Molecular dissection of the assembly pathway of spliceosomal U snRNPs

剪接体snRNPs的组装虽然在体外是自发的，但最近已证明依赖于体内大分子蛋白复合物的帮助。这种复合物以神经肌肉疾病脊髓性肌萎缩中受影响的蛋白质SMN命名，在atp依赖反应中促进snRNPs的细胞质组装。在这一过程中，剪接体Sm蛋白（构成U snRNA的蛋白核心）在转移到U snRNA上之前必须先与smn复合物结合。第二种复合物，以其甲基转移酶PRMT5命名，在功能上调节smn复合物。prmt5复合物催化Sm蛋白B/B′、D1和D3中精氨酸的对称二甲基化，从而增加了它们对smn复合物的亲和力。本项目旨在更好地了解组装反应的机理，并阐明在此过程中单个复杂组分的功能。这些研究的先决条件最近已经通过一个体外系统的发展完成，该系统再现了U snrnp组装，并通过产生突变的smn复合物。此外，将进行rna干扰研究，以验证SMN/ prmt5系统可能不仅作为组装因子，而且作为伴侣阻止Sm蛋白聚集和/或错组装到非snrna靶点的假设。最后，我们将讨论其他rnp的形成是否需要类似于SMN/PRMT5系统的辅助因子。

The assembly of spliceosomal snRNPs, albeit spontaneous in vitro, has recently been shown to depend on the aid of a macromolecular protein complex in vivo. This complex, named after SMN, the protein affected in the neuromuscular disease spinal muscular atrophy, facilitates the cytoplasmic assembly of snRNPs in an ATP-dependent reaction. In this process, spliceosomal Sm proteins, which constitute the protein core of U snRNPs, must bind to the SMN-complex prior to their transfer onto the U snRNA. A second com plex, termed after its methyltransferase PRMT5, functionally regulates the SMN-complex. The PRMT5-complex catalyses the symmetric dimethlyation of arginines in Sm proteins B/B', D1 and D3, thereby increasing their affinity for the SMN-complex. This project is designed to provide a better understanding of the mechanism of the assembly reaction, and to elucidate functions of individual complex components during this process. A prerequisite for these studies has recently been accomplished through the developme nt of an in-vitro-system that recapitulates U snRNP-assembly and through the generation of mutant SMN-complexes. In addition, RNA-interference studies will be conducted to test the hypothesis that the SMN/PRMT5-system may not only act as an assembly factor, but also as a chaperone preventing aggregation of Sm proteins and/or misassembly to non-snRNA targets. Finally, we will address whether the formation of other RNPs requires assisting factors akin to the SMN/PRMT5 system.