Domain Architectures and characterization of an RNA-binding protein, TLS
Domain Architectures and characterization of an RNA-binding protein, TLS
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DOI：
10.1074/jbc.m408552200
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发表时间：
2004-10-22
影响因子：
4.8
通讯作者：
Morikawa, K
中科院分区：
文献类型：
--
作者：
Iko, Y;Kodama, TS;Morikawa, K
脂肪肉瘤易位蛋白（Translocated in liposarcoma，TLS）是核内不均一核糖核蛋白复合物的重要组成部分，参与前体mRNA的剪接和完全加工的mRNA向胞浆的输出。我们研究了域组织的人TLS的结合使用有限的蛋白水解，基质辅助激光解吸电离飞行时间质谱，圆二色谱，电感耦合等离子体原子发射光谱，和NMR光谱的方法。我们发现，RNA识别基序（RRM）和锌指样结构域专门形成蛋白酶抗性核心结构内的分离的TLS蛋白片段，而其余地区，包括Arg-Gly-Gly重复，似乎是完全非结构化的。因此，TLS包含非结构化的N-末端一半，随后是RRM和锌指样结构域，它们通过柔性接头彼此连接。我们还进行了核磁共振分析，以获得更详细的了解个别RRM和锌指状结构域。Cd-113 NMR分析的锌指状结构域验证，锌是协调与四个半胱氨酸的C4型方案。我们还研究了每个结构域与含有GGUG序列的寡聚RNA的相互作用，这似乎是TLS剪接功能的关键。骨架酰胺NMR化学位移扰动分析表明，锌指结构域结合含GGUG的RNA，解离常数约为1.0 × 10（-5）M，而RRM结构域与该RNA没有可观察到的相互作用。这个令人惊讶的结果意味着锌指结构域在RNA识别中比RRM结构域起更主要的作用。
Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The Cd-113 NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0x10(-5) M, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.