Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels

Nascent RNA sequencing in 86 YRI LCLs. Cut&Tag from 95 YRI LCLs.Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that degrades transcripts containing premature termination codons. Alternative splicing (AS) is known to affect the expression levels of some human genes by producing transcripts rapidly degraded by NMD. However, the importance of AS in determining expression levels of most genes is unclear because rapidly degraded mRNA isoforms are difficult to quantify. To assess the impact of AS on gene expression levels, we analyzed population-scale data across eight molecular assays that capture gene regulation before, during, and after transcription and mRNA decay. Sequencing nascent RNA revealed that ~15% of all mRNA molecules are NMD targets, of which most result from low-usage cryptic splicing. Leveraging genetic variation across cell lines, we find that trait-associated loci explained by AS are similarly likely to associate with NMD-induced expression level differences as with differences in protein isoform usage, suggesting that much of the impact of AS is mediated by NMD. Finally, we used the splice-switching drug risdiplam to perturb AS at hundreds of genes, finding that ~3/4 of the splicing perturbations induce NMD. The remarkable prevalence of cryptic splice sites across the genome presents a rich opportunity for natural selection and therapeutics to shape the expression levels of nearly all human genes.

本数据集包含86株YRI淋巴母细胞系（YRI LCLs）的新生RNA测序数据，以及95株YRI淋巴母细胞系的Cut&Tag实验数据。无义介导的降解（Nonsense-mediated decay, NMD）是一种RNA监视机制，可降解携带提前终止密码子的转录本。已知可变剪接（Alternative splicing, AS）可通过产生被NMD快速降解的转录本，影响部分人类基因的表达水平。然而，由于快速降解的mRNA异构体难以定量，目前尚不清楚可变剪接对大多数基因表达水平的重要性。为评估可变剪接对基因表达水平的影响，我们分析了覆盖转录前、转录过程中、转录后及mRNA降解阶段的8种分子检测技术的群体规模数据。对新生RNA的测序分析显示，约15%的mRNA分子均为NMD底物，其中大多数源于低使用率的隐蔽剪接事件。利用细胞系间的遗传变异，我们发现由可变剪接解释的性状关联位点，其关联NMD诱导的表达水平差异与关联蛋白质异构体使用差异的概率相当，这表明可变剪接的大部分影响通过NMD通路介导。最后，我们使用剪接调控药物利司扑兰（risdiplam）对数百个基因的可变剪接进行扰动实验，发现约四分之三的剪接扰动会触发NMD通路激活。基因组中广泛分布的隐蔽剪接位点，为自然选择与治疗干预手段调控几乎所有人类基因的表达水平提供了广阔空间。