Coupling the iron-responsive element to GFP--an inducible system to study translation in a single living cell.
Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3'-untranslated region of calcium/calmodulin-dependent protein kinase IIalpha mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.
Goetze
Bernhard
B
Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany. bernhard.goetze@tuebingen.mpg.de
Grunewald
Barbara
B
Kiebler
Michael A
MA
Macchi
Paolo
P
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D005293:Ferritins / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D035925:Iron-Regulatory Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000737:chemistry / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D014162:Transfection
D035925:Iron-Regulatory Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins; D005293:Ferritins