2013 Rustbelt RNA Meeting
Alternative splicing is a robust mechanism for regulating the functional output of many important genes. In contrast to the well-studied regulation of tissue- and developmental stage-specific alternative splicing, the regulated alternative splicing changes in cells responding to environmental cues is poorly understood.
Using cardiomyocytes isolated from neonatal mice, we found that elevated intracellular calcium levels lead to significantly increased skipping of Nf1 exon 23a. Detailed dose-curve and time-course analyses indicated that the alternative splicing change is dynamic, as the splicing pattern is recovered after calcium levels are restored. These data have provided the first evidence for the robust regulation of alternative splicing by calcium signaling in cardiomyocytes.
Prolonged increase of cytoplasmic calcium levels has been shown to lead to histone hyperacetylation in cardiomyocytes. Indeed, western blots analysis indicated 2-2.5 fold higher total acetylation of histones H4 and H3, as well as H3K9 acetylation than that of control cells. Immuno-staining analysis showed that KCl-induced histone hyperacetylation is correlated with the translocation of class II HDACs from the cell nucleus to the cytoplasm. ChIP performed with pan-acetylated histone H3 antibodies indicated that histone hyperacetylation occurs throughout the Nf1 gene body. We hypothesize that the increase in histone acetylation results in increased rate of transcription leading to increased skipping of Nf1 exon 23a. Consistent with this hypothesis, the KCl-induced skipping of Nf1 exon 23a can be rescued by DRB treatment at low doses.
Importantly, the calcium mediated dynamic splicing pattern change is not limited to Nf1 exon 23a. To date, we have tested 8 alternative exons known to undergo fetal-to-adult splicing pattern switch and found increased skipping for 4 exons in the KCl-treated cardiomyocytes. Interestingly, the splicing patterns of these exons revert to the fetal patterns when the calcium level is abnormally high. Our studies have revealed a hidden network of alternative splicing that is regulated by calcium signaling in cardiomyocytes. We are currently studying the molecular basis of this novel mechanism and its biological relevance in normal and pathological processes.
Keywords: Alternative Splicing