RESUMO
Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.
Assuntos
Biossíntese de Proteínas , Proteínas , Animais , RNA Mensageiro/metabolismo , Códon/genética , Proteínas/genética , Estabilidade de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
Assuntos
RNA de Transferência , Animais , Humanos , Ratos , Anticódon , Linhagem Celular , Códon , Glicosilação , Nucleosídeo Q/química , Nucleosídeo Q/genética , Nucleosídeo Q/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Suínos , Peixe-Zebra/metabolismo , Conformação de Ácido NucleicoRESUMO
The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.
Assuntos
COVID-19/epidemiologia , Evolução Molecular , Mutação , Pandemias , SARS-CoV-2/genética , Sequência de Aminoácidos/genética , COVID-19/imunologia , COVID-19/transmissão , COVID-19/virologia , Códon/genética , Genes Virais , Deriva Genética , Adaptação ao Hospedeiro/genética , Humanos , Evasão da Resposta Imune , Filogenia , Saúde PúblicaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Assuntos
Neurônios/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biossíntese de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Animais , Axônios/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Códon/genética , Feminino , Glicina/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Tecido Nervoso/patologia , Consumo de Oxigênio , Neoplasias Pancreáticas/patologia , Pirazóis , Pirimidinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RatosRESUMO
Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
Assuntos
Miocárdio/metabolismo , Biossíntese de Proteínas , Adolescente , Adulto , Idoso , Animais , Códon/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ribossomos/genética , Ribossomos/metabolismo , Adulto JovemRESUMO
Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.
Assuntos
Elongação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Códon/genética , Epigênese Genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Humanos , Cinética , Modelos Biológicos , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Mensageiro/química , Ribossomos/metabolismoRESUMO
Transfer RNA (tRNA) is an adapter molecule that links a specific codon in mRNA with its corresponding amino acid during protein synthesis. tRNAs are enzymatically modified post-transcriptionally. A wide variety of tRNA modifications are found in the tRNA anticodon, which are crucial for precise codon recognition and reading frame maintenance, thereby ensuring accurate and efficient protein synthesis. In addition, tRNA-body regions are also frequently modified and thus stabilized in the cell. Over the past two decades, 16 novel tRNA modifications were discovered in various organisms, and the chemical space of tRNA modification continues to expand. Recent studies have revealed that tRNA modifications can be dynamically altered in response to levels of cellular metabolites and environmental stresses. Importantly, we now understand that deficiencies in tRNA modification can have pathological consequences, which are termed 'RNA modopathies'. Dysregulation of tRNA modification is involved in mitochondrial diseases, neurological disorders and cancer.
Assuntos
Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Animais , Códon/genética , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Arango et al. expand the repertoire of epitranscriptomic modifications by identifying N4-acetylcytidine in mRNA catalyzed by the known dual acetyltransferase NAT10. It occurs mainly in the coding sequence, likely in wobble positions of select codons, where it promotes stability and translation, possibly by safeguarding cognate codon-anticodon interaction.
Assuntos
Anticódon , Citidina , Acetilação , Códon , RNA MensageiroRESUMO
The pool of transfer RNA (tRNA) molecules in cells allows the ribosome to decode genetic information. This repertoire of molecular decoders is positioned in the crossroad of the genome, the transcriptome, and the proteome. Omics and systems biology now allow scientists to explore the entire repertoire of tRNAs of many organisms, revealing basic exciting biology. The tRNA gene set of hundreds of species is now characterized, in addition to the tRNA genes of organelles and viruses. Genes encoding tRNAs for certain anticodon types appear in dozens of copies in a genome, while others are universally absent from any genome. Transcriptome measurement of tRNAs is challenging, but in recent years new technologies have allowed researchers to determine the dynamic expression patterns of tRNAs. These advances reveal that availability of ready-to-translate tRNA molecules is highly controlled by several transcriptional and posttranscriptional regulatory processes. This regulation shapes the proteome according to the cellular state. The tRNA pool profoundly impacts many aspects of cellular and organismal life, including protein expression level, translation accuracy, adequacy of folding, and even mRNA stability. As a result, the shape of the tRNA pool affects organismal health and may participate in causing conditions such as cancer and neurological conditions.
Assuntos
Genoma/genética , Biossíntese de Proteínas , Proteômica/tendências , RNA de Transferência/genética , Anticódon/genética , Códon/genética , Genômica/tendências , Humanos , Transcriptoma/genéticaRESUMO
tRNAs are best known as basic modules for global regulation of protein synthesis. Goodarzi et al. now show that two tRNAs upregulated in metastatic breast cancer cells enhance stability and translation of transcripts enriched with these codons, leading to specific increase in production of pro-metastatic proteins.
Assuntos
Códon , RNA de Transferência/genética , Humanos , Biossíntese de ProteínasRESUMO
A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.
Assuntos
Códon/metabolismo , RNA Helicases DEAD-box/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Códon/genética , RNA Helicases DEAD-box/genética , Meia-VidaRESUMO
Francis' office window (at the Salk) commanded a panorama of the Pacific. "This grand natural scene was a physical correlate of Francis's intellectual world: wide-ranging, brilliantly lit, a little overawing, but also immensely inviting and above all an exciting place to be." (Mitchison, 2004).
