The transition of embryonic stem cell (ESC) pluripotency to differentiation is accompanied by an expansion of mRNA and proteomic diversity. Post-transcriptional regulation of ESCs is critically governed by cell type-specific splicing. However, little is known about the splicing factors and the molecular mechanisms directing ESC early lineage differentiation. Our study identifies RNA binding motif protein 24 (Rbm24) as a key splicing regulator that plays an essential role in controlling post-transcriptional networks during ESC transition into cardiac differentiation. Using an inducible mouse ESC line in which gene expression could be temporally regulated, we demonstrated that forced expression of Rbm24 in ESCs dramatically induced a switch to cardiac specification. Genome-wide RNA sequencing analysis identified more than 200 Rbm24-regulated alternative splicing events (AS) which occurred in genes essential for the ESC pluripotency or differentiation. Remarkably, AS genes regulated by Rbm24 composed of transcriptional factors, cytoskeleton proteins, and ATPase gene family members which are critical components required for cardiac development and functionality. Furthermore, we show that Rbm24 regulates ESC differentiation by promoting alternative splicing of pluripotency genes. Among the Rbm24-regulated events, Tpm1, an actin filament family gene, was identified to possess ESC/tissue specific isoforms. We demonstrated that these isoforms were functionally distinct and that their exon AS switch was essential for ESC differentiation. Our results suggest that ESC's switching into the differentiation state can be initiated by a tissue-specific splicing regulator, Rbm24. This finding offers a global view on how an RNA binding protein influences ESC lineage differentiation by a splicing-mediated regulatory mechanism. STEM CELLS 2016;34:1776-1789 SIGNIFICANCE STATEMENTThis study reports a novel function and post-transcriptional regulatory mechnism of an RNA binding protein Rbm24 in embryonic stem cells (ESC) differentiation. Our results suggest that ESC switching into the differentiation state can be initiated by a tissue-specific splicing regulator. To our knowledge, this is the first report describing an RNA binding protein controlling ESC commitment to cardiac lineage specification by engaging a splicing mechanism. Our findings would significantly add to the understanding of the mechanisms in ESC differentiation and heart development, and uncover potential novel pathways operating to direct differentiation of ESCs into cardiomyocytes for envisioned regenerative therapies.

RNA-binding protein Rbm24 is a key regulator of heart development and required for sarcomere assembly and heart contractility. Yet, its underlying mechanism remains unclear. Here, we link serine/threonine kinase 38 (Stk38) signaling to the regulation of Rbm24 by showing that Rbm24 phosphorylation and its function could be modulated by Stk38. Using co-immunoprecipitation coupled with mass spectrometry technique, we identified Stk38 as an endogenous binding partner of Rbm24. Stk38 knockdown resulted in decreased Rbm24 protein level in cardiomyocytes. Further studies using Stk38 kinase inhibitor or activator showed that Rbm24 protein stability was regulated in a kinase activity-dependent manner. Deficiency of Stk38 caused reduction of sarcomere proteins and disarrangement of sarcomere, suggesting that Stk38 is essential for Rbm24 to regulate sarcomere assembly. Our results revealed that Stk38 kinase catalyzes the phosphorylation of Rbm24 during sarcomerogensis and this orchestrates accurate sarcomere alignment. This furthers our understanding of the regulatory mechanism of cardiac sarcomere assembly in both physiologic and pathologic contexts, and uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity.

Scientific Reports 7: Article number: 44870; published online: 21 March 2017; updated: 30 June 2017. The original version of this Article contained errors. The original version contained errors in the spelling of the authors Yew Mun Lee, Meng Kai Zhang and Li Yan Guo, which were incorrectly given asLee Yew Mun, Zhang Meng Kai and Guo Li Yan.