Development of a fluorescent reporter to assess iron regulatory protein activity in living cells.
Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.
Henderson
Rebecca J
RJ
Department of Pharmacology, College of Medicine, Pennsylvania State University, 500 University Drive, Hershey, Pennsylvania 17033, USA.
Patton
Stephanie M
SM
Connor
James R
JR
D020816:Amino Acid Motifs; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D005293:Ferritins / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000378:metabolism*; D049408:Luminescent Agents / Q000494:pharmacology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C485182:Cyan Fluorescent Protein; D035925:Iron-Regulatory Proteins; D049408:Luminescent Agents; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D005293:Ferritins