Abstract
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 (http://www.AltAnalyze.org), 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.
Highlights
The differentiation of embryonic stem cells (ESCs) in vitro is a powerful system for identifying developmental cues required for lineage commitment
Alternative splicing of many of these genes, notably regulators of cell death and proliferation, were often predicted to impact protein domain or microRNA binding site inclusion, suggesting that the function or expression of these proteins is altered during differentiation
These results provide further evidence that alternative splicing is important in shaping the functional repertoire of ESCs and differentiated cells
Summary
The differentiation of embryonic stem cells (ESCs) in vitro is a powerful system for identifying developmental cues required for lineage commitment. Like their in vivo counterparts, the cells of the inner cell mass of the blastocyst, ESCs can self-renew and differentiate into all three adult germ layers. Wholegenome expression [1], microRNA (miRNA) [2], and epigenetic analyses [3,4] of ESC differentiation have identified additional factors that interact with these core transcription factors to regulate pluripotency. AS can alter domain composition and cellular localization, which can confer distinct signaling properties on the resulting protein. Disruption of AS of a single gene can have profound effects on cellular development, ranging from improper neonatal cardiac adaptation [7] to sex-determination [8] and synaptogenesis [9]
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