Mammalian splicing divergence is shaped by drift, buffering in trans, and a scaling law

We investigated the cis-regulatory divergences in alternative splicing and their relationship with tissue-dependent trans-regulation in multiple tissues of an F1 hybrid mouse. By obtaining more than 240 million read pairs on average in each sample from 5 organs and ESC as well as published data in liver, we comprehensively analyzed the allelic splicing patterns across tissues in hybrid mice. We find that tissue-dependent regulation causing large splicing differences is highly conserved and likely functional, while splicing divergence mainly affects genes under relaxed selective constraints. Although cis-divergence is in general associated with higher densities of sequence variants in regulatory regions, events with high usage of the dominant isoform could tolerate more mutations, which explains the paradoxical sequence conservation pattern in their exonic versus intronic splicing site flanking regions. Finally, we demonstrated that non-adaptive mutations are often masked in tissues where accurate splicing likely is more important, and experimentally attributed such buffering effect to trans-regulatory splicing efficiency. Overall design: High-throughput RNA-seq data of 5 organs (cerebral cortex, heart, kidney, lung, and spleen) and ESC from Female F1 hybrid mice (female C57BL/6J x male SPRET/EiJ). High-throughput RNA-seq data of fibroblast cells from F1 hybrid mice with and without Pladienolide B treatment.

本研究针对F1杂交小鼠（F1 hybrid mouse）多组织中的可变剪接（alternative splicing）顺式调控差异（cis-regulatory divergence），及其与组织依赖性反式调控（trans-regulation）的关联展开探究。通过对5种器官及胚胎干细胞（Embryonic Stem Cell, ESC）的每个样本平均获取超过2.4亿读对（read pairs），并结合已发表的肝脏组织数据，本研究对杂交小鼠跨组织的等位基因剪接模式开展了全面分析。研究发现，引发显著剪接差异的组织依赖性调控具有高度保守性且大概率具备功能；而剪接差异主要影响处于松弛选择约束下的基因。尽管顺式调控差异通常与调控区域内更高的序列变异密度相关，但主导转录本（isoform）使用率较高的剪接事件可耐受更多突变，这解释了其外显子与内含子剪接位点侧翼区域间矛盾的序列保守模式。最后，本研究证实，在精准剪接更为关键的组织中，非适应性突变常被掩盖，并通过实验将这种缓冲效应归因于反式调控的剪接效率。
研究设计：本研究包含两类高通量RNA测序（high-throughput RNA-seq）数据：一是来自雌性F1杂交小鼠（雌性C57BL/6J × 雄性SPRET/EiJ）的5种器官（大脑皮层、心脏、肾脏、肺脏及脾脏）与胚胎干细胞的测序数据；二是经普拉迪诺利德B（Pladienolide B）处理与未处理的F1杂交小鼠成纤维细胞的测序数据。