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1.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389711

RESUMO

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
2.
Nat Commun ; 12(1): 1836, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758175

RESUMO

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.


Assuntos
Bactérias/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Lipopeptídeos/imunologia , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Bactérias/metabolismo , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flagelina/farmacologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Hibridização in Situ Fluorescente , Glândulas Mamárias Animais , Camundongos , Organoides/efeitos dos fármacos , Organoides/imunologia , Organoides/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo
3.
Mol Cell ; 81(8): 1830-1840.e8, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581075

RESUMO

Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall. We monitor the resolution of these queues in real time and find that ribosome rescue is very slow compared to both elongation and termination. Modulation of pause strength, collision frequency, and the collision sensor ZNF598 reveals how the dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control. Our results establish that slow clearance of stalled ribosomes allows cells to distinguish between transient and deleterious stalls.


Assuntos
Elongação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , Ribossomos/genética , Proteínas de Transporte/genética , Células HEK293 , Humanos , Cinética , Peptídeos/genética , Poli A/genética , Controle de Qualidade , RNA Mensageiro/genética
4.
Elife ; 92020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32844748

RESUMO

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.


Assuntos
Núcleo Celular , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas , Puromicina , Animais , Caenorhabditis elegans , Núcleo Celular/química , Núcleo Celular/metabolismo , Emetina/metabolismo , Emetina/farmacologia , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/metabolismo , Puromicina/farmacologia , RNA de Transferência/metabolismo , Coelhos , Ribossomos/metabolismo , Análise de Célula Única
5.
Methods ; 162-163: 12-22, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30905747

RESUMO

The central dogma of molecular biology reaches a crescendo at its final step: the translation of an mRNA into its corresponding protein product. This process is highly regulated both spatially and temporally, requiring techniques to interrogate the subcellular translational status of mRNAs in both living and fixed cells. Single-molecule imaging of nascent peptides (SINAPs) and related techniques allow us to study this fundamental process for single mRNAs in live cells. These techniques enable researchers to address previously intractable questions in the central dogma, such as the origin of stochastic translational control and the role of local translation in highly polarized cells. In this review, we present the methodology and the theoretical framework for conducting studies using SINAPs in both established cell lines and primary cultured neurons.


Assuntos
Microscopia Intravital/métodos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Levivirus/genética , Camundongos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cultura Primária de Células/métodos , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Annu Rev Biophys ; 47: 85-106, 2018 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-29345990

RESUMO

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.


Assuntos
Regulação da Expressão Gênica/genética , RNA Mensageiro/química , Humanos , Cinética
7.
Methods ; 113: 64-71, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27794454

RESUMO

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.


Assuntos
Alanina-tRNA Ligase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Histidina-tRNA Ligase/química , RNA de Transferência Aminoácido-Específico/metabolismo , Treonina-tRNA Ligase/química , Alanina-tRNA Ligase/antagonistas & inibidores , Alanina-tRNA Ligase/genética , Alanina-tRNA Ligase/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Benzopiranos/química , Inibidores Enzimáticos/química , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Fluorometria/métodos , Histidina-tRNA Ligase/antagonistas & inibidores , Histidina-tRNA Ligase/genética , Histidina-tRNA Ligase/metabolismo , Muramidase/química , Muramidase/metabolismo , Transição de Fase , Ligação Proteica , Desdobramento de Proteína , RNA de Transferência Aminoácido-Específico/genética , Especificidade por Substrato , Treonina-tRNA Ligase/antagonistas & inibidores , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Aminoacilação de RNA de Transferência
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