RESUMO
The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.
Assuntos
Química Click , Imagem Molecular/métodos , Neurônios/metabolismo , RNA/genética , RNA/metabolismo , Animais , Encéfalo/metabolismo , Genes de RNAr , Proteínas de Ligação a Poli(A)/metabolismo , Células Piramidais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.
Assuntos
Perfilação da Expressão Gênica , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Células 3T3 , Animais , Técnicas de Cocultura , Epitopos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Especificidade da Espécie , Transcriptoma/genéticaRESUMO
MicroRNAs (miRNAs) regulate gene expression by binding to target messenger RNAs (mRNAs) and preventing their translation. In general, the number of potential mRNA targets in a cell is much greater than the miRNA copy number, complicating high-fidelity miRNA-target interactions. We developed an inducible fluorescent probe to explore whether the maturation of a miRNA could be regulated in space and time in neurons. A precursor miRNA (pre-miRNA) probe exhibited an activity-dependent increase in fluorescence, suggesting the stimulation of miRNA maturation. Single-synapse stimulation resulted in a local maturation of miRNA that was associated with a spatially restricted reduction in the protein synthesis of a target mRNA. Thus, the spatially and temporally regulated maturation of pre-miRNAs can be used to increase the precision and robustness of miRNA-mediated translational repression.
Assuntos
Dendritos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Animais , Células Cultivadas , Corantes Fluorescentes/química , Hipocampo/citologia , Masculino , Clivagem do RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonuclease III/genética , Ribonuclease III/metabolismo , Sinapses/metabolismoRESUMO
Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.