Shan J, Moran-Jones K, Munro T P, Kidd G J, Winzor D J, Hoek K S, Smith R
Biochemistry Department, The University of Queensland, Queensland 4072, Australia.
J Biol Chem. 2000 Dec 8;275(49):38286-95. doi: 10.1074/jbc.M007642200.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.
异质性核核糖核蛋白（hnRNP）A2可结合一段21个核苷酸的髓鞘碱性蛋白mRNA反应元件（A2RE）以及其他定位mRNA中的A2RE样序列，并且是少突胶质细胞胞质RNA转运中的反式作用因子。重组人hnRNP A1和A2被用于生物传感器中，以探究它们与A2RE及同源寡脱氧核苷酸的相互作用。这两种蛋白质都有一个结合寡核苷酸的位点，所结合的寡核苷酸序列明显不同，但在肝素存在的情况下不结合。它们还都有第二个特异性位点，该位点仅结合A2RE且不受肝素影响。hnRNP A2在后一个位点结合A2RE的解离常数（K(d)）接近50 nM，而hnRNP A1的K(d)高于10 μM。紫外线交联试验得出了类似的结论。在早期定性研究中似乎不结合hnRNP A2或不支持少突胶质细胞中RNA转运的突变A2RE序列，与该蛋白质的解离常数高于5 μM。两个串联的RNA识别基序（RRMs）而非单个RRMs模拟了hnRNP A2的结合行为。这些数据突出了A2RE与这些hnRNP相互作用的特异性，并表明hnRNP A2上序列特异性的A2RE结合位点是由两个顺式作用的RRMs形成的。
