ABSTRACT

Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository of new exon-intron boundaries induced by pathogenic mutations, termed DBASS, here we identify novel TE clusters that facilitated exon selection. To explore the extent to which such TE exons maintain RNA secondary structure of their progenitors, we carried out structural studies with a composite exon that was derived from a long terminal repeat (LTR78) and AluJ and was activated by a C > T mutation optimizing the 5ʹ splice site. Using a combination of SHAPE, DMS and enzymatic probing, we show that the disease-causing mutation disrupted a conserved AluJ stem that evolved from helix 3.3 (or 5b) of 7SL RNA, liberating a primordial GC 5ʹ splice site from the paired conformation for interactions with the spliceosome. The mutation also reduced flexibility of conserved residues in adjacent exon-derived loops of the central Alu hairpin, revealing a cross-talk between traditional and auxilliary splicing motifs that evolved from opposite termini of 7SL RNA and were approximated by Watson-Crick base-pairing already in organisms without spliceosomal introns. We also identify existing Alu exons activated by the same RNA rearrangement. Collectively, these results provide valuable TE exon models for studying formation and kinetics of pre-mRNA building blocks required for splice-site selection and will be useful for fine-tuning auxilliary splicing motifs and exon and intron size constraints that govern aberrant splice-site activation.

摘要

转座元件 (TE) 极大地影响了外显子-内含子结构的演变，并显着促进了发病率，但最近 TE 入侵旧 TE 如何协同产生新的编码序列却知之甚少。采用由致病突变引起的新外显子-内含子边界的更新存储库，称为 DBASS，在这里我们确定了促进外显子选择的新型 TE 簇。为了探索此类 TE 外显子在多大程度上维持其祖细胞的 RNA 二级结构，我们对源自长末端重复序列 (LTR78) 和Alu的复合外显子进行了结构研究J 并被优化 5ʹ 剪接位点的 C > T 突变激活。使用 SHAPE、DMS 和酶促探测的组合，我们表明致病突变破坏了从 7SL RNA 的螺旋 3.3（或 5b）进化而来的保守Alu J 茎，从配对构象中释放出原始 GC 5ʹ 剪接位点，用于与剪接体的相互作用。该突变还降低了中央Alu发夹的相邻外显子衍生环中保守残基的灵活性，揭示了传统和辅助剪接基序之间的串扰，这些基序从 7SL RNA 的相反末端进化而来，并且已经被 Watson-Crick 碱基配对逼近在没有剪接体内含子的生物体中。我们还确定了现有的铝由相同的 RNA 重排激活的外显子。总的来说，这些结果为研究剪接位点选择所需的前 mRNA 构件的形成和动力学提供了有价值的 TE 外显子模型，并将有助于微调辅助剪接基序以及控制异常剪接位点激活的外显子和内含子大小限制.