Crystallographic studies on UNR, a protein important for translation and decay of mRNA, and on the RNA-induced silencing complex RISC

RNA结合蛋白UNR参与了IRES介导的mRNA翻译的启动，MCRD依赖的mRNA的衰退，以及作为性致死蛋白的辅助抑制因子而抑制msl2-mRNA的翻译。UNR直接与mRNAs的IRES或MCRD元件结合，并且UNR还能够与其他蛋白质形成无RNA的复合体，如Poly(A)结合蛋白、UNR相互作用蛋白(UNIP)和性致死蛋白。UNR由五个冷激波结构域组成，但它们在不同功能复合体中的作用还有待阐明。为了了解UNR的多种功能的结构基础，我们希望通过X射线结晶学的方法来确定UNR和含有UNR的蛋白质或蛋白质-RNA复合体的三维结构。第二个项目涉及RNA诱导沉默复合体(RISC)的制备、结晶和晶体结构测定，RISC在通过RNA干扰调控基因表达方面发挥着核心作用。一个最小的人类RISC，由蛋白质DICER，ArgAerte2和TRBP形成，被证明具有dsRNA负载，链选择和靶向mRNA切割活性。我们将建立一个真核共表达系统，以获得合理数量的异三聚体蛋白复合体，用于结晶实验。主要目的是分析最小RISC在不同功能状态下的晶体结构。此外，这种重组的最小RISC将用于单粒子冷冻电子显微镜研究。

The RNA-binding protein UNR is involved in the IRES-mediated initiation of mRNA translation, the mCRD-dependent decay of mRNA, and in the inhibition of msl2-mRNA translation as co-repressor of the sex-lethal protein. UNR binds directly to the IRES- or the mCRD-element of mRNAs, and UNR is also capable to form RNA-free complexes with other proteins like the Poly(A)-binding protein, the UNR-interacting protein (UNRIP), and the sex-lethal protein. UNR consists of five cold shock domains, but their role with respect to the different functional complexes has to be elucidated. In order to understand the structural basis for the versatile functions of UNR we want to determine the three-dimensional structure of UNR and of functional UNR-containing proteinprotein or protein-RNA complexes by means of X-ray crystallography. A second project concerns the preparation, crystallization and crystal structure determination of an RNA-induced silencing complex (RISC), which plays a central role in the control of gene expression by RNA interference. A minimal human RISC, formed by the proteins Dicer, Argonaute2 and TRBP, was shown to exhibit dsRNA loading, strand selection, and target mRNA cleavage activities. We will establish a eukaryotic co-expression system in order to obtain reasonable amounts of the heterotrimeric protein complex for crystallization experiments. Major aim is the crystal structure analysis of the minimal RISC in different functional states. Additionally, this recombinant minimal RISC will be used for single particle cryo-electron microscopy studies.