Hartmuth K, Raker V A, Huber J, Branlant C, Lührmann R
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mankopff-Strasse 2, Marburg, D-35037, Germany.
J Mol Biol. 1999 Jan 8;285(1):133-47. doi: 10.1006/jmbi.1998.2300.
The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.
小核核糖核蛋白颗粒（snRNP）U1、U2、U4和U5含有一组共同的8种Sm蛋白，这些蛋白与其组成成分U小核RNA（small nuclear RNA）的保守单链5'-PuAU3-6GPu-3'（Sm结合位点）区域结合，形成Sm核心核糖核蛋白。利用天然的和体外重组的U1 snRNP，分析了Sm核心核糖核蛋白内RNA对化学结构探针的可及性。体外重组U1 snRNP的羟基自由基足迹分析表明，包括Sm结合位点在内的一个大的连续RNA区域内的核糖受到保护。这种保护依赖于Sm蛋白的结合。与核糖不同，Sm核心位点内的磷酸基团可被修饰试剂作用。5'端的不变腺苷残基以及Sm结合位点下游两个核苷酸处的一个腺苷，对硫酸二甲酯表现出意外的反应性。这种新的反应性可归因于腺苷的N7-甲基化，在裸露的RNA中未观察到，表明这是Sm核心核糖核蛋白内RNA-蛋白质相互作用的固有特性。此外，在Sm核心核糖核蛋白组装过程中，这种反应性与Sm亚核心中间体的形成同时出现。由于Sm亚核心可被视为该组装途径中的关键复合物，这些结果表明Sm位点腺苷碱基的特殊反应性可能是Sm核心核糖核蛋白正确组装的诊断标志。与此观点一致的是，发现Sm核心核糖核蛋白异常的N7-A甲基化敏感性与其导入非洲爪蟾卵母细胞核的能力之间存在很强的相关性。
