RNA‐Binding Protein QKI is a critical pre‐RNA splicing regulator for cardiac development and function

Abstract

RNA‐binding protein QKI (also known as Quaking) belongs to hnRNP K‐homology (KH)‐domain protein family and is implicated a novel regulator in pre‐RNA splicing. As previously shown, the QKI homolog how potentially regulates heart lumen formation and function in fruit fly. However, QKI’s function in mammalian heart development is largely unknown. We found that Qki specifically expressed in mouse developing hearts as early as E7.0 and this high‐level expression was maintained in the hearts to adulthood. Consistent with this finding, by using human embryonic stem cells (hESCs)‐cardiomyocyte differentiation system, QKI expression was found up‐regulated at Day 6, a critical time frame that cardiogenic progenitor transition into early cardiomyocytes. To determine the biological function of QKI in cardiogenesis, we generated hESCsQKIdef by using CRISPR/Cas9 gene editing technology and analyzed the physiological impact of QKI‐deficiency to cardiomyocyte differentiation and maturation. As expected, we did not find defect in pluripotency in hESCsQKIdef, but the contractile function was dramatically reduced in hESCsQKIdef‐derived cardiomyocytes. Based on scRNA‐seq and clustering analyses, there was no significant difference in the distribution of the clusters when compared hESCsQKIdef‐cardiomyocyte group to normal control group, suggesting that the cardiomyocyte differentiation was largely unaffected in hESCsQKIdef. However, scRNA‐seq demonstrated a handful of genes encoding sarcomere proteins were significantly altered in cardiomyocyte clusters, which included ACTN2, ACTA1, MYH7, and MYL2, etc., indicating a significant defect in cardiomyocyte maturation. Bulk RNA‐seq data not only further confirmed this notion, but also provided additional depth for detailed analysis on the QKI‐mediated pre‐RNA splicing events. In conjunction with QKI‐binding motif search and RNA immunoprecipitation (RIP) analysis, we were able to demonstrate that QKI was a key pre‐RNA splicing regulator for sets of genes involved in cardiac myofibrillogenesis and cardiac excitation‐contraction (E‐C) coupling, which ultimately impacted on cardiomyocyte maturation and function. Additional analysis of cardiac developmental defects in Qkibgeo mutant mice further validated that QKI was an indispensable splicing regulator in cardiovascular development. Our results not only revealed a novel mechanism by which QKI regulates pre‐mRNA splicing in cardiogenesis, but also implied that QKI was likely involved in the pathogenesis of certain forms of congenital heart defects and cardiomyopathies.Support or Funding InformationThis work was supported in part by National Institute of Health P01HL134599