Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.716
Filtrar
1.
Nat Commun ; 12(1): 2141, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837218

RESUMO

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.


Assuntos
Células Endoteliais/metabolismo , Mesângio Glomerular/metabolismo , Podócitos/metabolismo , Biossíntese de Proteínas/genética , Transcriptoma/fisiologia , Animais , Separação Celular , Biologia Computacional , Citometria de Fluxo , Heterogeneidade Genética , Mesângio Glomerular/citologia , Humanos , Masculino , Camundongos , RNA-Seq , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores da Fosfolipase A2/genética , Análise de Célula Única , Especificidade da Espécie
2.
Nat Commun ; 12(1): 1830, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758186

RESUMO

Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , Proteoma/genética , Ribossomos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrometria de Massas , Mutação de Sentido Incorreto , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Fator Tu de Elongação de Peptídeos/genética , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Proteínas Recombinantes , Ribossomos/efeitos dos fármacos , Estreptomicina/farmacologia , Estresse Fisiológico/genética
3.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670805

RESUMO

The major auxin, indole-3-acetic acid (IAA), is associated with a plethora of growth and developmental processes including embryo development, expansion growth, cambial activity, and the induction of lateral root growth. Accumulation of the auxin precursor indole-3-acetamide (IAM) induces stress related processes by stimulating abscisic acid (ABA) biosynthesis. How IAM signaling is controlled is, at present, unclear. Here, we characterize the ami1rooty double mutant, that we initially generated to study the metabolic and phenotypic consequences of a simultaneous genetic blockade of the indole glucosinolate and IAM pathways in Arabidopsisthaliana. Our mass spectrometric analyses of the mutant revealed that the combination of the two mutations is not sufficient to fully prevent the conversion of IAM to IAA. The detected strong accumulation of IAM was, however, recognized to substantially impair seed development. We further show by genome-wide expression studies that the double mutant is broadly affected in its translational capacity, and that a small number of plant growth regulating transcriptional circuits are repressed by the high IAM content in the seed. In accordance with the previously described growth reduction in response to elevated IAM levels, our data support the hypothesis that IAM is a growth repressing counterpart to IAA.


Assuntos
Redes Reguladoras de Genes , Ácidos Indolacéticos/metabolismo , Biogênese de Organelas , Ribossomos/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Germinação , Ácidos Indolacéticos/química , Redes e Vias Metabólicas , Modelos Moleculares , Mutação/genética , Fenótipo , Biossíntese de Proteínas/genética , Reprodutibilidade dos Testes , Sementes/metabolismo , Transcrição Genética
4.
Nat Commun ; 12(1): 1678, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33723245

RESUMO

Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.


Assuntos
Longevidade , Mutagênese , Mutação , Biossíntese de Proteínas/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosforilação , RNA Mensageiro , Receptor de Insulina/genética , eIF-2 Quinase/metabolismo
5.
Cell Rep ; 34(11): 108872, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33730572

RESUMO

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines.


Assuntos
Biossíntese de Proteínas/genética , Viroses/genética , Vírus/genética , Linhagem Celular , Linhagem Celular Tumoral , Uso do Códon/genética , Genes Neoplásicos/genética , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Alvéolos Pulmonares/virologia , RNA de Transferência/genética , Infecções Respiratórias/virologia , Tropismo/genética , Proteínas Virais/genética , Viroses/virologia
6.
Nat Commun ; 12(1): 1778, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741954

RESUMO

Memory reconsolidation, the process by which memories are again stabilized after being reactivated, has strengthened the idea that memory stabilization is a highly plastic process. To date, the molecular and cellular bases of reconsolidation have been extensively investigated particularly within the hippocampus. However, the role of adult neurogenesis in memory reconsolidation is unclear. Here, we combined functional imaging, retroviral and chemogenetic approaches in rats to tag and manipulate different populations of rat adult-born neurons. We find that both mature and immature adult-born neurons are activated by remote memory retrieval. However, only specific silencing of the adult-born neurons immature during learning impairs remote memory retrieval-induced reconsolidation. Hence, our findings show that adult-born neurons immature during learning are required for the maintenance and update of remote memory reconsolidation.


Assuntos
Aprendizagem/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Neurônios/fisiologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Microscopia Confocal , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Ratos Sprague-Dawley , Fatores de Tempo
7.
Nat Commun ; 12(1): 1799, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741965

RESUMO

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.


Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Células CACO-2 , Feminino , Gonorreia/microbiologia , Gonorreia/prevenção & controle , Humanos , Camundongos , Neisseria gonorrhoeae/genética , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
8.
Int J Mol Med ; 47(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33604677

RESUMO

The Fos proto­oncogene, activator protein­1 (AP­1) transcription factor subunit (c­fos) gene, a member of the immediate early gene family, encodes c­Fos, which is a subunit of the AP­1 transcription factor. The present study aimed to investigate the mechanism by which the translation efficiency of c­fos mRNA is upregulated when cellular protein synthesis is shut off. The result of western blotting revealed that the protein expression levels of c­Fos were increased in rhabdomyosarcoma cells infected with enterovirus 71 (EV71) compared with uninfected cells. PCR was used to get the c­fos 5'­untranslated region (UTR). The luciferase assay of a bicistronic vector containing the c­fos 5'UTR revealed that the c­fos 5'UTR contains an internal ribosome entry site (IRES) sequence and a 175 nucleotide sequence (between 31 and 205 nt) that is essential for IRES activity. Analysis of potential IRES trans­acting factors revealed that poly(C)­binding protein 2 (PCBP2) negatively regulated the activity of the c­fos IRES, whereas the La autoantigen (La) positively regulated its activity. The results of RNA­protein immunoprecipitation demonstrated that both PCBP2 and La bound to the c­fos 5'UTR. Furthermore, the IRES activity of in vitro­transcribed c­fos mRNA was upregulated during EV71 infection. The present study suggested a mechanism for the effect of viral infection on host genes, and provided a novel target for gene translation regulation.


Assuntos
Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Genes fos/genética , Sítios Internos de Entrada Ribossomal/genética , Proteínas Proto-Oncogênicas c-fos/genética , Autoantígenos/metabolismo , Sequência de Bases/genética , Linhagem Celular Tumoral , Enterovirus Humano A/metabolismo , Células HEK293 , Células HeLa , Humanos , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/virologia , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismo , Fator de Transcrição AP-1/genética , Transcrição Genética/genética
9.
Nat Rev Mol Cell Biol ; 22(5): 307-325, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33594280

RESUMO

Mitochondria are cellular organelles responsible for generation of chemical energy in the process called oxidative phosphorylation. They originate from a bacterial ancestor and maintain their own genome, which is expressed by designated, mitochondrial transcription and translation machineries that differ from those operating for nuclear gene expression. In particular, the mitochondrial protein synthesis machinery is structurally and functionally very different from that governing eukaryotic, cytosolic translation. Despite harbouring their own genetic information, mitochondria are far from being independent of the rest of the cell and, conversely, cellular fitness is closely linked to mitochondrial function. Mitochondria depend heavily on the import of nuclear-encoded proteins for gene expression and function, and hence engage in extensive inter-compartmental crosstalk to regulate their proteome. This connectivity allows mitochondria to adapt to changes in cellular conditions and also mediates responses to stress and mitochondrial dysfunction. With a focus on mammals and yeast, we review fundamental insights that have been made into the biogenesis, architecture and mechanisms of the mitochondrial translation apparatus in the past years owing to the emergence of numerous near-atomic structures and a considerable amount of biochemical work. Moreover, we discuss how cellular mitochondrial protein expression is regulated, including aspects of mRNA and tRNA maturation and stability, roles of auxiliary factors, such as translation regulators, that adapt mitochondrial translation rates, and the importance of inter-compartmental crosstalk with nuclear gene expression and cytosolic translation and how it enables integration of mitochondrial translation into the cellular context.


Assuntos
Eucariotos/genética , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Transcrição Genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Fosforilação Oxidativa , RNA Mensageiro/genética , RNA de Transferência/genética
10.
Nat Commun ; 12(1): 1076, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597535

RESUMO

Upstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze ~17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (Ne), has shaped the contents of uORFs in eukaryotes. Besides, gene expression level is important in influencing uORF occurrences across genes in a species. Our analyses suggest that most uORFs might play regulatory roles rather than encode functional peptides. We also show that the Kozak sequence context of uORFs has evolved across eukaryotic clades, and that noncanonical uORFs tend to have weaker suppressive effects than canonical uORFs in translation regulation. This study provides insights into the driving forces underlying uORF evolution in eukaryotes.


Assuntos
Eucariotos/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Fases de Leitura Aberta/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Códon de Iniciação/genética , Eucariotos/classificação , Evolução Molecular , Filogenia , Biossíntese de Proteínas/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
11.
Methods Mol Biol ; 2218: 367-380, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606246

RESUMO

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.


Assuntos
Biossíntese de Proteínas/genética , Peixe-Zebra/genética , Animais , Citoplasma/genética , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/genética , Oócitos/fisiologia , Oogênese/genética , Poli A/genética , Poliadenilação/genética , RNA Mensageiro Estocado
12.
Nat Chem Biol ; 17(4): 421-427, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542534

RESUMO

Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated messenger RNAs, inevitably generating nonfunctional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA-RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6- to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant protein production but also efficiencies of metabolic pathways.


Assuntos
Engenharia Genética/métodos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Bactérias/genética , Sequência de Bases/genética , Escherichia coli/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Controle de Qualidade , RNA Mensageiro/química , RNA Mensageiro/metabolismo
13.
Nat Struct Mol Biol ; 28(1): 103-117, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398172

RESUMO

Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.


