Modified nucleotides at the 5′ end of human U2 snRNA are required for spliceosomal E-complex formation
Modified nucleotides at the 5′ end of human U2 snRNA are required for spliceosomal E-complex formation
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DOI：
10.1261/rna.7186504
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发表时间：
2004-12-01
期刊：
影响因子：
4.5
通讯作者：
Lührmann, R
中科院分区：
文献类型：
--
作者：
Dönmez, G;Hartmuth, K;Lührmann, R
U2 snRNA是核前mrna剪接的关键角色，包含一个5'端m(3)G帽和许多内部修饰。后者在脊椎动物中被证明通常是U2在剪接中的功能所必需的，但确切地说，哪些残基是必不可少的，它们在snRNP和/或剪接体组装中的作用目前尚不清楚。在这里，我们研究了HeLa U2 snRNA的单个修饰核苷酸在mrna前剪接中的作用，使用了两步体外重建/互补实验。我们发现U2 snRNA的前20个核苷酸中的3个假嘌呤和5个2' o -甲基，而不是M(3)G帽，是有效的pre-mRNA剪接所必需的。单个假尿嘧啶不是必需的，但对U2功能有累积影响。相反，5个2' o甲基化中的4个（位置1、2、12和19）是剪接所必需的。17S U2 snRNPs的体外组装不依赖于修饰U2残基的存在。然而，独立于atp的早期剪接体E复合体的形成需要单独的内部修饰。我们的数据强烈表明，U2前20个核苷酸的修饰在促进U2与U1 snRNP和/或E复合体内其他因子的相互作用中起着重要作用。
U2 snRNA, a key player in nuclear pre-mRNA splicing, contains a 5'-terminal m(3)G cap and many internal modifications. The latter were shown in vertebrates to be generally required for U2 function in splicing, but precisely which residues are essential and their role in snRNP and/or spliceosome assembly is presently not clear. Here, we investigated the roles of individual modified nucleotides of HeLa U2 snRNA in pre-mRNA splicing, using a two-step in vitro reconstitution/complementation assay. We show that the three pseudouridines and five 2'O-methyl groups within the first 20 nucleotides of U2 snRNA, but not the M(3)G cap, are required for efficient pre-mRNA splicing. Individual pseudouridines were not essential, but had cumulative effects on U2 function. In contrast, four of five 2'O-methylations (at positions 1, 2, 12, and 19) were individually required for splicing. The in vitro assembly of 17S U2 snRNPs was not dependent on the presence of modified U2 residues. However, individual internal modifications were required for the formation of the ATP-independent early spliceosomal E complex. Our data strongly suggest that modifications within the first 20 nucleotides of U2 play an important role in facilitating the interaction of U2 with U1 snRNP and/or other factors within the E complex.