Transposon clusters as substrates for aberrant splice-site activation

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 <i>Alu</i>J and was activated by a C &gt; 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 <i>Alu</i>J 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 <i>Alu</i> 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 <i>Alu</i> 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.转座因子（Transposed elements, TEs）极大地塑造了外显子-内含子结构的演化，并显著参与了疾病致病过程，但近期转座因子入侵旧有转座因子如何协同产生新型编码序列的机制仍不甚明晰。本研究依托更新后的、由致病突变诱导产生的新型外显子-内含子边界数据库（DBASS），鉴定出一批可促进外显子选择的新型转座因子簇。为探究此类转座因子外显子保留其祖先RNA二级结构的程度，我们针对一个由长末端重复序列（LTR78）和AluJ元件衍生而来、并通过C>T突变优化5'剪接位点而被激活的复合外显子开展了结构分析。结合SHAPE、DMS以及酶促探测技术，我们证实该致病突变破坏了一条源自7SL RNA的3.3号螺旋（或称5b亚域）的保守AluJ茎环结构，将原始的GC型5'剪接位点从配对构象中释放，使其可与剪接体相互作用。该突变同时降低了中心Alu发夹结构中相邻外显子衍生保守残基所在环区的柔性，揭示了传统剪接基序与辅助剪接基序之间的串扰——二者分别源自7SL RNA的两端，并早在无剪接体内含子的生物中就已通过沃森-克里克碱基配对形成近似结构。我们还鉴定出一批由相同RNA重排激活的现有Alu外显子。综上，本研究结果为研究剪接位点选择所需的前体mRNA（pre-mRNA）结构单元的形成过程与动力学特征提供了极具价值的转座因子外显子模型，同时可为微调辅助剪接基序以及调控异常剪接位点激活的外显子、内含子大小限制提供理论参考。