Assuntos
Regulação da Expressão Gênica/genética , Complexo Repressor Polycomb 2/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Polimerase II/metabolismo , RNA/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA/genética , Transcrição Genética/genética , Ativação Transcricional/genética
14.
Nat Commun ; 12(1): 599, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500394

RESUMO

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.


Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Biologia Sintética/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Óperon de RNAr/genética
15.
Gene ; 766: 145130, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32911030

RESUMO

The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona.


Assuntos
Proteínas de Bactérias/genética , Biossíntese de Proteínas/genética , Saccharopolyspora/genética , Animais , Regulação para Baixo/genética , Metabolismo Energético/genética , Insetos/microbiologia , Proteômica/métodos , Regulação para Cima/genética
16.
Gene ; 766: 145032, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32771387

RESUMO

Control of gene expression by epigenetic regulators is fundamental to tissue development and homeostasis. Loss-of-function (LOF) studies using siRNAs for epigenetic regulators require that RNA interference rapidly reduces the cellular levels of the corresponding mRNAs and/or proteins. The most abundant chromatin structural proteins (i.e., the core histones H2A, H2B, H3 and H4) have relatively long half-lives and do not turn over rapidly, although their mRNAs are labile. The question arises whether epigenetic regulatory enzymes (e.g., Ezh2) or proteins that interact with histones via selective modifications (e.g., Cbx1 to Cbx8, Brd4) are stable or unstable. Therefore, we performed classical α-amanitin and cycloheximide inhibition assays that block, respectively, mRNA transcription and protein translation in mouse MC3T3 osteoblasts, ATDC5 chondrocytes and C2C12 myoblasts. We find that mRNA levels of Cbx proteins and Ezh2 were significantly depleted after 24 hrs, while their corresponding proteins remained relatively stable. As positive control, the half-life of the labile cyclin D1 protein was found to be less than 1 hr. Our study suggests that histone code readers and writers are relatively stable chromatin-related proteins, which is consistent with their long-term activities in maintaining chromatin organization and phenotype identity. These findings have conceptual ramifications for the interpretation of RNAi experiments that reduce the mRNA but not protein levels of epiregulatory proteins. We propose that siRNAs for at least some epigenetic regulatory proteins may exert their biological effects by blocking translation and new protein synthesis rather than by decreasing pre-existing protein pools.


Assuntos
Epigênese Genética/genética , Sistema Musculoesquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células 3T3 , Animais , Linhagem Celular , Cromatina/genética , Epigenômica/métodos , Histonas/genética , Camundongos , Biossíntese de Proteínas/genética , Estabilidade Proteica , Transcrição Genética/genética
17.
EMBO J ; 40(2): e106696, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346941

RESUMO

Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine-tRNAGUA fragments in human cells-causing significant depletion of the precursor tRNA. Tyrosine-tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation-revealing a dedicated tRNA-regulated growth-suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.


Assuntos
Códon/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Tirosina/genética , Animais , Caenorhabditis elegans/genética , Linhagem Celular , Proliferação de Células/genética , Células HEK293 , Humanos , Estresse Oxidativo/genética , Proteases Específicas de Ubiquitina/genética
18.
Nature ; 590(7845): 332-337, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33328638

RESUMO

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.


Assuntos
Apresentação do Antígeno , Mutação da Fase de Leitura , Melanoma/imunologia , Peptídeos/genética , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , Linfócitos T/imunologia , Linhagem Celular , Códon/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/imunologia , Interferon gama/farmacologia , Melanoma/patologia , Peptídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteoma , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Triptofano/deficiência , Triptofano/genética , Triptofano/metabolismo
19.
Nucleic Acids Res ; 49(1): 458-478, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332560

RESUMO

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.


Assuntos
Autoantígenos/fisiologia , Proteína I de Ligação a Poli(A)/fisiologia , Polirribossomos/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteínas/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Regiões 5' não Traduzidas/genética , Autoantígenos/genética , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Naftiridinas/farmacologia , Mutação Puntual , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética
20.
EMBO J ; 40(2): e107097, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346912

RESUMO

Transfer RNAs (tRNAs) are central adaptors that decode genetic information during translation and have been long considered static cellular components. However, whether dynamic changes in tRNAs and tRNA-derived fragments actively contribute to gene regulation remains debated. In this issue, Huh et al (2020) highlight tyrosine tRNAGUA fragmentation at the nexus of an evolutionarily conserved adaptive codon-based stress response that fine-tunes translation to restrain growth in human cells.


Assuntos
Biossíntese de Proteínas , RNA de Transferência , Ciclo Celular , Códon/genética , Humanos , Biossíntese de Proteínas/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Tirosina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...