A GFP-based system to uncouple mRNA transport from translation in a single living neuron.
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 Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) 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 CaMKIIalpha 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.
Macchi
Paolo
P
Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
Hemraj
Indradeo
I
Goetze
Bernhard
B
Grunewald
Barbara
B
Mallardo
Massimo
M
Kiebler
Michael A
MA
D020121:5' Untranslated Regions; D000818:Animals; D001693:Biological Transport, Active; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003712:Dendrites / Q000378:metabolism; D005293:Ferritins / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005293:Ferritins; D007501:Iron