Mougin Annie, Gottschalk Alexander, Fabrizio Patrizia, Lührmann Reinhard, Branlant Christiane
UMR 7567 CNRS-UHP Nancy I, Maturation des ARN et Enzymologie Moléculaire, Université H. Poincaré B.P. 239, 54506 Vandoeuvre-les Nancy Cédex, France.
J Mol Biol. 2002 Apr 12;317(5):631-49. doi: 10.1006/jmbi.2002.5451.
The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.
U4/U6.U5三小核核糖核蛋白颗粒是剪接体的关键组成部分。我们使用化学试剂和核糖核酸酶，首次对三小核核糖核蛋白颗粒中U4和U6小核核糖核酸（snRNAs）的结构及可及性进行了全面的实验分析。这些三小核核糖核蛋白颗粒是从HeLa细胞核提取物和酿酒酵母细胞提取物中纯化得到的。我们还研究了U5的可及性。对于这两种生物，数据表明形成了U4/U6 Y形结构。在人类三小核核糖核蛋白颗粒和U4/U6小核核糖核蛋白颗粒中，U6形成了长程相互作用，此前有人提出这种相互作用负责去蛋白化的U4/U6双链体的解离。在酵母和人类三小核核糖核蛋白颗粒中，U5比U4和U6受到的保护更多，这表明U5小核核糖核蛋白特异性蛋白复合物和三小核核糖核蛋白颗粒的其他成分包裹了U5的5'茎环。U5的环I部分可及，并且酵母和人类三小核核糖核蛋白颗粒中环I的化学修饰是相同的。这反映了蛋白质与功能性环I相互作用的高度保守性。只有U4/U6 Y形基序的某些部分（U4的5'茎环和螺旋II）受到保护。由于酵母和人类三小核核糖核蛋白颗粒蛋白质组成的差异，酵母中连接5'茎环与Y形结构的U6片段和U4中央单链片段比人类三小核核糖核蛋白颗粒中更易接近，特别是U6的系统发育保守的ACAGAG序列。我们结合关于酵母和人类小核核糖核蛋白颗粒的RNA和蛋白质成分及其在剪接体组装中的作用的知识对数据进行了讨论。
