Nelissen R L, Will C L, van Venrooij W J, Lührmann R
Department of Biochemistry, University of Nijmegen, The Netherlands.
EMBO J. 1994 Sep 1;13(17):4113-25. doi: 10.1002/j.1460-2075.1994.tb06729.x.
The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.
已知U1小核核糖核蛋白颗粒（snRNP）特异性的70K和A蛋白可直接结合到U1 snRNA的茎环结构上，而U1-C蛋白并不结合裸露的U1 snRNA，其与U1 snRNP的结合依赖于其他U1 snRNP蛋白成分。以U1-70K和U1-C蛋白为研究对象，对这些颗粒特异性蛋白与U1 snRNP结合过程中的蛋白质-蛋白质相互作用进行了研究。用体外翻译的U1-C蛋白与裸露的U1 snRNA或缺乏其特异性蛋白的纯化U1 snRNP（核心U1 snRNP）孵育后形成的复合物进行免疫沉淀，结果显示，U1-C与U1 snRNP结合既需要常见的snRNP蛋白，也需要U1-70K蛋白。对各种体外翻译的U1-70K突变体进行结合研究表明，U1-70K的N端结构域对于U1-C与核心U1 snRNP的相互作用是必需且充分的。令人惊讶的是，U1-70K蛋白的几个N端片段，虽然缺乏U1-70K的RNP-80基序且不结合裸露的U1 RNA，但却能与核心U1 snRNP稳定结合。这表明，常见的U1 snRNP蛋白与U1 RNA结合后会产生一个新的U1-70K结合位点。U1-70K的N端结构域与核心RNP结构域之间的相互作用对U1 snRNP具有特异性；与核心U2或U5 snRNP未观察到稳定结合，这表明snRNP核心结构域之间存在本质的结构差异。使用纯化的U1 snRNP进行的化学交联实验支持了U1特异性蛋白与常见snRNP蛋白之间存在直接蛋白质-蛋白质相互作用的证据。检测到U1-70K与常见的D2或B'/B蛋白之间以及U1-C与B'/B之间存在单独的交联。本文提出了一个U1 snRNP组装模型，其中RNA骨架上的常见蛋白复合物作为U1特异性蛋白结合的平台。
