Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.925
Filtrar
1.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672790

RESUMO

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons-where an intermediate step is a nonsense substitution-show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.


Assuntos
Códon sem Sentido , Códon de Terminação/genética , Fases de Leitura Aberta/genética , Células Procarióticas/metabolismo , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Bases , Evolução Molecular , Modelos Genéticos , Filogenia , Mutação Puntual , Pseudogenes/genética , Seleção Genética , Homologia de Sequência do Ácido Nucleico
2.
BMC Bioinformatics ; 22(1): 156, 2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33765913

RESUMO

BACKGROUND: Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. RESULTS: We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. CONCLUSIONS: The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/ . The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.


Assuntos
Biossíntese de Proteínas , Ribossomos , Códon de Terminação , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
3.
Biochem Biophys Res Commun ; 550: 8-14, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33676232

RESUMO

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.


Assuntos
/transmissão , Monitoramento Epidemiológico , Deleção de Genes , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética , /epidemiologia , Códon sem Sentido , Códon de Terminação/genética , Evolução Molecular , Genes Virais/genética , Humanos , Filogenia , Seleção Genética , Fatores de Tempo , Reino Unido/epidemiologia
4.
Emerg Microbes Infect ; 10(1): 252-255, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33525998

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated from the oro/pharyngeal swabs of two Italian COVID-19 patients, physicians in a COVID-19 division hospital, with different courses of the disease. The complete genome sequences show that the two isolates belong to the B1.1 lineage, but contain a nucleotide mutation in the ORF6, leading to a stop codon and to the deletion of 6 amino acids in the C terminus. This deletion was unique, compared to the currently available sequences deposited in the GISAID and GenBank database. It did not affect the in vitro viral replication, neither the neutralizing activities of the patients' antibodies. Based on homology analysis with other Coronaviruses, the two isolated lacked the ORF6 aminoacidic portion responsible for the inhibition of the antiviral Interferon (IFN)-based host response. IFN seems to have a dual role of in SARS-CoV-2 infected patients: not only antiviral activity, but also a detrimental role in case of excessive production. A deletion in the SARS-CoV-2 ORF6 protein might have a specific, still unknown role in the viral pathogenesis.


Assuntos
/virologia , Códon de Terminação/genética , Mutação Puntual , Proteínas Virais/genética , /diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , /isolamento & purificação
5.
Nucleic Acids Res ; 49(5): 2684-2699, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33561188

RESUMO

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.


Assuntos
Adenosina/análogos & derivados , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Adenosina/metabolismo , Códon , Códon de Terminação , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptídeos/metabolismo , Ribossomos/metabolismo
6.
Expert Opin Investig Drugs ; 30(2): 167-176, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393390

RESUMO

INTRODUCTION: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin (DMD) gene. Most patients die from respiratory failure or cardiomyopathy. There are significant unmet needs for treatments for DMD as the standard of care is principally limited to symptom relief through treatments including steroids. AREAS COVERED: This review summarizes safety and efficacy in promising areas of DMD therapeutics - small molecules, stop codon readthrough, gene replacement, and exon skipping - under clinical examination from 2015-2020 as demonstrated in the NIH Clinical Trials and PubMed search engines. EXPERT OPINION: Currently, steroids persist as the most accessible medicine for DMD. Stop-codon readthrough, gene replacement, and exon-skipping therapies all aim to restore dystrophin expression. Of these strategies, gene replacement therapy has recently gained momentum while exon-skipping retains great traction. The  FDA approval of three exon-skipping antisense oligonucleotides illustrate this regulatory momentum, though the effectiveness and sequence design of eteplirsen remain controversial. Cell-penetrating peptides promise to more efficaciously treat DMD-related cardiomyopathy.The recent success of antisense therapies, however, poses major regulatory challenges. To fully realize the benefits of exon-skipping, including cocktail oligonucleotide-mediated multiple exon-skipping and oligonucleotide drugs for very rare mutations, regulatory challenges need to be addressed in coordination with scientific advances.


Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Distrofina/genética , Terapia Genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos/uso terapêutico , Animais , Peptídeos Penetradores de Células/efeitos adversos , Códon de Terminação , Desenvolvimento de Medicamentos , Drogas em Investigação/efeitos adversos , Drogas em Investigação/uso terapêutico , Éxons , Regulação da Expressão Gênica , Predisposição Genética para Doença , Terapia Genética/efeitos adversos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Mutação , Oligonucleotídeos/efeitos adversos , Esteroides/efeitos adversos , Esteroides/uso terapêutico , Resultado do Tratamento
7.
Food Chem ; 335: 127663, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738540

RESUMO

Dissecting the functions of high molecular weight glutenin subunits (HMW-GSs) is helpful for improving wheat quality via breeding. In this study, we used a wheat mutant AS273 in which HMW-GS 1Dy12 was silenced to investigate the silencing mechanism of 1Dy12 and its effects on gluten accumulation and flour-processing quality. Results suggested that the expression of 1Dy12 in AS273 was decreased by one fifth during grain development; a stop codon produced by a base mutation (C/T) led to truncated translation; the absence of 1Dy12 stimulated the accumulation of low molecular weight glutenin subunits (LMW-GSs), gliadins, and glutenin macropolymers, and was resulted in larger protein bodies; AS273 had an inferior flour-processing performance. Based on the outputs achieved in this study it is concluded that 1Dy12 makes important contributions to bread, sponge cake and biscuit-processing quality.


Assuntos
Pão , Glutens/genética , Glutens/metabolismo , Triticum/genética , Triticum/metabolismo , Pão/análise , Códon de Terminação , Eletroforese em Gel de Poliacrilamida , Farinha , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Gliadina/metabolismo , Peso Molecular , Mutação , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Triticum/crescimento & desenvolvimento
8.
Brain ; 144(2): 411-419, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33313762

RESUMO

Claudin-11, a tight junction protein, is indispensable in the formation of the radial component of myelin. Here, we report de novo stop-loss variants in the gene encoding claudin-11, CLDN11, in three unrelated individuals presenting with an early-onset spastic movement disorder, expressive speech disorder and eye abnormalities including hypermetropia. Brain MRI showed a myelin deficit with a discrepancy between T1-weighted and T2-weighted images and some progress in myelination especially involving the central and peripheral white matter. Exome sequencing identified heterozygous stop-loss variants c.622T>C, p.(*208Glnext*39) in two individuals and c.622T>G, p.(*208Gluext*39) in one individual, all occurring de novo. At the RNA level, the variant c.622T>C did not lead to a loss of expression in fibroblasts, indicating this transcript is not subject to nonsense-mediated decay and most likely translated into an extended protein. Extended claudin-11 is predicted to form an alpha helix not incorporated into the cytoplasmic membrane, possibly perturbing its interaction with intracellular proteins. Our observations suggest that stop-loss variants in CLDN11 expand the genetically heterogeneous spectrum of hypomyelinating leukodystrophies.


Assuntos
Anodontia/genética , Anodontia/patologia , Ataxia/genética , Ataxia/patologia , Encéfalo/patologia , Claudinas/genética , Hipogonadismo/genética , Hipogonadismo/patologia , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Adolescente , Encéfalo/diagnóstico por imagem , Criança , Códon de Terminação/genética , Feminino , Variação Genética , Humanos , Imagem por Ressonância Magnética , Masculino , Linhagem
9.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322589

RESUMO

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.


Assuntos
Códon sem Sentido/genética , Códon de Terminação/genética , Animais , Humanos , Mutação/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo
10.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
11.
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
12.
PLoS One ; 15(8): e0237405, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817702

RESUMO

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.


Assuntos
Chlamydomonas reinhardtii/genética , Códon de Terminação/genética , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos/genética
13.
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
14.
Proc Natl Acad Sci U S A ; 117(36): 22167-22172, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839318

RESUMO

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.


Assuntos
Códon de Terminação , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Mutação
15.
PLoS One ; 15(7): e0232559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658922

RESUMO

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.


Assuntos
Códon de Terminação/genética , Perfilação da Expressão Gênica , Mitocôndrias/genética , Mutação , Presenilina-2/genética , Ribossomos/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Contagem de Células , Homozigoto , Hipóxia/genética , Neurônios/citologia , Estabilidade de RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
16.
Mol Cell ; 79(4): 588-602.e6, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32615089

RESUMO

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.


