PURIFICATION OF A RNA-BINDING PROTEIN FROM RAT-LIVER - IDENTIFICATIONAS FERRITIN-L CHAIN AND DETERMINATION OF THE RNA PROTEIN BINDING CHARACTERISTICS/

In cultured rat hepatocytes the degradation of phosphoenolpyruvate car boxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability, The 3'-untranslated region of phosphoeno lpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation as says, A cytosolic protein was isolated, which bound to the phosphoenol pyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs, The protein was purified to homogeneity; it had an a pparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa, Sequence analysis of a tryptic pept ide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphoryl ation with cAMP-dependent protein kinase and was augmented after depho sphorylation with alkaline phosphatase. Weak binding was observed in e xtracts from okadaic acid-treated cultured hepatocytes compared with u ntreated cells, Preincubation of ferritin with an anti-phosphoserine o r an anti-phosphothreonine antibody attenuated binding to RNA, while a n anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues w ere in close proximity to the RNA-binding region, Ferritin is the iron storage protein in the Liver, Binding of ferritin to RNA was diminish ed in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect, It is concluded that ferritin m ight function as RNA-binding protein and that it may have important fu nctions in the general regulation of cellular RNA metabolism.