Cloning of a novel RNA binding polypeptide (RA301) induced by hypoxia/reoxygenation.
Astrocytes have a critical role in the neuronal response to ischemia, as their production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of their function as RNA splicing factor. Northern analysis displayed striking induction only in cultured astrocytes within 15 min of reoxygenation and reached a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrated expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA was mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function of astrocytes was suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues.
D000332:Aerobiosis; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression / Q000187:drug effects; D015850:Interleukin-6 / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D017386:Sequence Homology, Amino Acid; D000068103:Serine-Arginine Splicing Factors
D000906:Antibodies; D018076:DNA, Complementary; D015850:Interleukin-6; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; C490770:Tra2b protein, rat; D000068103:Serine-Arginine Splicing Factors