Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2
Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2
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DOI：
10.1074/jbc.m007642200
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发表时间：
2000-12-08
影响因子：
4.8
通讯作者：
Smith, B
中科院分区：
文献类型：
--
作者：
Shan, JG;Moran-Jones, K;Smith, B
异质核核糖核蛋白（hnRNP）A2结合21个核苷酸的髓鞘碱性蛋白mRNA反应元件，A2RE，和A2RE样序列在其他本地化的mRNA，是一个反式作用因子在少突胶质细胞胞质RNA运输。重组人hnRNP A1和A2用于生物传感器中，以探索与A2RE和同源寡脱氧核糖核苷酸的相互作用。这两种蛋白质都有一个单一的位点，结合寡核苷酸与显着不同的序列，但不结合肝素的存在。两者还具有仅结合A2RE且不受肝素影响的第二特异性位点，hnRNP A2在后一位点结合A2RE，其Kd接近50 nM，而hnRNP A1的Kd高于10 μ M。紫外线交联试验得出了类似的结论。突变A2RE序列，在早期的定性研究中似乎不结合hnRNP A2或支持少突胶质细胞中的RNA运输，对于这种蛋白质具有高于5 μ M的解离常数。两个串联的RNA识别基序（RRM），但不是单独的RRM，模仿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上的序列特异性A2RE结合位点由两种RRM顺式作用形成。
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 muM. 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 muM 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.