Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non- canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.解析剪接体(spliceosome)在活体内功能的研究工具，其发展进程已落后于该领域结构生物学的相关进展。我们开发了可在核苷酸分辨率下，对与剪接体结合的RNA前体、中间产物及终产物进行全局图谱分析的方法。我们将这些工具应用于三个演化跨度达6亿年的不同酵母物种中。该方法具备高灵敏度，可检测到包括间断剪接、递归剪接与嵌套剪接在内的新型非经典催化案例。通过构建统计模型解析RNA特征与实验数据间的定量关系，我们揭示了内含子大小、位置与数量在底物历经两个催化阶段的进程中各自独立的调控作用。其中还包含物种特异性的调控因素，提示剪接体与转录组存在共演化现象。进一步研究发现，在RNA前体与套索中间体阶段，存在大量依赖ATP的内源性底物清除过程，且该清除过程与内含子滞留这一剪接调控形式密切相关。剪接体图谱分析是一种可量化、可推广的全局技术，可用于研究真核基因表达的核心组分——核糖核蛋白复合物(RNP)。