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

Coverage-dependent bias creates the appearance of binary splicing in single cells.

Single cell RNA sequencing provides powerful insight into the factors that determine each cell9s unique identity, including variation in transcription and RNA splicing among diverse cell types. Previous studies led to the surprising observation that alternative splicing outcomes among single cells are highly variable and follow 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 analyzed single cell alternative splicing in human and mouse single cell RNA-seq datasets, and modeled them with a probablistic simulator. Our simulations show that low gene expression and low capture efficiency distort the observed distribution of isoforms in single cells. This gives the appearance of a binary isoform distribution, 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.

https://biorxiv.org/content/biorxiv/early/2019/12/20/2019.12.19.883256.full-text.pdf

Coverage-dependent bias creates the appearance of binary splicing in single cells

Thyroid hormone, gene expression, and Central Nervous System: Where we are.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice. STRAP (serine threonine kinase receptor-associated protein) promotes tumorigenicity. Here the authors report that STRAP associates with spliceosome and regulates alternative splicing during embryonic stem cell lineage commitment and early mouse embryo organogenesis.

/pdf/strap-regulates-alternative-splicing-fidelity-during-lineage-2sy26bq924.pdf

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model.

https://www.genetics.org/content/genetics/207/2/593.full.pdf

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

Alternative pre-mRNA splicing (AS) produces multiple isoforms of mRNAs and proteins from a single gene. It is most prevalent in the mammalian brain and is thought to contribute to the formation and/or maintenance of functional complexity of the brain. Increasing evidence has documented the significant changes of AS between different regions or different developmental stages of the brain, however, the dynamics of AS and the possible function of it during neural progenitor cell (NPC) differentiation is less well known. Here, using purified NPCs and their progeny neurons isolated from the embryonic mouse cerebral cortex, we characterized the global differences of AS events between the 2 cell types by deep sequencing. The sequencing results revealed cell type-specific AS in NPCs and neurons that are important for distinct functions pertinent to the corresponding cell type. Our data may serve as a resource useful for further understanding how AS contributes to molecular regulations in NPCs and neurons during cortical development.

/pdf/alternative-rna-splicing-associated-with-mammalian-neuronal-1ys7g9jxuv.pdf

Anqi Geng

Papers

Alternative RNA Splicing Associated With Mammalian Neuronal Differentiation