Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation

Abstract

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.

Stk38 Modulates Rbm24 Protein Stability to Regulate Sarcomere Assembly in Cardiomyocytes

“…To examine whether the effect of Stk38 on Rbm24 protein stability would influence Rbm24′s splicing, we analyzed Rbm24-dependent splicing both in HL-1-shRbm24 and HL-1-shStk38 cells. Similar to the alternative splicing (AS) in previous studies910, Rbm24-regulated AS pattern was observed in HL-1-shRbm24 cells in all cases. As shown in Fig.…”

“…Previous studies showed that Rbm24 governs muscle-specific splicing events that are critically involved in cardiac development910. To examine whether the effect of Stk38 on Rbm24 protein stability would influence Rbm24′s splicing, we analyzed Rbm24-dependent splicing both in HL-1-shRbm24 and HL-1-shStk38 cells.…”

“…Alternative splicing assay was performed as previously described9. Specific primers flanking predicted sites of alternative splicing were used for RT-PCR amplifications.…”

“…It is tissue-specifically expressed in the heart and muscle78. Most recently, we reported that Rbm24 played an important role in regulating ESC cardiac differentiation by a splicing-mediated regulatory mechanism9. Yang et al .…”

Expression patterns of Rbm24 in lens, nasal epithelium, and inner ear during mouse embryonic development

“…Rbm24‐dependent splicing related to exon inclusion have been also highlighted in skeletal muscle and cardiac differentiation in vitro (Cardinali et al, ; Zhang et al, ). This tissue specific role of Rbm24 in the regulation of cardiac gene expression, sarcomeric assembly, and cardiac contractility seems to be conserved among vertebrates because Rbm24 deficiency using morpholino oligonucleotides in zebrafish resulted in a reduction in sarcomeric proteins expression and diminished heart contractility in embryos (Poon et al, ).…”