Hamilton B J, Burns C M, Nichols R C, Rigby W F
Section of Connective Tissue Diseases, Departments of Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
J Biol Chem. 1997 Nov 7;272(45):28732-41. doi: 10.1074/jbc.272.45.28732.
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A)+ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.
不均一核核糖核蛋白A1（hnRNP A1）穿梭于细胞质和细胞核之间，在RNA代谢中发挥重要作用。虽然已证明细胞核中的hnRNP A1能结合内含子序列并调节剪接，但细胞质中的hnRNP A1与聚腺苷酸（poly(A)+）RNA相关联，这表明其具有不同的RNA配体特异性。先前的研究表明，细胞质中的hnRNP A1能够与重复的AUUUA序列（ARE）进行高亲和力结合，这些序列已被证明可调节mRNA的周转和翻译。通过二维凝胶电泳和蛋白水解研究相结合的方法，我们确定hnRNP A1（或翻译后以相同方式调控的结构相关蛋白）是人类T淋巴细胞中能够与全长粒细胞-巨噬细胞集落刺激因子mRNA背景下的ARE相互作用的主要细胞质蛋白。我们还在交联和迁移率变动实验中证明，相对于前体mRNA，细胞质中的hnRNP A1更优先结合ARE。RNA聚合酶II抑制作用增加了细胞质hnRNP A1对ARE（AUBP活性）和聚尿苷酸-琼脂糖的结合，而细胞核中的hnRNP A1结合不受影响。细胞核和细胞质中的hnRNP A1可通过其RNA结合对二酰胺和N-乙基马来酰亚胺的不同敏感性来区分。通过抑制剂和代谢标记研究确定，RNA聚合酶II抑制后细胞质hnRNP A1的AUBP活性增加与丝氨酸-苏氨酸去磷酸化相关。因此，细胞质和细胞核中的hnRNP A1表现出不同的RNA结合谱，可能是通过丝氨酸-苏氨酸磷酸化来传导的。这些发现与hnRNP A1在细胞核和细胞质中基因表达的转录后调控中发挥不同作用的特定能力相关。
