Structure and function of U5 snRNP

 描述（由申请人提供）：前体mRNA剪接对于所有真核生物中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。对前体mRNA剪接的分子机制的透彻理解有可能为人类疾病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，剪接体是由5个snRNA和100多个蛋白质因子组成的大型RNA/蛋白质复合物。5个snRNA（U1、U2、U4、U 5、U6）和相关蛋白形成snRNP，snRNP在内含子识别、剪接位点定义和剪接反应中起重要作用。U 5 snRNP是一个核心剪接体成分，含有U 5 snRNA和三个蛋白因子（Prp 8，Brr 2和Snu 114），即使在离液盐处理后也能形成稳定的核心。U 5 snRNA与两个外显子相互作用，并且对于在第二催化反应中对齐用于连接的外显子至关重要。Prp 8位于剪接体的中心，并且被假设帮助形成/稳定活性位点，甚至为催化反应贡献官能团。Brr 2是一种DExD/H-box蛋白，负责U4/U6解旋，这是剪接体激活的重要步骤。Snu 114是剪接体中唯一的GTP酶，被认为调节Brr 2的活性。Prp 8和Brr 2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U 5 snRNA的结构。是 尚不清楚U 5 snRNP中的蛋白质如何相互作用，与U 5 snRNA或剪接体催化核心相互作用。该提案的目标是确定U 5 snRNP组分的结构和功能，以及它们与其他蛋白质和RNA（包括剪接体催化核心）的复合物，使用尖端冷冻电子显微镜，晶体学，生物化学和酵母遗传学方法的组合。深入了解U 5 snRNP的结构和功能，将大大推进我们对前体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.