Trzcińska-Daneluti Agata M, Górecki Adam, Czubaty Alicja, Kowalska-Loth Barbara, Girstun Agnieszka, Murawska Magdalena, Lesyng Bogdan, Staroń Krzysztof
Institute of Biochemistry, Faculty of Biology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland.
J Mol Biol. 2007 Jun 15;369(4):1098-112. doi: 10.1016/j.jmb.2007.04.017. Epub 2007 Apr 12.
RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.
RNA识别基序（RRM）结构域既能结合核酸，也能结合蛋白质。先前在人拓扑异构酶I的帽区形成的蛋白质复合物中发现了几种含有两个紧密间隔的RRM结构域的蛋白质，人拓扑异构酶I是一种核酶，负责DNA松弛或SR剪接蛋白的磷酸化。为了深入了解RRM蛋白与拓扑异构酶I帽区之间的特异性相互作用，我们使用酵母双杂交系统研究了它们的二元相互作用。已确定hnRNP A1、p54（nrb）和SF2/ASF存在相互作用，但hnRNP L或HuR不存在相互作用。为了确定负责结合的氨基酸模式，采用了实验诱变并对这些过程进行了计算建模。这些研究表明，与帽区结合需要两个RRM结构域和共有序列的六个残基。基于上述数据，提出了hnRNP A1-拓扑异构酶I复合物的结构模型。hnRNP A1结合位点的主要成分是第一个RRM结构域β表面上的一个疏水口袋，类似于与Mago相互作用的Y14蛋白所描述的口袋。我们证明RRM结构域与帽区之间的相互作用对于拓扑异构酶I催化的激酶反应很重要。连同先前描述的SF2/ASF的RRM结构域对DNA切割的抑制作用，上述结果表明RRM蛋白的结合可能调节拓扑异构酶I的活性。
