A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151.

The putative iron-responsive element in the human erythroid 5-aminolevulinate synthase mRNA mediates translational control.

The 52-nucleotide 5'-untranslated region of the human erythroid 5-aminolevulinate synthase mRNA contains a 28-nucleotide iron-responsive element-like stem-loop motif. We fused the 5'-untranslated region upstream to the coding sequence of the human growth hormone cDNA. A chimeric construct containing a mutated variant of the presumptive iron-responsive element was similarly synthesized. Translation of the wild type chimeric transcript was markedly repressed (approximately 95%) in rabbit reticulocyte lysates as opposed to the mutant. Both transcripts translated with comparable efficiency in wheat germ extracts. Purified placental iron regulatory factor selectively and markedly inhibited translation of the wild type chimeric transcript (> 90%) when tested in wheat germ extracts. By contrast, translations of either the mutant chimeric transcript or other control mRNA species were unaffected. The proximal position of the iron-responsive element relative to the cap site was shown to be important for translational control, in vitro. Our studies suggest that interaction of the iron regulatory factor with the iron-responsive element sterically hinders formation of the preinitiation complex, resulting in translational repression. Thus inactivation of the repressor protein by critical levels of iron or heme would trigger translation of this mRNA in erythroid cells. Consequently, protoporphyrin and heme synthesis would be subtly coordinated with iron supply.

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.

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta.

We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.

A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA.

Iron regulates human transferrin receptor (hTR) expression by modulating the stability of cytoplasmic hTR mRNA. This regulation requires a distinct secondary structure in the mRNA 3' untranslated region. We identified a specific cytoplasmic factor that binds simultaneously to four homologous palindromes within the regulatory domain. Iron chelator induced the RNA binding activity 25-fold in parallel with mRNA. Upon the addition of iron salts, a rapid decay of factor activity closely preceded hTR mRNA degradation, indicating a causal relation. Induction and decay occurred posttranscriptionally. Binding of the factor to hTR mRNA palindromes was competed by 5' regulatory sequences of ferritin mRNA, which are responsible for iron-dependent translational control. These results suggest that cellular iron maintains its homeostasis by coordinate regulation of hTR and ferritin expression via a common factor.

The repressor sequence upstream of c-mos acts neither as polyadenylation site nor as transcription termination region.

Recently we reported that the c-mos(rat) coding region is preceded by sequences (RIS) which repress accumulation of c-mos RNA in the cytoplasm. To investigate the effect of RIS on RNA transcription or processing a retroviral promoter was inserted at different positions relative to RIS. Cotransfection was used to establish cell lines with high copy number of the plasmids and to avoid any selection for c-mos expression or RIS function. Analysis of RNA in the cell lines indicated that RIS does not provide a poly(A) site and allowed characterization of the c-mos(rat) poly(A) site. Surprisingly, RIS contains sequences homologous to elements involved in eucaryotic RNA cleavage/polyadenylation. To determine an effect of RIS on transcription, RNA was elongated in vitro in nuclei isolated from the cell lines and used to analyze the number of RNA polymerase II molecules transcribing different regions of the plasmid. The analysis showed that RIS does not act as transcription termination region.