In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site
In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site
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DOI：
10.1017/s1355838200000200
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发表时间：
2000-05-01
期刊：
影响因子：
4.5
通讯作者：
Bass, BL
中科院分区：
文献类型：
--
作者：
Öhman, M;Källman, AM;Bass, BL
RNA 编辑酶 ADAR 家族可将完全或大部分双链的 RNA 中的腺苷脱氨。在哺乳动物中，大多数特征底物编码参与神经传递的受体，并且这些底物被认为是哺乳动物酶 ADAR1 和 ADAR2 的目标。尽管某些 ADAR 底物的脱氨基非常混杂，但哺乳动物谷氨酸受体 B (gluR-B) mRNA 前体在一些特定的腺苷处发生脱氨基。与大多数双链 RNA (dsRNA) 结合蛋白一样，ADAR 与许多不同的序列结合，但很少有研究直接测量和比较结合亲和力。我们试图确定 ADAR 脱氨特异性是否发生是因为酶以更高的亲和力与目标区域结合。为了探索这个问题，我们研究了大鼠 ADAR2 与含有 R/G 编辑位点的大鼠 gluR-B 前 mRNA 区域的结合，并将野生型分子与含有减少 R/G 位点编辑的突变的分子进行了比较。尽管与两个序列的结合亲和力几乎相同，但足迹研究表明 ADAR2 在编辑位点周围的离散区域与野生型 RNA 结合，而与突变体的结合似乎是非特异性的。
The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.