HuR蛋白介导的人类mRNA出核调控过程的结构生物学研究

During the past two years, we have solved the crystal structure of human CF Im, comprising CF Im25 and the RNA recognition motif domain of CF Im68 (CF Im68RRM), and the crystal structure of the CF Im-RNA complex. These structures show that two CF Im68RRM molecules bind to the CF Im25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF Im25-CF Im68RRM heterotetramer, providing structural basis for the mechanism by which CF Im binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF Im68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF Im68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF Im68 makes an essential contribution to pre-mRNA binding. The above results have been published in the Cell Research journal in 2011. In the next phase of our research work, we plan to focus on the structural basis and functional mechanism of the exporting process from nucleus of the mature mRNA. We would like to resolve the complex structures in the HuR mediated mRNA exporting process, such as HuR(RRM1/RRM2)-poly(U) and HuR(RRM3)-poly(A) protein-RNA complexes, and HuR-Trn2, HuR-PP32-Crm1 and HuR(N226)-Trn2 protein-protein complexes. The structural details of the interaction relationships between above complexes and the corresponding cell function verifications would help us to reveal the mechanisms of the HuR mediated mRNA exporting process, and also would enhance our understanding about the Human cell response regulation mechanism in the post-transcription level.

经过两年多的研究探索，我们已完成人类前体mRNA剪切因子Im(CF Im)25KDa亚基与68KDa亚基RRM结构域蛋白质-蛋白质复合物晶体结构的解析；获得了CF Im 复合物与RNA的2.9?分辨率三元复合物结构；阐明了人类CF Im复合物识别前体mRNA“UGUAA”序列的分子机制；相关研究结果已发表在2011年的Cell Research杂志上。下一阶段我们拟针对完成3′端剪切加工修饰后的成熟mRNA的出核机制展开研究，通过解析HuR(RRM1/RRM2)-poly(U)、HuR(RRM3)-poly(A)等蛋白-RNA复合物及HuR-Trn2、HuR-PP32-Crm1、HuR(N226)-Trn2等蛋白-蛋白复合物结构，阐明该过程各效应因子间的相互作用关系，结合细胞功能实验揭示该过程分子运作机理。上述工作的开展将大大增强我们对真核细胞瞬时应答过程在转录后水平的调控机制的理解。

真核生物中，前体mRNA经历内含子切除、转录子重新拼接及5′末端“加帽”和3′末端加尾修饰后，成为具备完整功能的成熟mRNA。由于蛋白质的合成必须在细胞质中完成，携带蛋白质编码的成熟mRNA须跨越核孔复合物进入细胞质，才能实现其生物学功能。对于3′末端含有ARE元件的mRNA而言，HuR蛋白介导的出核机制是此类mRNA最主要的出核转运方式。本项目拟通过结构生物学、生物化学、细胞生物学手段揭示HuR蛋白与mRNA中ARE区段及poly(A)之间的相互识别模式；揭示HuR与Trn2、PP32及Crm1蛋白间的相互作用关系及上述相互作用在mRNA穿越核孔过程中发挥的作用。在课题执行过程中，我们先后顺利完成了HuR蛋白单体及HuR(RRM1/RRM2)-poly(U)复合物结构-功能关系研究、RNA跨核运输重要蛋白hnRNP-L功能机制研究、RNA加工过程tRNA甲基转移酶Trm10及Trm6/Trm61复合物的结构与功能关系研究，还有RNA降解通路新成员Dis3l2-RNA的功能机制研究。上述研究结果大大加深了我们对于人体mRNA出核转运过程中蛋白质-RNA相互作用及别构调节机制的了解，为深入理解RNA加工全过程提供了重要线索。相关研究结果已发表SCI论文22篇，向PDB数据库中提交蛋白质三维结构39个，同时依托课题研究工作培养硕士4人，圆满完成项目计划任务。