A GFP-based System to Uncouple mRNA Transport from Translation in a Single Living NeuronV⃞

AUTOR(ES)

Macchi, Paolo

FONTE

The American Society for Cell Biology

RESUMO

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5′-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3′-UTR of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected—in addition to iron—by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3′-UTR of CaMKIIα confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.

Documentos Relacionados

• A GFP-based assay for rapid screening of compounds affecting ARE-dependent mRNA turnover
• gfp-Based N-Acyl Homoserine-Lactone Sensor Systems for Detection of Bacterial Communication
• A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins
• Development of an enhanced GFP-based dual-color reporter to facilitate genetic screens for the recovery of mutations in mice
• mRNA analysis of single living cells