Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.
Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.
Hirling
H
H
Swiss Institute for Experimental Cancer Research, Genetics Unit, Epalinges.
Emery-Goodman
A
A
Thompson
N
N
Neupert
B
B
Seiser
C
C
Kühn
L C
LC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D005293:Ferritins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D035925:Iron-Regulatory Proteins; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D035925:Iron-Regulatory Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011990:Receptors, Transferrin; D005293:Ferritins; D010450:Endopeptidases; D014357:Trypsin