Multiple RNA-RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome.
Multiple RNA-RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome.
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DOI：
10.1093/nar/gky966
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发表时间：
2018-12-14
影响因子：
14.9
通讯作者：
Lührmann R
中科院分区：
文献类型：
--
作者：
Bao P;Boon KL;Will CL;Hartmuth K;Lührmann R
剪接体催化的U2/U6 RNA核心的活性三维构象由二级和三级RNA相互作用网络稳定，但也依赖于剪接体蛋白的形成。为了确定U2/U6 RNA核心中特定RNA的二级和第三级相互作用对剪接的贡献，我们引入了关键U6核苷酸的突变，并使用酵母体外U6耗尽/互补系统测试了它们对剪接的影响。去除含有U6催化三联体的RNA三级相互作用，或删除U6- u80或U6- a59碱基，对剪接没有影响，表明受影响的二级和三级相互作用不是剪接催化所必需的。然而，去除催化三联体U6-G60的碱基完全阻断了剪接，但不影响活化剪接体的组装或随后转化为B*状配合物。我们的数据表明，允许催化金属M1结合的RNA核心的催化结构可以通过蛋白质- RNA接触来维持。然而，U2/U6 RNA核心中的RNA堆叠相互作用是金属M2生产配位所必需的。U2/U6 RNA核心的功能构象因此得到了高度缓冲，RNA - RNA和蛋白质- RNA相互作用的重叠贡献。
The active 3D conformation of the spliceosome's catalytic U2/U6 RNA core is stabilised by a network of secondary and tertiary RNA interactions, but also depends on spliceosomal proteins for its formation. To determine the contribution towards splicing of specific RNA secondary and tertiary interactions in the U2/U6 RNA core, we introduced mutations in critical U6 nucleotides and tested their effect on splicing using a yeast in vitro U6 depletion/complementation system. Elimination of selected RNA tertiary interactions involving the U6 catalytic triad, or deletions of the bases of U6-U80 or U6-A59, had moderate to no effect on splicing, showing that the affected secondary and tertiary interactions are not required for splicing catalysis. However, removal of the base of U6-G60 of the catalytic triad completely blocked splicing, without affecting assembly of the activated spliceosome or its subsequent conversion into a B*-like complex. Our data suggest that the catalytic configuration of the RNA core that allows catalytic metal M1 binding can be maintained by Protein–RNA contacts. However, RNA stacking interactions in the U2/U6 RNA core are required for productive coordination of metal M2. The functional conformation of the U2/U6 RNA core is thus highly buffered, with overlapping contributions from RNA–RNA and Protein–RNA interactions.
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