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1.
Nucleic Acids Res ; 48(10): 5201-5216, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32382758

RESUMO

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 fármacos
2.
Science ; 368(6488)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32299921

RESUMO

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.


Assuntos
Códon , Elongação Traducional da Cadeia Peptídica , Estabilidade de RNA , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Crioeletrônica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
3.
PLoS Comput Biol ; 16(2): e1007618, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32049979

RESUMO

Computational modelling of in vivo protein synthesis is highly complicated, as it requires the simulation of ribosomal movement over the entire transcriptome, as well as consideration of the concentration effects from 40+ different types of tRNAs and numerous other protein factors. Here I report on the development of a stochastic model for protein translation that is capable of simulating the dynamical process of in vivo protein synthesis in a prokaryotic cell containing several thousand unique mRNA sequences, with explicit nucleotide information for each, and report on a number of biological predictions which are beyond the scope of existing models. In particular, I show that, when the complex network of concentration dependent interactions between elongation factors, tRNAs, ribosomes, and other factors required for protein synthesis are included in full detail, several biological phenomena, such as the increasing peptide elongation rate with bacterial growth rate, are predicted as emergent properties of the model. The stochastic model presented here demonstrates the importance of considering the translational process at this level of detail, and provides a platform to interrogate various aspects of translation that are difficult to study in more coarse-grained models.


Assuntos
Simulação por Computador , Ribossomos/metabolismo , Processos Estocásticos , Cinética , Elongação Traducional da Cadeia Peptídica , Reprodutibilidade dos Testes
4.
Nucleic Acids Res ; 48(3): 1043-1055, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31598688

RESUMO

In recent years translation elongation has emerged as an important contributor to the regulation of gene expression. There are multiple quality control checkpoints along the way of producing mature proteins and targeting them to the right cellular compartment, or associating them correctly with their partners. Ribosomes pause to allow co-translational protein folding, protein targeting or protein interactions, and the pausing is dictated by a combination of the mRNA sequence and structure, the tRNA availability and the nascent peptide. However, ribosome pausing can also lead to ribosome collisions and co-translational degradation of both mRNA and nascent chain. Understanding how the translating ribosome tunes the different maturation steps that nascent proteins must undergo, what the timing of these maturation events is, and how degradation can be avoided when pausing is needed, is now possible by the emergence of methods to follow ribosome dynamics in vivo. This review summarizes some of the recent studies that have advanced our knowledge about co-translational events using the power of ribosome profiling, and some of the questions that have emerged from these studies.


Assuntos
Elongação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Regulação da Expressão Gênica , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA Mensageiro/metabolismo
5.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
6.
Proc Natl Acad Sci U S A ; 116(46): 23068-23074, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31672910

RESUMO

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNAPhe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.


Assuntos
Biossíntese de Proteínas , Pseudouridina/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , Thermus thermophilus/genética , Códon/genética , Códon/metabolismo , Fases de Leitura Aberta , Elongação Traducional da Cadeia Peptídica , Pseudouridina/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Uridina/metabolismo
7.
Nature ; 573(7775): 605-608, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31534220

RESUMO

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Enzimática , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Microscopia de Fluorescência , Ligação Proteica , Conformação Proteica , Ribossomos/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
8.
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
9.
EMBO Mol Med ; 11(10): e10018, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31468715

RESUMO

Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end-stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti-hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co-factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy.


Assuntos
Acetiltransferases/metabolismo , Cardiomegalia/fisiopatologia , Elongação Traducional da Cadeia Peptídica , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Miócitos Cardíacos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(30): 15023-15032, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31292258

RESUMO

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.


