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.
All sequencing data reanalyzed in this study were acquired from GEO.
Single-cell analysis of allelic gene expression in pluripotency, differentiation and X-chromosome inactivationNCBI Gene Expression Omnibus, GSE74155.
Defining the early steps of cardiovascularlineage segregation by single cell RNA-seqNCBI Gene Expression Omnibus, GSE100471.
Pseudo-temporal ordering of individual cells reveals regulators of differentiationNCBI Gene Expression Omnibus, GSE52529.
Single-cell alternative splicing analysis with Expedition reveals splicing dynamics during neuron differentiationNCBI Gene Expression Omnibus, GSE85908.
Olfactory stem cell differentiation: horizontal basal cell (HBC) lineageNCBI Gene Expression Omnibus, GSE95601.
- Carlos F Buen Abad Najar
- Nir Yosef
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- L Stirling Churchman, Harvard Medical School, United States
© 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.
The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in Caenorhabditis elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin–DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.
Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.