Lelay-Taha M N, Reveillaud I, Sri-Widada J, Brunel C, Jeanteur P
J Mol Biol. 1986 Jun 5;189(3):519-32. doi: 10.1016/0022-2836(86)90321-9.
Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.
利用氯化铯密度梯度离心和DEAE-琼脂糖离子交换色谱相结合的方法，对来自HeLa细胞的含有U1和U5 snRNA的小核核糖核蛋白（snRNP）进行了分级分离。该方法基于Mg2+赋予snRNP的极高稳定性，使其能够耐受氯化铯中极高的离子强度。用这种方法制备的U1 snRNP包含所有9种主要蛋白质（68K、A、B、B'、C、D、E、F、G），与先前通过免疫沉淀鉴定的蛋白质相对应，因此可被抗RNP和抗Sm抗体沉淀。以这种方式纯化的U5 snRNP包含常见的D到G蛋白，并且还富含一种可能是U5 snRNP特异性的25×10³ Mr蛋白。由D到G蛋白覆盖的抗核心U5 snRNA序列（核苷酸84至3' OH）仅延伸了6个核苷酸。在U4-U6 snRNP中也观察到类似情况，我们已获得足够纯的形式来检测受保护的序列。然而，U4-U6 snRNP中U4的抗核心序列（核苷酸116至3' OH）延伸了37个核苷酸，这表明该颗粒的蛋白质组成可能比U5 snRNP更复杂。鉴于我们目前对受蛋白质保护的snRNA序列的了解，对snRNP的核糖核蛋白组织进行了总结和讨论。
