Nakamaki T, Imamura J, Brewer G, Tsuruoka N, Koeffler H P
Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine 90048, USA.
J Cell Physiol. 1995 Dec;165(3):484-92. doi: 10.1002/jcp.1041650306.
The adenosine-uridine (AU)-rich sequences within the 3' untranslated region (UTR) of many short-lived mRNAs are important in their rapid degradation. We present evidence that human embryonic lung fibroblasts (W138) contain five major proteins of 70, 45, 40, 38, 32.5 kd, which specifically bind the AU-rich region of human granulocyte-macrophage colony-stimulating factor (GM-CSF) 3'UTR containing 7 x AUUUA motifs. The 40 and 38 kd proteins also bound the 3x and 5 x AUUUA cassettes and even more strongly bound to the AUUUUUUUA motif. All five of these proteins showed more abundant localization in the nucleus than the cytoplasm. 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, accompanied by decreased binding activity of the 32.5 kd protein in the nucleus. 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 cytoplasm as well as the nucleus, but another group of AU-binding proteins (AUF1 [45, 40, 38 kd], and 32.5 kd), which are not hnRNP, have characteristics related to those of hnRNPs. Taken together with our previous results (Akashi et al., 1994, Blood, 83:3182-3187), AU-binding factors including 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 GM-CSF mRNA, indicating that binding of these proteins to their cognate RNA is not responsible for the stabilization of these transcripts.
许多短寿命mRNA的3'非翻译区（UTR）内富含腺苷酸-尿苷酸（AU）的序列在其快速降解过程中起着重要作用。我们提供的证据表明，人胚肺成纤维细胞（W138）含有5种主要蛋白质，分子量分别为70、45、40、38、32.5kd，它们能特异性结合人粒细胞-巨噬细胞集落刺激因子（GM-CSF）3'UTR的富含AU区域，该区域含有7个AUUUA基序。40kd和38kd的蛋白质也能结合3个和5个AUUUA盒式结构，并且与AUUUUUUUA基序的结合更强。这5种蛋白质在细胞核中的定位均比细胞质中更为丰富。32.5kd的蛋白质是主要的细胞质AU结合蛋白。用放线菌素D孵育后，细胞质中45、40、38和32.5kd蛋白质的结合活性显著增加，同时细胞核中32.5kd蛋白质的结合活性降低。抗不均一核核糖核蛋白C（hnRNP C）抗体可免疫沉淀40kd和38kd的蛋白质，抗富含AU元件RNA结合蛋白（AUF1）抗体可免疫沉淀45、40和38kd的蛋白质。目前的结果不仅表明hnRNP C是存在于细胞质和细胞核中的AU结合蛋白，而且另一组非hnRNP的AU结合蛋白（AUF1[45、40、38kd]和32.5kd）具有与hnRNPs相关的特性。结合我们之前的结果（赤ashi等人，1994年，《血液》，83:3182 - 3187），包括hnRNP C和AUF1在内的能结合超过3个AUUUA基序的AU结合因子可能参与了这些转录本的快速降解。在几种能稳定GM-CSF mRNA的刺激下，这些蛋白质与富含AU序列的结合活性没有显著的定量变化，这表明这些蛋白质与其同源RNA的结合并不负责这些转录本的稳定。
