Heinrichs V, Hackl W, Lührmann R
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
J Mol Biol. 1992 Sep 5;227(1):15-28. doi: 10.1016/0022-2836(92)90678-d.
The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.
主要的小核核糖核蛋白（snRNP）U1、U2、U5和U4/U6参与前体mRNA的剪接。U1、U2、U4和U5 RNA共享一个高度保守的序列基序PuA(U)nGPu，称为Sm位点，其通常两侧为两个发夹环。Sm位点为剪接体snRNP共有的一组常见蛋白质B'、B、D1、D2、D3、E、F和G提供主要结合位点。我们通过在U1 snRNP颗粒内使用紫外线诱导的RNA-蛋白质交联，研究了常见snRNP蛋白质识别snRNA的Sm位点的能力。体外重建的U1 snRNP颗粒含有用32P标记的U1 snRNA。蛋白质与这种U1 snRNA的交联仅在构成保守Sm位点的snRNA单链延伸存在时发生。通过一维和二维凝胶电泳对交联蛋白质的表征表明，snRNP蛋白质G已与U1 snRNA交联。用抗G抗血清对交联的RNA-蛋白质复合物进行特异性免疫沉淀证实了这一点。通过用核糖核酸酶T1和A进行指纹分析，交联位于U1 snRNA上；这表明蛋白质G已与Sm位点5'端一半（AAUUUGUGG）内的AAU延伸交联。这些结果表明，snRNP蛋白质G可能参与Sm位点的直接识别。
