AKTA Pure Chromatography System

 描述(申请人提供)：前-信使核糖核酸剪接对于所有真核生物的基因表达是必不可少的，剪接错误会导致遗传疾病和许多其他疾病。深入了解前mRNA剪接的分子机制有可能为人类疾病的治疗提供有用的方法。内含子的剪接是通过剪接体催化的两个酯交换反应进行的，剪接体是一个由5个SnRNA和100多个蛋白质因子组成的大型RNA/蛋白质复合体。5个SnRNAs(U1、U2、U4、U5、U6)及其相关蛋白形成SnRNPs，在内含子识别、剪接位点定义和剪接反应中发挥重要作用。U5SnRNP是一个核心剪接体成分，它包含U5SnRNA和三个蛋白质因子(Prp8、Brr2和Snu114)，即使在杂乱盐处理后也形成一个稳定的核心。U5SnRNA与两个外显子相互作用，在第二催化反应中对连接外显子起关键作用。Prp8位于剪接体的中心，被认为有助于形成/稳定活性部位，甚至为催化反应贡献官能团。Brr2是一种DExD/H-box蛋白，负责U4/U6的解离，这是剪接体激活的关键步骤。Snu114是剪接体中唯一的GTP酶，被认为调节Brr2的活性。Prp8和Brr2的几个结构域的晶体结构是已知的，但没有任何全长蛋白质或U5 SnRNA的结构。它是 不清楚U5 SnRNP中的蛋白质如何相互作用，如何与U5 SnRNA或剪接体催化核心相互作用。这项建议的目标是利用尖端冷冻电子显微镜、结晶学、生物化学和酵母遗传学方法相结合的方法，确定U5 SnRNP组件的结构和功能，以及它们与其他蛋白质和RNA的复合体(包括剪接体催化核心的那些)。深入了解U5SnRNP的结构和功能将极大地促进我们对Pre-mRNA剪接的分子机制的理解。

 DESCRIPTION (provided by applicant): Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. The five snRNAs (U1, U2, U4, U5, U6) and associated proteins form snRNPs, which play important roles in intron recognition, splice site definition, and the splicing reaction. U5 snRNP is a core spliceosomal component that contains U5 snRNA and three protein factors (Prp8, Brr2, and Snu114) that form a stable core even after chaotropic salt treatment. U5 snRNA interacts with both exons and is critical for aligning the exons for ligation in the second catalytic reaction. Prp8 lies in the heart of the spliceosome and is hypothesized to help form/stabilize the active site or even contribute functional groups to the catalytic reaction. Brr2 is a DExD/H-box protein responsible for U4/U6 unwinding, an essential step for spliceosomal activation. Snu114 is the only GTPase in the spliceosome and is thought to regulate the activity of Brr2. Crystal structures of several domains of Prp8 and Brr2 are known, but there is no structure of any full-length protein or U5 snRNA. It is not clear how proteins in U5 snRNP interact with each other, with U5 snRNA, or with the spliceosome catalytic core. The goal of this proposal is to determine the structure and function of components of the U5 snRNP, as well as their complexes with other proteins and RNAs (including those at the spliceosome catalytic core), using a combination of cutting edge cryo-electron microscopy, crystallography, biochemistry, and yeast genetics approaches. A thorough understanding of the structure and function of U5 snRNP will significantly advance our understanding of the molecular mechanisms of pre-mRNA splicing in general.