Assuntos
Elongação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Códon/química , Códon/metabolismo , Marcação por Isótopo , Cinética , Modelos Genéticos , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese
11.
Genome Biol Evol ; 11(9): 2439-2456, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290967

RESUMO

Hepatoviruses show an intriguing deviated codon usage, suggesting an evolutionary signature. Abundant and rare codons in the cellular genome are scarce in the human hepatitis A virus (HAV) genome, while intermediately abundant host codons are abundant in the virus. Genotype-phenotype maps, or fitness landscapes, are a means of representing a genotype position in sequence space and uncovering how genotype relates to phenotype and fitness. Using genotype-phenotype maps of the translation efficiency, we have shown the critical role of the HAV capsid codon composition in regulating translation and determining its robustness. Adaptation to an environmental perturbation such as the artificial induction of cellular shutoff-not naturally occurring in HAV infection-involved movements in the sequence space and dramatic changes of the translation efficiency. Capsid rare codons, including abundant and rare codons of the cellular genome, slowed down the translation efficiency in conditions of no cellular shutoff. In contrast, rare capsid codons that are abundant in the cellular genome were efficiently translated in conditions of shutoff. Capsid regions very rich in slowly translated codons adapt to shutoff through sequence space movements from positions with highly robust translation to others with diminished translation robustness. These movements paralleled decreases of the capsid physical and biological robustness, and resulted in the diversification of capsid phenotypes. The deviated codon usage of extant hepatoviruses compared with that of their hosts may suggest the occurrence of a virus ancestor with an optimized codon usage with respect to an unknown ancient host.


Assuntos
Proteínas do Capsídeo/genética , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Elongação Traducional da Cadeia Peptídica , Adaptação Fisiológica , Proteínas do Capsídeo/metabolismo , Códon , Humanos , Mutação , Biossíntese de Proteínas , Dobramento de Proteína , RNA de Transferência/metabolismo
12.
Biol Trace Elem Res ; 192(1): 18-25, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31342342

RESUMO

The fact that selenocysteine (Sec) is delivered to the elongating ribosome by a tRNA that recognizes a UGA stop codon makes it unique and a thorn in the side of what was originally thought to be a universal genetic code. The mechanism by which this redefinition occurs has been slowly coming to light over the past 30 years, but key questions remain. This review seeks to highlight the prominent mechanistic questions that will guide the direction of work in the near future. These questions arise from two major aspects of Sec incorporation: (1) novel functions for the Sec insertion sequence (SECIS) that resides in all selenoprotein mRNAs and (2) the myriad of RNA-binding proteins, both known and yet to be discovered, that act in concert to modify the translation elongation process to allow Sec incorporation.


Assuntos
Códon de Terminação , Elongação Traducional da Cadeia Peptídica/genética , Selenocisteína , Selenoproteínas , Animais , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/biossíntese , Selenoproteínas/genética
13.
Sci Adv ; 5(6): eaav0184, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31223645

RESUMO

Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Nutrientes/genética , Elongação Traducional da Cadeia Peptídica/genética , RNA de Transferência/genética , Schizosaccharomyces/genética , Transdução de Sinais/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação Fúngica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Mitose/genética , Fosforilação/genética
14.
Nature ; 570(7761): 400-404, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31108498

RESUMO

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2-5 and 70S4,6-8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.


Assuntos
Microscopia Crioeletrônica , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Iniciação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Escherichia coli/química , Elongação Traducional da Cadeia Peptídica , Conformação Proteica , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/química , Fatores de Tempo
15.
PLoS Comput Biol ; 15(5): e1007070, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120880

RESUMO

Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. By applying our methods to data from S. cerevisiae and mouse embryonic stem cells, we find that the extracted rates reproduce expected correlations with various molecular properties, and we also find that mouse embryonic stem cells have a global translation speed of 5.2 AA/s, in agreement with previous reports that used other approaches. Our analysis further reveals that a codon can exhibit up to 26-fold variability in its translation rate depending upon its context within a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated, and it suggests that translational regulation might be used by cells to a greater degree than previously thought.


