QKI is a critical pre-mRNA alternative splicing regulator of cardiac myofibrillogenesis and contractile function

Xinyun Chen,Hongrui Ji,Chen-Leng Cai,Xiuya Li,Yunlong Liu,Dayan Cao,Zhuo Liu,Hongyu Gao,Wei Sheng,Jie Na,Lina Ba,Guoying Huang,Ying Liu,Ken‐Ichi Yamamura ,Chen Xu,Jie Huang,Hanping Qi,Maria Sanderson,Edward Simpson ,Lei Yang,Xiaohui Li,Weinian Shou,Ninghui Sun

doi:10.1038/s41467-020-20327-5

Abstract

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.

Highlights

The RNA-binding protein QKI belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders
To determine the temporal expression pattern of QKI during human embryonic stem cell (hESC)-to-cardiomyocyte differentiation (Supplementary Fig. 1A), we analyzed QKI-5, -6, and -7 mRNA expression levels in undifferentiated hESCs and differentiating cells at Day-1, -3, -6, -8, and -10 by using quantitative reverse transcription PCR (Fig. 1A). In this in vitro differentiation system, cells at Day-1 were considered to be early nascent mesodermal cells with high levels of expression of T (Brachyury); cells at Day-3 were considered to be cardiogenic mesodermal cells with a specific upregulation of MESP1; cells at Day-6 were considered to be cardiac progenitor cells with upregulation of ISL1; and cells at Day-8 to -10 were considered to be well-committed differentiating cardiomyocytes positive for a series of cardiomyocyte markers, such as NKX2-5 and TNNT2. Using these markers as internal references (Fig. 1B), we were able to demonstrate that QKI-5 transcripts were present in undifferentiated hESCs and there was a quick downregulation at the early differentiation stage
Western blot analysis further supported this observation that QKI-5 is the main isoform in hESCs and during hESC-to-cardiomyocyte differentiation (Fig. 1C and Supplementary Fig. 1B–E)

Summary

Introduction

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. One particular member of the STAR (signal transduction and activation of RNA) gene family, known as Quaking (QKI), belongs to the hnRNP K-homology (KH)-domain family of proteins, and it is a sequence-specific RBP that is enriched in the heart and central nervous system[29,30,31,32]. QKI-6 and QKI-7 lack NLS and apparently have different biological functions[38,40,47,48] They seem to play more important roles in regulating mRNA stability and other posttranscriptional mRNA processing and intracellular transportation[49,50,51,52]. Despite the evidence of Qki expression in developing hearts, the role of QKI in regulating normal cardiogenesis and cardiac physiology has not been carefully studied

Methods

Results

Conclusion