Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

University of California Santa Cruz, United States of America
PLoS Computational Biology (Impact Factor: 4.62). 11/2009; 5(11):e1000553. DOI: 10.1371/journal.pcbi.1000553
Source: PubMed


The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

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Available from: Nathan Salomonis,
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    • "4.1. Hypertrophy is not the cause of altered expression or splice variants Coordinated control of alternative splicing modifies the transcriptome and increases the complexity of the proteome during most developmental processes [29] [30].Studies of animal models have shown the importance of appropriate splicing for proper heart development [8] [12] [31] [32]. Still, knowledge of the sequence of events leading to tissue specific alternative splicing is incomplete. "

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    • "To detect alternative exons regulated from these arrays, the expression of pairs of opposing (reciprocal) exon-junctions were compared using a previously described linear regression approach (Sugnet et al., 2006) in the program AltAnalyze (Salomonis et al., 2009) for ~25,000 detected transcripts. This analysis revealed 478 disease-associated inclusion and exclusion events that occurred in 332 distinct genes, along with exons containing predicted miRNA binding sites (Table S9). "
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    ABSTRACT: MicroRNAs (miRNAs) are key post transcriptional regulators of their multiple target genes. However, the detailed profile of miRNA expression in Parkinson's disease, the second most common neurodegenerative disease worldwide and the first motor disorder has not been charted yet. Here, we report comprehensive miRNA profiling by next-generation small-RNA sequencing, combined with targets inspection by splice-junction and exon arrays interrogating leukocyte RNA in Parkinson's disease patients before and after deep brain stimulation (DBS) treatment and of matched healthy control volunteers (HC). RNA-Seq analysis identified 254 miRNAs and 79 passenger strand forms as expressed in blood leukocytes, 16 of which were modified in patients pre-treatment as compared to HC. 11 miRNAs were modified following brain stimulation 5 of which were changed inversely to the disease induced changes. Stimulation cessation further induced changes in 11 miRNAs. Transcript isoform abundance analysis yielded 332 changed isoforms in patients compared to HC, which classified brain transcriptomes of 47 PD and control independent microarrays. Functional enrichment analysis highlighted mitochondrion organization. DBS induced 155 splice changes, enriched in ubiquitin homeostasis. Cellular composition analysis revealed immune cell activity pre and post treatment. Overall, 217 disease and 74 treatment alternative isoforms were predictably targeted by modified miRNAs within both 3' and 5' untranslated ends and coding sequence sites. The stimulation-induced network sustained 4 miRNAs and 7 transcripts of the disease network. We believe that the presented dynamic networks provide a novel avenue for identifying disease and treatment-related therapeutic targets. Furthermore, the identification of these networks is a major step forward in the road for understanding the molecular basis for neurological and neurodegenerative diseases and assessment of the impact of brain stimulation on human diseases.
    Frontiers in Molecular Neuroscience 05/2013; 6:10. DOI:10.3389/fnmol.2013.00010 · 4.08 Impact Factor
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    • "For example, in the Drosophila male germline lineage, splicing factors are highly enriched in undifferentiated cells, but they are downregulated in differentiating cells, suggesting that their expression levels are controlled by developmental programming (Gan et al., 2010). In addition, during both neural and cardiac differentiation of ESCs, genes regulating RNA splicing themselves undergo robust AS (Salomonis et al., 2009), for example, PTB and nPTB which have mutually exclusive expression in the nervous system. This expression pattern is partly regulated by AS. "
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    ABSTRACT: The application of stem cells to regenerative medicine depends on a thorough understanding of the molecular mechanisms underlying their pluripotency. Many studies have identified key transcription factor-regulated transcriptional networks and chromatin landscapes of embryonic and a number of adult stem cells. In addition, recent publications have revealed another interesting molecular feature of stem cells- a distinct alternative splicing pattern. Thus, it is possible that both the identity and activity of stem cells are maintained by stem cell-specific mRNA isoforms, while switching to different isoforms ensures proper differentiation. In this review, we will discuss the generality of mRNA isoform switching and its interaction with other molecular mechanisms to regulate stem cell pluripotency, as well as the reprogramming process in which differentiated cells are induced to become pluripotent stem cell-like cells (iPSCs).
    02/2013; 8(1):50-59. DOI:10.1007/s11515-012-1198-y
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