Black D L, Pinto A L
Howard Hughes Medical Institute, New Haven, Connecticut.
Mol Cell Biol. 1989 Aug;9(8):3350-9. doi: 10.1128/mcb.9.8.3350-3359.1989.
To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.
为了解U5小核核糖核蛋白（snRNP）如何与其他剪接体成分相互作用，对其结构以及与U4/U6 snRNP的结合进行了分析。通过在甘油梯度上分离HeLa细胞核提取物中的snRNP，研究了U5 snRNP与U4/U6 snRNP的相互作用。鉴定出一种在25S处迁移且包含U4、U5和U6但不包含U1或U2 snRNA的复合物。与用天然凝胶电泳分离snRNP的结果不同，这种U4/U5/U6 snRNP复合物需要ATP才能从单个snRNP组装而成。然后比较了U5 snRNP以及U4/5/6 snRNP复合物中U5 RNA的结构。寡核苷酸靶向的RNase H消化鉴定出U5 snRNP中一个能够与其他核酸序列碱基配对的RNA序列。化学修饰实验确定了该序列以及另外两个U5 RNA序列在U5 RNP中可被修饰。其中一个区域是U5 RNA二级结构中的一个大环，其序列从酿酒酵母到人类都保守。有趣的是，未观察到游离U5 snRNP与U4/U5/U6 snRNP复合物中的U5在修饰上存在差异，这表明U4/U5/U6 snRNP组装不需要识别U5 snRNP中的特定RNA序列。
