CHARACTERIZATION OF ADENOSINE-URIDINE-RICH RNA-BINDING FACTORS

The adenosine-uridine (AU)-rich sequences within the 3' untranslated r egion (UTR) of many short-lived mRNAs are important in their rapid deg radation. We present evidence that human embryonic lung fibroblasts (W 138) contain five major proteins of 70, 45, 40, 38, 32.5 kd, which spe cifically bind the AU-rich region of human granulocyte-macrophage colo ny-stimulating factor (CM-CSF) 3'UTR containing 7 x AUUUA motifs. The 40 and 38 kd proteins also bound the 3 x and 5 x AUUUA cassettes and e ven mere strongly bound to the AUUUUUUUA motif. All five of these prot eins showed mere abundant localization in the nucleus than the cytopla sm. The 32.5 kd protein was the major cytoplasmic AU-binding protein. Incubation with actinomycin D resulted in a marked increase in binding activity of 45, 40, 38, and 32.5 kd proteins in the cytoplasm, accomp anied by decreased binding activity of the 32.5 kd protein in the nucl eus. Antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) immunoprecipitated the 40 and 38 kd proteins, and antibody against the AU-rich element RNA-binding protein (AUF1) immunoprecipitated the 45, 40, and 38 kd proteins. The present results not only demonstrated that hnRNP C are AU-binding proteins which are present in the cytopla sm as well as the nucleus, but another group of AU-binding proteins (A UF1 [45, 40, 38 kd], and 32.5 kd), which are net hnRNP, have character istics related to those of hnRNPs. Taken together with our previous re sults (Akashi et al., 1994, Blood, 83:3182-3187), AU-binding factors i ncluding hnRNP C and AUF1, which bind more than 3 x AUUUA motifs, may be involved in rapid degradation of these transcripts. No significant quantitative changes of these proteins in their binding activity to AU -rich sequences occurred in response to several stimuli that stabilize CM-CSF mRNA, indicating that binding of these proteins to their cogna te RNA is not responsible for the stabilization of these transcripts. (C) 1995 Wiley-Liss. Inc.