European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge, UK.
European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
Genome Biol. 2019 Feb 11;20(1):30. doi: 10.1186/s13059-019-1644-0.
Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.
We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results show that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on genomic features as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons. These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.
Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
可变剪接是真核细胞的一种关键调控机制，可增加功能不同的基因产物的有效数量。通过批量 RNA 测序，已在人类组织和遗传多样化的人群中研究了剪接变异。这确定了与疾病相关的剪接事件，以及剪接与基因组特征（包括序列组成和保守性）之间的关联。然而，同一组织或细胞类型的单个细胞之间剪接的可变性及其决定因素仍知之甚少。
我们应用平行的 DNA 甲基化和转录组测序来对分化的人类诱导多能干细胞进行特征分析，以研究剪接变异（外显子跳跃）及其决定因素。我们的结果表明，基于局部序列组成和基因组特征，可以准确预测单细胞剪接的变异。我们观察到，局部 DNA 甲基化图谱对跨细胞剪接变异的贡献适中但一致。基于基因组特征以及 DNA 甲基化信息构建的组合模型可以准确预测个体盒式外显子的不同剪接模式。这些类别包括常规的包含和排除模式，但也包括细胞间剪接变异的更细微模式。最后，我们确定并描述了 DNA 甲基化与细胞分化过程中剪接变化之间的关联。
我们的研究为单细胞水平的可变剪接提供了新的见解，并揭示了 DNA 甲基化变异与剪接之间以前未被充分认识的联系。
