Rimmele M E, Belasco J G
Skirball Institute of Biomolecular Medicine and Department of Microbiology, New York University School of Medicine, New York 10016, USA.
RNA. 1998 Nov;4(11):1386-96. doi: 10.1017/s1355838298981171.
The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".
剪接体蛋白U1A和U2B''各自利用同源的RNA识别基序（RRM）结构域特异性结合其各自的小核RNA（snRNA）靶标，即U1hpll和U2hpIV，这两个茎环在序列上相似但又不同。先前的研究表明，辅助蛋白U2A'促进U2B''与U2hpIV的结合，而U1A与U1hpll的特异性结合则不需要辅因子。在此我们报告，U2A'使U2B''能够区分U2hpIV的环序列与U1hpll的环序列，但在茎序列区分中不起作用。虽然U2A'也能促进U1A与U2hpIV的异源特异性结合，但由于U2A'对U2B''的亲和力大约高500倍，因此需要更高浓度的辅助蛋白。额外的实验已经确定了U1A中的一个亮氨酸残基（Leu-44），它对于该蛋白优先识别U1hpll的环序列而非U2hpIV的环序列的内在特异性至关重要。我们的数据表明，U1A和U2B''之间RNA结合特异性的大部分差异可由这个亮氨酸残基以及辅助蛋白U2A'对U2B''特异性的贡献来解释。
