He Guang-Jun, Yan Yong-Bin
State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Biochem Biophys Rep. 2019 Mar 19;18:100626. doi: 10.1016/j.bbrep.2019.100626. eCollection 2019 Jul.
Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.
聚腺苷酸特异性核糖核酸酶（PARN）催化mRNA聚腺苷酸尾巴的降解，以调节高等真核细胞中的翻译效率和mRNA衰变。全长PARN是一种多结构域蛋白，包含催化核酸酶结构域、R3H结构域、RRM结构域和C端固有无序结构域（CTD）。三个结构良好的RNA结合结构域的作用已得到广泛研究，而关于CTD的了解却很少。在本研究中，通过比较全长PARN与两个缺失CTD的N端片段的热失活和变性行为，研究了CTD对PARN稳定性和聚集能力的影响。我们的结果表明，钾离子诱导了额外的规则二级结构，并增强了PARN对热诱导失活、解折叠和聚集的稳定性。CTD阻止了PARN的热失活，但促进了热聚集在比失活和解折叠所需温度低得多的温度下开始。热转变过程中色氨酸荧光的蓝移表明热处理诱导了结构域组织的重排。CTD增强了钾离子的稳定作用，这意味着CTD的作用主要是通过静电相互作用实现的。这些结果表明，CTD可能与分子主体动态相互作用，CTD的释放通过静电相互作用促进了自我缔合。
