Coverage-dependent bias creates the appearance of binary splicing in single cells
- University of California, Berkeley, United States
Abstract
Single cell RNA sequencing provides powerful insight into the factors that determine each cell's unique identity. Previous studies led to the surprising observation that alternative splicing among single cells is highly variable and follows a bimodal pattern: a given cell consistently produces either one or the other isoform for a particular splicing choice, with few cells producing both isoforms. Here we show that this pattern arises almost entirely from technical limitations. We analyze alternative splicing in human and mouse single cell RNA-seq datasets, and model them with a probabilistic simulator. Our simulations show that low gene expression and low capture efficiency distort the observed distribution of isoforms. This gives the appearance of binary splicing outcomes, even when the underlying reality is consistent with more than one isoform per cell. We show that accounting for the true amount of information recovered can produce biologically meaningful measurements of splicing in single cells.
Data availability
All sequencing data reanalyzed in this study were acquired from GEO.
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Defining the early steps of cardiovascularlineage segregation by single cell RNA-seqNCBI Gene Expression Omnibus, GSE100471.
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Pseudo-temporal ordering of individual cells reveals regulators of differentiationNCBI Gene Expression Omnibus, GSE52529.
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Olfactory stem cell differentiation: horizontal basal cell (HBC) lineageNCBI Gene Expression Omnibus, GSE95601.
Article and author information
Author details
Funding
UC MEXUS-Conacyt (Doctoral Fellowship)
- Carlos F Buen Abad Najar
Chan Zuckerberg Biohub
- Nir Yosef
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Buen Abad Najar et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Coverage-dependent bias creates the appearance of binary splicing in single cells
eLife 9:e54603.
https://doi.org/10.7554/eLife.54603
Further reading
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O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.
