Structural analysis of the assembly machinery of spliceosomal U snRNPs

剪接体 U snRNP 组装机制的结构分析

• 批准号: 91932152
• 负责人: Professor Dr. Utz Fischer
• 金额: --
• 依托单位: Lehrstuhl für Biomolekulare NMR-Spektroskopie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/91932152?language=en
• 关键词: Structural; analysis; assembly; machinery; spliceosomal

The spliceosome is a macromolecular machine that catalyzes splicing of eukaryotic pre-mRNA. Four uridyl-rich small nuclear ribonucleoproteins (U snRNPs) are major building blocks of spliceosomes with various functions including identification of intronic sequences and the formation of its catalytic center. Each U snRNP contains a specific set of proteins dedicated to its specific task in splicing. However, they also have a set of seven Sm proteins in common that form the structural core of snRNPs. The formation of this Sm core, although spontaneously in vitro, follows a highly regulated and assisted pathway in vivo. It starts with the nuclear export of newly synthesized snRNAs and their assembly with Sm proteins in the cytoplasm. U snRNPs that have formed the Sm core translocate to the nucleus where additional particle-specific proteins are added. The mature particles are eventually incorporated into the spliceosome to enable splicing. Several labs including ours have identified a sophisticated machinery that assists the assembly of splicesomal U snRNPs in vivo. This machinery consists of at least 12 proteins that are organized in two cooperating units termed SMN-complex and PRMT5-complex, respectively. While the SMN-complex acts as RNP assembler and loads Sm proteins onto the snRNA, the PRMT5 complex functions upstream in this pathway. Its main task is committed by the assembly chaperone pICln, which forces Sm proteins into higher order structures (termed 6S complex and pICln-D3/B) required for the subsequent transfer onto the SMN-complex. In the past funding period, we have solved the atomic structure of two key intermediates of the snRNP assembly machinery. The 6S complex structure identifies the PRMT5 complex subunit pICln as an assembly chaperone and Sm-protein mimic, which enables the topological organization of five Sm proteins into a closed ring. A second structure of 6S bound to the SMN-complex components SMN and Gemin2 uncovers important mechanistic aspects of pICln elimination and Sm protein activation for snRNA binding. In this grant application, we wish to extend our studies on the assembly machinery focusing on the structural analysis of the entire SMN-complex and its dynamics during snRNP formation. Furthermore, the structural basis of 6S-formation on the PRMT5 complex will be investigated. From these studies we expect detailed structural and functional insight into critical steps of snRNP formation in vivo.

剪接体是催化真核生物前体mRNA剪接的大分子机器。四种富含尿苷酰的小核核糖核蛋白（U snRNP）是剪接体的主要结构单元，具有多种功能，包括识别内含子序列和形成其催化中心。每个U snRNP包含一组特定的蛋白质，专门用于其特定的剪接任务。然而，它们也有一组共同的七种Sm蛋白，形成snRNP的结构核心。 这种Sm核心的形成，虽然自发地在体外，遵循高度调节和协助的途径在体内。它始于新合成的snRNA的核输出及其与细胞质中的Sm蛋白的组装。已经形成Sm核心的U snRNP易位到细胞核，在细胞核中添加额外的颗粒特异性蛋白。成熟的颗粒最终并入剪接体中以实现剪接。包括我们在内的几个实验室已经确定了一种复杂的机制，可以帮助体内剪接体U snRNP的组装。这种机制由至少12种蛋白质组成，它们分别组织在两个合作单位中，称为SMN复合物和PRMT5复合物。虽然SMN复合物充当RNP组装器并将Sm蛋白加载到snRNA上，但PRMT5复合物在该途径的上游起作用。它的主要任务是由组装分子伴侣pICln承担，其迫使Sm蛋白进入后续转移到SMN复合物上所需的更高级结构（称为6S复合物和pICln-D3/B）。在过去的资助期间，我们已经解决了snRNP组装机器的两个关键中间体的原子结构。6S复合物结构鉴定PRMT5复合物亚基pICln作为组装分子伴侣和Sm-蛋白模拟物，这使得五个Sm蛋白的拓扑组织成为一个闭环。结合到SMN复合物组分SMN和Gemin2的6S的第二结构揭示了pICln消除和Sm蛋白激活snRNA结合的重要机制方面。在这项资助申请中，我们希望扩大我们的研究集中在整个SMN复合物的结构分析和snRNP形成过程中的动态装配机械。此外，6S-形成的PRMT5复合物的结构基础进行了研究。从这些研究中，我们期望详细的结构和功能的洞察snRNP在体内形成的关键步骤。