Assuntos
Pegada de DNA/métodos , Genoma Fúngico , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/metabolismo , Regiões 3' não Traduzidas , Anisomicina/farmacologia , Códon de Terminação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mutação , Fosforilação , Estabilidade de RNA , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
17.
PLoS One ; 15(6): e0225563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32570272

RESUMO

To evaluate the impact of hypermutation on the HIV-1 dissemination at the population level we studied 7072 sequences HIV-1 gene vif retrieved from the public databank. From this dataset 854 sequences were selected because they had associated values of CD4+ T lymphocytes counts and viral loads and they were used to assess the correlation between clinical parameters and hypermutation. We found that the frequency of stop codons at sites 5, 11 and 79 ranged from 2.8x10-4 to 4.2x10-4. On the other hand, at codons 21, 38, 70, 89 and 174 the frequency of stop codons ranged from 1.4x10-3 to 2.5x10-3. We also found a correlation between clinical parameters and hypermutation where patients harboring proviruses with one or more stop codons at the tryptophan sites of the gene vif had higher CD4+ T lymphocytes counts and lower viral loads compared to the population. Our findings indicate that A3 activity potentially restrains HIV-1 replication because individuals with hypermutated proviruses tend to have lower numbers of RNA copies. However, owing to the low frequency of hypermutated sequences observed in the databank (44 out of 7072), it is unlikely that A3 has a significant impact to curb HIV-1 dissemination at the population level.


Assuntos
Códon/genética , HIV-1/genética , Triptofano , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Contagem de Linfócito CD4 , Códon de Terminação/genética , HIV-1/fisiologia , Mutação , Carga Viral/genética
18.
Gene ; 754: 144879, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531458

RESUMO

Gelsolin is an actin-binding protein that plays a significant role in sustaining cell motility and cell metabolism. Investigations of the mutations present in the key regions of gelsolin provide extensive information to further understand the mechanism by which gelsolin causes variation in the phenotype [e.g., residual feed intake (RFI) or feed efficiency ]of pigs. However, there have been no investigations of the variation in functional binding regions or research on Chinese native pigs. In this study, three key regions of gelsolin were investigated in 144 pigs from six breeds using a sequencing method. The results revealed 16 nucleotide substitutions, eight of which (c.42-13G/T, c.59 T/C, c.86C/T, c.87G/T, c.104C/T, c.144 T/C, c.206G/C, and c.237 + 21A/G) were novel and identified in intron 1, exon 2, and intron 2. Two variants (c.87G/T and c.144 T/C) resulted in a premature stop codon (p.Gly16Uga(Stop)) and an amino acid change (p.Tyr35His), respectively. In region 1, c.144 T/C was the most common (at a total frequency of 46.5%), followed by c.42-13G/T (at a total frequency of 41.7%). In region 2, two variants (c.350A/G and c.374A/G) were most common (both at a total frequency of 36.1%). There were significant differences (P < 0.05) in variant frequencies between Chinese indigenous pigs and overseas pigs. Our findings revealed one novel premature stop codon and eight novel variations in re-sequencing regions, which suggest that these variations of gelsolin may influence its mRNA expression and consequently affect production traits in swine.


Assuntos
Códon de Terminação/genética , Gelsolina/genética , Variação Genética , Suínos/genética , Animais , Suínos/classificação
19.
PLoS Genet ; 16(6): e1008830, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502192

RESUMO

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Códon de Terminação , Conjuntos de Dados como Assunto , Embrião não Mamífero , Éxons/genética , Redes Reguladoras de Genes/genética , Homozigoto , Humanos , Íntrons/genética , Camundongos , Músculo Esquelético/inervação , Mutagênese , Mutação , Crescimento Neuronal/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Alinhamento de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
20.
J Pharmacol Exp Ther ; 374(2): 264-272, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32376628

RESUMO

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 µg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.


Assuntos
Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Furanos/farmacologia , Proteômica , Linhagem Celular Tumoral , Genes p53/genética , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...