Assuntos
DNA/química , Genética/história , Biologia Molecular/história , Animais , Caenorhabditis elegans , Códon , Biologia do Desenvolvimento/história , Inglaterra , História do Século XX , PesquisaRESUMO
The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.
Assuntos
Códon , Eucariotos/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/química , Ribossomos/fisiologia , Imagem Molecular , Células Procarióticas/fisiologia , RNA Mensageiro/fisiologia , RNA de Transferência/fisiologiaRESUMO
Translation elongation efficiency is largely thought of as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we have identified 17 pairs of adjacent codons associated with reduced expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated by adjacent codons occurs during translation itself as GFP expression is restored by increased tRNA levels or by non-native tRNAs with exact-matching anticodons. Inhibition operates in endogenous genes, based on analysis of ribosome profiling data. Our findings suggest translation efficiency is modulated by an interplay between tRNAs at adjacent sites in the ribosome and that this concerted effect needs to be considered in predicting the functional consequences of codon choice.
Assuntos
Códon , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Genes Fúngicos , RNA Fúngico/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/biossínteseRESUMO
Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a pro-metastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , RNA de Transferência/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Códon , Progressão da Doença , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Pulmão/patologia , Camundongos , Invasividade Neoplásica/genética , Micrometástase de Neoplasia/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Ribonucleico , Ribossomos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CRISPR screens have empowered the high-throughput dissection of gene functions; however, more explicit genetic elements, such as codons of amino acids, require thorough interrogation. Here, we establish a CRISPR strategy for unbiasedly probing functional amino acid residues at the genome scale. By coupling adenine base editors and barcoded sgRNAs, we target 215,689 out of 611,267 (35%) lysine codons, involving 85% of the total protein-coding genes. We identify 1,572 lysine codons whose mutations perturb human cell fitness, with many of them implicated in cancer. These codons are then mirrored to gene knockout screen data to provide functional insights into the role of lysine residues in cellular fitness. Mining these data, we uncover a CUL3-centric regulatory network in which lysine residues of CUL3 CRL complex proteins control cell fitness by specifying protein-protein interactions. Our study offers a general strategy for interrogating genetic elements and provides functional insights into the human proteome.
Assuntos
Lisina , Proteoma , Humanos , Proteoma/genética , Lisina/genética , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , CódonRESUMO
For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here, we assess the role of mRNA stability in localization by combining the isolation of the soma and neurites of mouse primary cortical and mESC-derived neurons, SLAM-seq, m6A-RIP-seq, the perturbation of mRNA destabilization mechanisms, and the analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m6A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons.
Assuntos
Neuritos , Neurônios , Animais , Camundongos , RNA Mensageiro/genética , Códon , Estabilidade de RNARESUMO
In addition to the main, protein-coding, open reading frame (mORF), many eukaryotic mRNAs contain upstream ORFs (uORFs) initiated at AUG or near-cognate codons residing 5' of the mORF start site. Whereas translation of uORFs generally represses translation of the mORFs, a subset of uORFs serves as a nexus for regulating translation of the mORF. In this review, we summarize the mechanisms by which uORFs can repress or stimulate mRNA translation, highlight uORF-mediated translational repression involving ribosome queuing, and critically evaluate recently described alternatives to the delayed reinitiation model for uORF-mediated regulation of the GCN4/ATF4 mRNAs.
Assuntos
Biossíntese de Proteínas , Ribossomos , Códon de Iniciação/genética , Códon/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fases de Leitura Aberta/genéticaRESUMO
Macrophages initiate inflammatory responses via the transcription factor NFκB. The temporal pattern of NFκB activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NFκB activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic workflow identified six dynamical features-termed signaling codons-that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NFκB signaling codons. Thus, the dynamics of NFκB signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some inflammatory diseases.
Assuntos
Códon/genética , Macrófagos/fisiologia , NF-kappa B/genética , Transdução de Sinais/genética , Animais , Células Cultivadas , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Sjogren/genética , Fator de Transcrição RelA/genéticaRESUMO
The advent of ribosome profiling and other tools to probe mRNA translation has revealed that codon bias - the uneven use of synonymous codons in the transcriptome - serves as a secondary genetic code: a code that guides the efficiency of protein production, the fidelity of translation and the metabolism of mRNAs. Recent advancements in our understanding of mRNA decay have revealed a tight coupling between ribosome dynamics and the stability of mRNA transcripts; this coupling integrates codon bias into the concept of codon optimality, or the effects that specific codons and tRNA concentrations have on the efficiency and fidelity of the translation machinery. In this Review, we first discuss the evidence for codon-dependent effects on translation, beginning with the basic mechanisms through which translation perturbation can affect translation efficiency, protein folding and transcript stability. We then discuss how codon effects are leveraged by the cell to tailor the proteome to maintain homeostasis, execute specific gene expression programmes of growth or differentiation and optimize the efficiency of protein production.