Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
Nucleic Acids Res. 2011 Feb;39(3):1117-30. doi: 10.1093/nar/gkq837. Epub 2011 Jan 13.
TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.
TIAR 和 HuR 是 mRNA 结合蛋白，在翻译调控中发挥重要作用。它们都具有三个 RNA 识别基序（RRMs），并与富含 AU 的元件（AREs）结合，具有明显重叠的特异性。在这里，我们使用 SPR 表明 TIAR 和 HuR 以纳摩尔范围结合 U 丰富和 AU 丰富的 RNA，对 U 丰富的 RNA 具有更高的整体亲和力。然而，对 U 丰富序列的更高亲和力主要是由于更快地与 U 丰富的 RNA 结合，我们认为这反映了更高的结合概率。TIAR 和 HuR 在结合 RNA 的模式上存在差异。TIAR 能够以纳摩尔亲和力结合脱氧寡核苷酸，而 HuR 的亲和力降低到微摩尔水平。对富含 U 的 DNA 的研究表明，TIAR 结合对 RNA 2'-羟基的依赖性小于 HuR 结合。最后，我们表明，对于 TIAR 与 RNA 结合的前两个结构域，首次记录的 SAXS 数据更符合灵活的、伸长的形状，而不是 Hu 蛋白的前两个结构域结合 RNA 时采用的紧凑形状。因此，我们提出这些竞争细胞中相同结合位点的三 RRM 蛋白以根本不同的方式与其靶标相互作用。
