Distinct binding properties of TIAR RRMs and linker region
Distinct binding properties of TIAR RRMs and linker region
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DOI：
10.4161/rna.24341
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发表时间：
2013-04-01
期刊：
影响因子：
4.1
通讯作者：
Wilce, Jacqueline A.
中科院分区：
文献类型：
--
作者：
Kim, Henry S.;Headey, Stephen J.;Wilce, Jacqueline A.
RNA结合蛋白TIAR是一种mRNA结合蛋白，其作为翻译阻遏物，在细胞应激条件下特别重要。它通过其RNA识别基序（RRM）结构域与靶mRNA和DNA结合，并通过形成应激颗粒参与剪接调节和翻译抑制。TIAR也被证明可以结合ssDNA并在转录调控中发挥作用。在这里，我们显示，使用表面等离子体共振和核磁共振光谱，具体的作用，个别TIAR域的高亲和力结合RNA和DNA的目标。我们证实，RRM 2的TIAR是主要的RNA和DNA结合域。然而，与富含U的RNA和富含T的DNA的强纳摩尔亲和力结合取决于RRM 2的C末端的接头区中发现的六个氨基酸残基的存在。RRM 1本身显示出与DNA的结合优于与RNA的结合。我们进一步表征RRM 2与C-末端延伸和富含AU的靶RNA序列之间的相互作用，使用NMR光谱来鉴定参与结合的氨基酸残基。我们证明了TIAR RRM 2及其C-末端延伸是TIAR与靶RNA序列高亲和力（nM）相互作用的主要贡献者。
The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of stress granules. TIAR has also been shown to bind ssDNA and play a role in the regulation of transcription. Here we show, using surface plasmon resonance and nuclear magnetic resonance spectroscopy, specific roles of individual TIAR domains for high-affinity binding to RNA and DNA targets. We confirm that RRM2 of TIAR is the major RNA- and DNA-binding domain. However, the strong nanomolar affinity binding to U-rich RNA and T-rich DNA depends on the presence of the six amino acid residues found in the linker region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. We further characterize the interaction between RRM2 with the C-terminal extension and an AU-rich target RNA sequence using NMR spectroscopy to identify the amino acid residues involved in binding. We demonstrate that TIAR RRM2, together with its C-terminal extension, is the major contributor for the high-affinity (nM) interactions of TIAR with target RNA sequences.