Assuntos
Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Animais , Códon/genética , Códon/metabolismo , Biologia Computacional , Simulação por Computador , Cinética , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma
16.
Commun Biol ; 2: 154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069264

RESUMO

Lysine acetylation is critical in regulating important biological processes in many organisms, yet little is known about acetylome evolution and its contribution to phenotypic diversity. Here, we compare the acetylomes of baker's yeast and the three deadliest human fungal pathogens, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using mass spectrometry enriched for acetylated peptides together with public data from Saccharomyces cerevisiae, we show that fungal acetylomes are characterized by dramatic evolutionary dynamics and limited conservation in core biological processes. Notably, the levels of protein acetylation in pathogenic fungi correlate with their pathogenicity. Using gene knockouts and pathogenicity assays in mice, we identify deacetylases with critical roles in virulence and protein translation elongation. Finally, through mutational analysis of deactylation motifs we find evidence of positive selection at specific acetylation motifs in fungal pathogens. These results shed new light on the pathogenicity regulation mechanisms underlying the evolution of fungal acetylomes.


Assuntos
Amidoidrolases/genética , Criptococose/microbiologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/genética , Processamento de Proteína Pós-Traducional , Acetilação , Amidoidrolases/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Candida albicans/genética , Candida albicans/metabolismo , Candida albicans/patogenicidade , Criptococose/mortalidade , Criptococose/patologia , Cryptococcus neoformans/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Elongação Traducional da Cadeia Peptídica , Peptídeos/genética , Peptídeos/metabolismo , Proteômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sobrevida , Virulência
17.
NPJ Syst Biol Appl ; 5: 12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30962948

RESUMO

The ability to dynamically control mRNA translation has a great impact on many intracellular processes. Whereas it is believed that translational control in eukaryotes occurs mainly at initiation, the condition-specific changes at the elongation level and their potential regulatory role remain unclear. Using computational approaches applied to ribosome profiling data, we show that elongation rate is dynamic and can change considerably during the yeast meiosis to facilitate the selective translation of stage-specific transcripts. We observed unique elongation changes during meiosis II, including a global inhibition of translation elongation at the onset of anaphase II accompanied by a sharp shift toward increased elongation for genes required at this meiotic stage. We also show that ribosomal proteins counteract the global decreased elongation by maintaining high initiation rates. Our findings provide new insights into gene expression regulation during meiosis and demonstrate that codon usage evolved, among others, to optimize timely translation.


Assuntos
Regulação da Expressão Gênica/genética , Meiose/genética , Elongação Traducional da Cadeia Peptídica/genética , Biologia Computacional/métodos , Elongação Traducional da Cadeia Peptídica/fisiologia , Iniciação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/fisiologia , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética
18.
Cell Rep ; 26(12): 3313-3322.e5, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893603

RESUMO

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome.


Assuntos
Córtex Cerebral , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil , Elongação Traducional da Cadeia Peptídica , Transdução de Sinais , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Camundongos , Camundongos Knockout
19.
Cancer Res ; 79(10): 2480-2493, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30914432

RESUMO

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.


Assuntos
Glucose/genética , Glucose/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , RNA Longo não Codificante/genética , Serina-Treonina Quinases TOR/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Pulmão/genética , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Camundongos , Regulação para Cima/genética
20.
mBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914506

RESUMO

As the protozoan parasite Toxoplasma gondii disseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution. Toxoplasma expresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created a Toxoplasma strain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O2, TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O2 tensions as well as resistance to reactive oxygen species. In vivo, reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O2 levels, in contrast to TgPHYa, which supports the parasite at low O2 IMPORTANCE Because oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O2-sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O2-sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasite Toxoplasma gondii, we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology of Toxoplasma.


Assuntos
Regulação da Expressão Gênica , Estresse Oxidativo , Elongação Traducional da Cadeia Peptídica , Prolil Hidroxilases/metabolismo , Estresse Fisiológico , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Moléculas de Adesão Celular/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo
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