Abstract
BackgroundAlternative 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.ResultsWe 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.ConclusionsOur study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
Highlights
Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products
We quantified splicing rates for between 1386 and 4917 cassette exons in each cell, estimating splicing rates (PSI) as the fraction of reads that include the alternative exon versus the total number of reads at the cassette exon
Whereas DNA methylation did not contribute to improving the splicing prediction, we observe that DNA methylation levels of underdispersed cassette exons were significantly reduced in all genomic contexts, most significantly in the upstream exon
Summary
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. MeCP2 recruits histone deacetylases in methylated contexts that wrap the DNA more tightly around the histones This interplay between MeCP2 and DNA methylation slows down Pol II, leading to an increased exon inclusion rate [10]. Binding of HP1 to the alternative exon leads to increased exon skipping [11] These alternative mechanisms point to a complex regulation of splicing via an interplay between DNA sequence and DNA methylation, both in proximal as well as distal contexts of the alternative exon
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