Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics.
In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3'-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.
Heise
T
T
Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Nath
A
A
Jungermann
K
K
Christ
B
B
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005293:Ferritins / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008099:Liver / Q000737:chemistry*; D008297:Male; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014018:Tissue Distribution
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D005293:Ferritins; D007501:Iron; D017868:Cyclic AMP-Dependent Protein Kinases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)