RESUMEN
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RESUMEN
Chemical inhibitors have revealed requirements for protein synthesis that drive cellular plasticity. We developed a genetically encodable protein synthesis inhibitor (gePSI) to achieve cell-type-specific temporal control of protein synthesis. Controlled expression of the gePSI in neurons or glia resulted in rapid, potent and reversible cell-autonomous inhibition of protein synthesis. Moreover, gePSI expression in a single neuron blocked the structural plasticity induced by single-synapse stimulation.
Asunto(s)
Ingeniería Genética , Hipocampo/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Sinapsis/metabolismo , Animales , Células Cultivadas , Células HeLa , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Ratas , Sinapsis/efectos de los fármacosRESUMEN
Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.
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Cumarinas/química , Sondas Moleculares/química , Neuronas/metabolismo , Proteínas/química , Puromicina/análogos & derivados , Animales , Supervivencia Celular/efectos de la radiación , Cumarinas/síntesis química , Cumarinas/efectos de la radiación , Hipocampo/citología , Sondas Moleculares/síntesis química , Sondas Moleculares/efectos de la radiación , Puromicina/síntesis química , Puromicina/efectos de la radiación , Ratas , Rayos UltravioletaRESUMEN
Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.
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Fibroblastos/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Anticuerpos , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Ratones , Neuronas/metabolismo , Ratas , Coloración y EtiquetadoRESUMEN
Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.
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Regiones no Traducidas 3'/fisiología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Células HEK293 , Hipocampo/química , Hipocampo/metabolismo , Humanos , Masculino , Neuronas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Neurons are amongst the most structurally complex cells and exhibit a high degree of spatial compartmentalization. Also, neurons exhibit rapid and dynamic signaling by processing information in a precise and, sometimes, spatially-restricted manner. The signaling that occurs in axons and dendrites necessitates the maintenance and modification of their local proteomes. Local translation of mRNAs into protein is one solution that neurons use to meet synaptic demand and activity. Here we review some of the key findings and recent discoveries that have shaped our understanding of local translation in neuronal function and highlight important new techniques that might pave the way for new insights.