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

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.

人类胚胎干细胞（human embryonic stem cells, hESCs）的可变剪接在其自我更新、多能性及组织谱系特化过程中所发挥的作用，目前尚未完全明确。为了更清晰地阐明这类调控线索，我们对H9人胚胎干细胞系进行了遗传改造，使其可通过新霉素抗性筛选多能性人胚胎干细胞，通过嘌呤霉素抗性筛选心脏祖细胞。我们利用未分化人胚胎干细胞、心脏及神经前体细胞的外显子水平微阵列表达数据，筛选出具有心脏限制性表达模式，或在心脏/神经分化中呈现普遍表达模式的剪接异构体。上述两组剪接事件所涉及的生物学通路包括细胞骨架重塑、RNA剪接、肌肉细胞特化以及细胞周期检查点调控，同时也涵盖了编码丝氨酸/苏氨酸激酶和解旋酶的基因。我们借助一款名为AltAnalyze的全新分析工具（访问网址：http://www.AltAnalyze.org），鉴定出了一批可预测影响蛋白质功能与表达的新型蛋白质结构域及微小RNA（microRNA, miRNA）结合位点结构特征。这些改变包括：在人胚胎干细胞中可对抗细胞周期停滞的剪接异构体富集，以及在心脏前体细胞中可促进钙信号通路激活与心脏发育的剪接异构体富集。通过将全基因组范围的可变剪接预测结果与全新的功能注释相结合，本研究数据揭示了一系列潜在调控机制——这些机制可通过特定剪接异构体及微小RNA调控，影响细胞谱系定型与人类胚胎干细胞的干性维持。