U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III.
U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III.
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DOI：
10.1038/srep31393
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发表时间：
2016-08-11
影响因子：
4.6
通讯作者：
Shukla GC
中科院分区：
文献类型：
--
作者：
Singh J;Sikand K;Conrad H;Will CL;Komar AA;Shukla GC
U12依赖型剪接体催化核心的形成分别涉及U6atac和U12与U12依赖型内含子的5′剪接位点和分支位点区域的相互作用。除了U6atac和U12 snRNAs之间分子间螺旋I区域的形成外，这些RNA分子内的其他几个区域预计会形成茎环结构。我们之前的工作表明，U6atac snRNA的3′茎环区域包含一种U12依赖型剪接体特异性靶向活性。在此，我们展示了对U6atac 3′茎环的一个亚结构在U12依赖型体内剪接中的详细结构 - 功能分析以及其需求。我们表明，p65（一种U12 snRNA结合蛋白）的C末端RNA识别模体也与U6atac的远端3′茎环结合。通过使用双剪接位点突变抑制子分析，我们证明U12 snRNA的p65蛋白结合的顶端茎环可被这个U6atac远端3′茎环替代。此外，我们在人类U6atac背景下测试了来自系统发育上距离较远的物种的U6atac 3′末端的兼容性，以确定这些结构的进化相关性以及体内功能。总之，我们证明与主要剪接体相比，次要剪接体中的RNA - RNA和RNA - 蛋白质相互作用具有高度的可塑性。
Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome.