A NOVEL U2-U6 SNRNA STRUCTURE IS NECESSARY FOR MAMMALIAN MESSENGER-RNA SPLICING
A NOVEL U2-U6 SNRNA STRUCTURE IS NECESSARY FOR MAMMALIAN MESSENGER-RNA SPLICING
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DOI：
10.1101/gad.9.7.843
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发表时间：
1995-04-01
影响因子：
10.5
通讯作者：
MANLEY, JL
中科院分区：
文献类型：
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作者：
SUN, JS;MANLEY, JL
剪接mRNA前体需要一系列复杂而动态的RNA-RNA碱基配对相互作用，其中U2和U6单链RNA发挥核心作用。使用基因抑制分析，我们提炼和扩展了U2-U6 SnRNA结构，该结构可能包括剪接体的催化中心。我们首先证明了在酵母中被证明的一个关键的U2-U6螺旋，螺旋Ia，对于哺乳动物的剪接也是必不可少的。然而，相邻螺旋Ib上的突变不能被类似地抑制，U2和U6中的相关残基被证明参与了分子内，而不是分子间的碱基配对。接下来，我们证明了对新的U2-U6螺旋的需求，该螺旋III包括从U2中的分支位点识别序列延伸3‘的碱基和从U6中的进化不变序列延伸的5’碱基，该序列先前与5‘剪接位点识别有关。这种构型表明，螺旋III可能有助于并列前mRNA5‘剪接位点和分支位点。我们通过证明分支位点的突变可以被5‘剪接位点的突变抑制来提供证据，前提是在U2和U6的适当碱基上进行了补偿性改变。我们的结果为U2和U6 SnRNA如何相互作用以及如何与Pre-mRNA启动剪接的第一个催化步骤提供了新的见解。
Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles. Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome. We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing. Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site. We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6. Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.