Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU
Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU
复制标题
DOI：
10.1371/journal.pone.0144510
复制
发表时间：
2015-12-09
期刊：
影响因子：
3.7
通讯作者：
McKenna, Sean A.
中科院分区：
文献类型：
--
作者：
Ariyo, Emmanuel O.;Booy, Evan P.;McKenna, Sean A.
富含鸟嘌呤的核酸能够折叠成独特的结构，称为g -四联体。g -四重复合物由平行或反平行排列在堆叠的g -四重复合物平面上的四束鸟苷酸组成。Hoogsteen氢键和以平面为中心的一价阳离子进一步稳定了结构。RHAU （RNA解旋酶与AU-rich element相关）是atp依赖的DExH/D RNA解旋酶家族的成员，可以结合和分解g -四联体。RHAU包含一个核心解旋酶结构域，具有n端延伸，能够识别RNA和DNA g -四联体并具有完全结合亲和力。PITX1是双头体同源盒蛋白中的一员，是脊椎动物发育过程中活跃的转录激活因子，主要在垂体前叶和其他几个器官中活跃。我们之前已经证明，RHAU通过与PITX1 mRNA 3'端的g -四联体相互作用调节PITX1水平。为了了解g -四重体识别的结构基础，研究人员利用多种生物物理技术，包括电泳迁移率位移测定、紫外-可见光谱、圆二色性、动态光散射、小角x射线散射和核磁共振光谱，对纯化的最小PITX1 g -四重体进行了表征。我们的生物物理分析提供了证据，证明RNA g -四重体，而不是其DNA对应物，可以采用平行取向，并且只有RNA可以通过g -四重体的四面与RHAU的n端结构域相互作用。这项工作扩展了我们对RHAU的n端区域如何识别平行g -四联体的见解。
Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element) is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.