Department of Biological Sciences and RNA Institute, University at Albany, Albany, NY 12222, USA.
Nucleic Acids Res. 2018 Sep 19;46(16):8542-8556. doi: 10.1093/nar/gky416.
Group II introns, the putative progenitors of spliceosomal introns and retrotransposons, are ribozymes that are capable of self-splicing and DNA invasion. In the cell, group II introns form ribonucleoprotein (RNP) complexes with an intron-encoded protein, which is essential to folding, splicing and retromobility of the intron. To understand the structural accommodations underlying splicing, in preparation for retromobility, we probed the endogenously expressed Lactococcus lactis Ll.LtrB group II intron RNP using SHAPE. The results, which are consistent in vivo and in vitro, provide insights into the dynamics of the intron RNP as well as RNA-RNA and RNA-protein interactions. By comparing the excised intron RNP with mutant RNPs in the precursor state, confined SHAPE profile differences were observed, indicative of rearrangements at the active site as well as disengagement at the functional RNA-protein interface in transition between the two states. The exon-binding sequences in the intron RNA, which interact with the 5' exon and the target DNA, show increased flexibility after splicing. In contrast, stability of major tertiary and protein interactions maintains the scaffold of the RNA through the splicing transition, while the active site is realigned in preparation for retromobility.
内含子 II 类，剪接体内含子和逆转座子的假定前体，是能够自我剪接和 DNA 入侵的核酶。在细胞中，内含子 II 类与内含子编码蛋白形成核糖核蛋白（RNP）复合物，这对于内含子的折叠、剪接和逆行移动是必不可少的。为了理解剪接的结构适应，为逆行移动做准备，我们使用 SHAPE 对内源性表达的乳球菌乳脂亚种 Ll.LtrB 内含子 II 类 RNP 进行了探测。这些结果在体内和体外都是一致的，为内含子 RNP 的动力学以及 RNA-RNA 和 RNA-蛋白质相互作用提供了深入的了解。通过将切除的内含子 RNP 与前体状态下的突变 RNP 进行比较，观察到受限的 SHAPE 谱差异，表明在两个状态之间的转换过程中，活性部位发生重排，以及功能 RNA-蛋白质界面脱离。内含子 RNA 中与 5' 外显子和靶 DNA 相互作用的外显子结合序列在剪接后显示出增加的灵活性。相比之下，主要的三级和蛋白质相互作用的稳定性通过剪接过渡维持 RNA 的支架，而活性部位则重新排列以准备逆行移动。
