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1.
PLoS One ; 15(10): e0239700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33017414

RESUMO

In the past two decades, research into the biochemical, biophysical and structural properties of the ribosome have revealed many different steps of protein translation. Nevertheless, a complete understanding of how they lead to a rapid and accurate protein synthesis still remains a challenge. Here we consider a coarse network analysis in the bacterial ribosome formed by the connectivity between ribosomal (r) proteins and RNAs at different stages in the elongation cycle. The ribosomal networks are found to be dis-assortative and small world, implying that the structure allows for an efficient exchange of information between distant locations. An analysis of centrality shows that the second and fifth domains of 23S rRNA are the most important elements in all of the networks. Ribosomal protein hubs connect to much fewer nodes but are shown to provide important connectivity within the network (high closeness centrality). A modularity analysis reveals some of the different functional communities, indicating some known and some new possible communication pathways Our mathematical results confirm important communication pathways that have been discussed in previous research, thus verifying the use of this technique for representing the ribosome, and also reveal new insights into the collective function of ribosomal elements.


Assuntos
Bactérias/genética , Redes Reguladoras de Genes/genética , Ribossomos/genética , Bactérias/metabolismo , Biologia Computacional/métodos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Elongação da Transcrição Genética/fisiologia
2.
Nat Commun ; 11(1): 5260, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067428

RESUMO

Protein synthesis is the most expensive process in fast-growing bacteria. Experimentally observed growth rate dependencies of the translation machinery form the basis of powerful phenomenological growth laws; however, a quantitative theory on the basis of biochemical and biophysical constraints is lacking. Here, we show that the growth rate-dependence of the concentrations of ribosomes, tRNAs, mRNA, and elongation factors observed in Escherichia coli can be predicted accurately from a minimization of cellular costs in a mechanistic model of protein translation. The model is constrained only by the physicochemical properties of the molecules and has no adjustable parameters. The costs of individual components (made of protein and RNA parts) can be approximated through molecular masses, which correlate strongly with alternative cost measures such as the molecules' carbon content or the requirement of energy or enzymes for their biosynthesis. Analogous cost minimization approaches may facilitate similar quantitative insights also for other cellular subsystems.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
3.
Nat Commun ; 11(1): 5096, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037221

RESUMO

Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.


Assuntos
Fator G para Elongação de Peptídeos/biossíntese , Fator G para Elongação de Peptídeos/química , Dobramento de Proteína , Luminescência , Fator G para Elongação de Peptídeos/genética , Biossíntese de Proteínas , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Imagem Individual de Molécula
4.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
5.
Nucleic Acids Res ; 48(17): 9433-9448, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890397

RESUMO

The fragments that derive from transfer RNAs (tRNAs) are an emerging category of regulatory RNAs. Known as tRFs, these fragments were reported for the first time only a decade ago, making them a relatively recent addition to the ever-expanding pantheon of non-coding RNAs. tRFs are short, 16-35 nucleotides (nts) in length, and produced through cleavage of mature and precursor tRNAs at various positions. Both cleavage positions and relative tRF abundance depend strongly on context, including the tissue type, tissue state, and disease, as well as the sex, population of origin, and race/ethnicity of an individual. These dependencies increase the urgency to understand the regulatory roles of tRFs. Such efforts are gaining momentum, and comprise experimental and computational approaches. System-level studies across many tissues and thousands of samples have produced strong evidence that tRFs have important and multi-faceted roles. Here, we review the relevant literature on tRF biology in higher organisms, single cell eukaryotes, and prokaryotes.


Assuntos
Neoplasias/genética , Doenças do Sistema Nervoso/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Viroses/genética , Animais , Enzimas/metabolismo , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Neoplasias/mortalidade , Estabilidade de RNA , RNA de Transferência/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Estresse Fisiológico/genética
6.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
7.
Nucleic Acids Res ; 48(18): 10342-10352, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32894284

RESUMO

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.


Assuntos
DNA Ribossômico/genética , Proteínas Hedgehog/genética , RNA Polimerase I/genética , Neoplasias de Mama Triplo Negativas/radioterapia , Proteína GLI1 em Dedos de Zinco/genética , Proteínas de Ciclo Celular/genética , Nucléolo Celular/genética , Nucléolo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , DNA Ribossômico/efeitos da radiação , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Humanos , RNA Polimerase I/efeitos da radiação , Radiação Ionizante , Ribossomos/genética , Ribossomos/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Transcrição Genética/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
8.
Nucleic Acids Res ; 48(18): 10259-10279, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941650

RESUMO

To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3' UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3' UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3' UTR, where it can resume translation in-frame as well as out-of-frame.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Códon de Terminação/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Telomerase/genética , Regiões 3' não Traduzidas/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Ribossomos/genética
9.
Nucleic Acids Res ; 48(18): 10441-10455, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941651

RESUMO

Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.


Assuntos
Fator de Iniciação 4G em Eucariotos/genética , GTP Cicloidrolase/genética , Fases de Leitura Aberta/genética , Serina-Treonina Quinases TOR/genética , Regiões 3' não Traduzidas/genética , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Naftiridinas/farmacologia , Poliovirus/genética , Biossíntese de Proteínas/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , Ribossomos/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores
10.
Nucleic Acids Res ; 48(18): 10602-10613, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976557

RESUMO

Currently, predictive translation tuning of regulatory elements to the desired output of transcription factor (TF)-based biosensors remains a challenge. The gene expression of a biosensor system must exhibit appropriate translation intensity, which is controlled by the ribosome-binding site (RBS), to achieve fine-tuning of its dynamic range (i.e. fold change in gene expression between the presence and absence of inducer) by adjusting the translation level of the TF and reporter. However, existing TF-based biosensors generally suffer from unpredictable dynamic range. Here, we elucidated the connections and partial mechanisms between RBS, translation level, protein folding and dynamic range, and presented a design platform that predictably tuned the dynamic range of biosensors based on deep learning of large datasets cross-RBSs (cRBSs). In doing so, a library containing 7053 designed cRBSs was divided into five sub-libraries through fluorescence-activated cell sorting to establish a classification model based on convolutional neural network in deep learning. Finally, the present work exhibited a powerful platform to enable predictable translation tuning of RBS to the dynamic range of biosensors.


Assuntos
Técnicas Biossensoriais , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/genética , Fatores de Transcrição/genética , Sítios de Ligação/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/isolamento & purificação
11.
Mol Cell ; 79(6): 1024-1036.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871103

RESUMO

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/ultraestrutura , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Ribossomos/ultraestrutura , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/ultraestrutura
12.
Nucleic Acids Res ; 48(16): 9301-9319, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813020

RESUMO

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.


Assuntos
Complexos Multiproteicos/genética , Mapas de Interação de Proteínas/genética , Pequeno RNA não Traduzido/genética , RNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Fator Proteico 1 do Hospedeiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Ribossomos/genética
14.
Nucleic Acids Res ; 48(17): 9521-9537, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32766783

RESUMO

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.


Assuntos
Regiões 5' não Traduzidas , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Iniciação 4A em Eucariotos/genética , Esteróis/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mutação , Ribossomos/genética , Ribossomos/metabolismo
15.
Nucleic Acids Res ; 48(17): 9478-9490, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32821926

RESUMO

One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome's stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.


Assuntos
Biologia Computacional/métodos , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Códon de Iniciação , Modelos Genéticos , RNA Mensageiro , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Nat Commun ; 11(1): 4134, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807779

RESUMO

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Códon de Terminação/genética , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética
17.
PLoS Genet ; 16(8): e1008967, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813698

RESUMO

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.


Assuntos
Deficiências do Desenvolvimento/genética , Desenvolvimento Embrionário/genética , Fator de Transcrição PAX9/genética , Ribossomos/genética , Animais , Nucléolo Celular/genética , Deficiências do Desenvolvimento/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Crista Neural/patologia , Biossíntese de Proteínas/genética , RNA Polimerase II/genética , Ribossomos/patologia , Xenopus/genética , Xenopus/crescimento & desenvolvimento
18.
Proc Natl Acad Sci U S A ; 117(34): 20785-20793, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778592

RESUMO

Transfer RNA (tRNA) activity is tightly regulated to provide a physiological protein translation, and tRNA chemical modifications control its function in a complex with ribosomes and messenger RNAs (mRNAs). In this regard, the correct hypermodification of position G37 of phenylalanine-tRNA, adjacent to the anticodon, is critical to prevent ribosome frameshifting events. Here we report that the tRNA-yW Synthesizing Protein 2 (TYW2) undergoes promoter hypermethylation-associated transcriptional silencing in human cancer, particularly in colorectal tumors. The epigenetic loss of TYW2 induces guanosine hypomodification in phenylalanine-tRNA, an increase in -1 ribosome frameshift events, and down-regulation of transcripts by mRNA decay, such as of the key cancer gene ROBO1. Importantly, TYW2 epigenetic inactivation is linked to poor overall survival in patients with early-stage colorectal cancer, a finding that could be related to the observed acquisition of enhanced migration properties and epithelial-to-mesenchymal features in the colon cancer cells that harbor TYW2 DNA methylation-associated loss. These findings provide an illustrative example of how epigenetic changes can modify the epitranscriptome and further support a role for tRNA modifications in cancer biology.


Assuntos
Neoplasias do Colo/genética , Mudança da Fase de Leitura do Gene Ribossômico , RNA de Transferência/genética , Ribossomos/genética , tRNA Metiltransferases/deficiência , Adulto , Idoso , Anticódon/genética , Anticódon/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/metabolismo , Ilhas de CpG , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Conformação de Ácido Nucleico , Fenilalanina/genética , Fenilalanina/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
19.
Nat Commun ; 11(1): 4304, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855412

RESUMO

Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.


Assuntos
Aminoácidos Cíclicos/metabolismo , Biossíntese Peptídica , Ribossomos/metabolismo , Aminoacilação de RNA de Transferência , Engenharia Genética , Mutação , Polimerização , RNA de Transferência/metabolismo , Ribossomos/genética
20.
Proc Natl Acad Sci U S A ; 117(33): 19879-19887, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747536

RESUMO

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.


Assuntos
Escherichia coli/genética , RNA Bacteriano/química , RNA Ribossômico 23S/química , Ribossomos/genética , Escherichia coli/química , Escherichia coli/metabolismo , Evolução Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/química , Ribossomos/metabolismo
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