptídica , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Clonagem Molecular , Códon/química , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lisina/metabolismo , Modelos Moleculares , Príons/química , Príons/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
19.
EMBO J ; 38(16): e100727, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31330067

RESUMO

Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.


Assuntos
Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/genética , Biossíntese de Proteínas , Proteínas Argonauta/química , Autoantígenos/metabolismo , Sítios de Ligação , Códon de Terminação , Fatores de Iniciação em Eucariotos/química , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais
20.
Nat Protoc ; 14(8): 2279-2317, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332354

RESUMO

A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor's nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8-10 working days, and initial data analysis can be completed within 1-2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.


Assuntos
Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos , RNA Mensageiro , Ribossomos , Saccharomyces cerevisiae , Biblioteca Gênica , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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