Quantitative analysis of CUG-BP1 binding to RNA repeats
Quantitative analysis of CUG-BP1 binding to RNA repeats
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DOI：
10.1093/jb/mvm230
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发表时间：
2008-03-01
影响因子：
2.7
通讯作者：
Ishiura, Shoichi
中科院分区：
文献类型：
--
作者：
Mori, Daisuke;Sasagawa, Noboru;Ishiura, Shoichi
CUG结合蛋白1（CUG-BP 1）是RNA结合蛋白的CUG-BP 1和ETR-3样因子（CELF）家族的成员，并且参与强直性肌营养不良1型（DM 1）。已经鉴定了CUG-BP 1的几种mRNA靶点，包括胰岛素受体、肌肉氯离子通道和心肌肌钙蛋白T。另一方面，CUG-BP 1对CUG重复序列仅具有弱亲和力。我们进行了定量结合试验，以评估CUG-BP 1的亲和力几个重复RNA的表面等离子体共振（SPR）。尽管我们检测到了CUG-P1和CUG重复之间的相互作用，但其他富含UG的序列实际上表现出更强的相互作用。CUG-BP 1与RNA的结合常数表明，UG重复序列的亲和力远强于CUG重复序列。在酵母三杂交系统中，我们还发现CUG-BP 1的N端缺失突变体具有UG重复序列结合活性，而C端缺失突变体则没有。我们的数据表明，CUG-BP 1特异性识别UG重复，可能是通过合作结合的RNA识别基序在蛋白质的两端。这是第一个报告的结合常数CUG-BP 1计算在体外。
CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-P1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.