Demonstration and characterization of the iron regulatory protein in human brain

Jing Hu, James Connor

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5-aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem-loop structures called iron-responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA-protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA- protein complexes are specific to the IRE structure. UV cross-linking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA-protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA-protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of ~100 kDa and is capable of forming two RNA-protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.

Original languageEnglish (US)
Pages (from-to)838-844
Number of pages7
JournalJournal of Neurochemistry
Volume67
Issue number2
StatePublished - Aug 1 1996

Fingerprint

Iron-Regulatory Proteins
Brain
Demonstrations
RNA
Iron
Ferritins
Immune Sera
Proteins
Complementary DNA
Molecular mass
Aminolevulinic Acid
Transferrin Receptors
RNA-Binding Proteins
Astrocytoma
Post Translational Protein Processing
Gene Library
Assays
Clone Cells
Western Blotting
Observation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

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abstract = "Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5-aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem-loop structures called iron-responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA-protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA- protein complexes are specific to the IRE structure. UV cross-linking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA-protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA-protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of ~100 kDa and is capable of forming two RNA-protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.",
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Demonstration and characterization of the iron regulatory protein in human brain. / Hu, Jing; Connor, James.

In: Journal of Neurochemistry, Vol. 67, No. 2, 01.08.1996, p. 838-844.

Research output: Contribution to journalArticle

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