DDX21 RNA解旋酶不同结构域的功能特点解析

DEAD-box家族是在生物体内普遍存在的一类高度保守的RNA解旋酶，在RNA的合成和加工、细胞发育和细胞代谢等过程中都发挥着重要作用。DDX21 RNA解旋酶是DEAD-box家族成员之一，而目前为止DDX21的酶学功能及结构特征尚未被完全了解。本研究运用生物化学与生物物理学前沿技术，系统地研究了DDX21各结构域在不同功能中发挥的作用。首先重组构建并纯化了人的DDX21 RNA解旋酶及不同的截短蛋白质，利用动态激光散射和凝胶层析技术分析各蛋白质的寡聚形态，发现N-端的非功能区（N-端181aa）与C-端的4个FRGQR重复结构域对其结构有较大的影响；利用荧光偏振技术比较分析了各蛋白质与单链RNA的结合反应，结果显示，仅保留DEADc和HELICc结构域的截短蛋白质与单链RNA完全没有亲和性，缺失N-端181aa的截短蛋白质对ssRNA的结合能力与全长蛋白质基本一致，而仅缺失C-端的4个重复FRGQR结构域的截短蛋白质与单链RNA的亲和能力将显著下降；利用快速停流检测技术分析各截短蛋白质的解旋及退火活性，发现DEADc、HELICc及GUCT_RHII三个结构域共同参与DDX21的解旋功能，另一方面，缺失C-端4个FRGQR重复结构域的截短蛋白质导致退火能力的丧失。本研究揭示了DDX21的GUCT_RHII结构域及C-端4个FRGQR重复结构域在其结构及功能中发挥的重要作用，为今后研究DDX21的结构及其细胞功能提供了重要的理论依据。

Functional Characteristics Of Different Domains Of DDX21

The DEAD-box family contains ubiquitous, highly conserved RNA helicases that not only take part in the generation and processing of RNAs, but also play critical roles in development and cellular metabolic processes. In humans, the biological functions and enzymatic characteristics of DDX21, the nuclear RNA helicase belongs to the DEAD-box protein family. However, the biological functions and enzymology of DDX21 are not completely understood. In this study, we applied a series of new biochemical and biophysical techniques to systematically analyze the functions of the different DDX21 domains. Firstly, we report the expression and purification of recombinant DDX21 and DDX21 variants with truncations of several structural domains in a bacterial system. Full-length DDX21 is a multimer according to dynamic light scattering and size-exclusion chromatography, and truncated proteins have different oligomeric forms. It was found that the structure of N-terminal non-functional region (N-terminal 181aa) and the C-terminal four FRGQR repeated ddomains had a great influence on its structure. Besides, we evaluated the nucleotide-binding properties of the wild-type DDX21 and the different truncated DDX21 using steady state fluorescence anisotropy with ssRNA ssubstrate. The results showed that the DEADc and HELICc domains had no affinity with ssRNA, while DDX21 without N-terminal 181aa it displayed very similar ssRNA-binding ability as the full-length DDX21. Meanwhile, when the four FRGQR repeated domains were deleted, the affinity of ssRNA decreased significantly. Furthermore, helicase and annealing activity of each protein were analyzed by stopped flow FRET assays. The analysis revealed that DEADc, HELICc, and GUCT_RHII domains cooperatively regulated the helicase activity of DDX21. At the same time, proteins without four repeated FRGQR domains had no annealing ability. This study reveals that the GUCT_RHII domain and four repeated FRGQR domains play crucial roles in nucleic acids binding, unwinding and annealing activity, and provides a theoretical basis for further studies on the structure and cellular functions of DDX21.