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-QKI^del) using
   CRISPR/Cas9 gene editing technology, we analyze the physiological role
   of QKI in cardiomyocyte differentiation, maturation, and contractile
   function. hESCs-QKI^del 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.

   Subject terms: RNA, Heart development, Cardiology
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   RNA binding protein Quaking (QKI) is known for its broad function in
   pre-mRNA splicing and modification and its association with several
   neurodevelopmental disorders. Here the authors reveal that QKI-mediated
   regulation of RNA splicing is indispensable to cardiac development and
   contractile physiology.

Introduction

   The transcriptional and posttranscriptional modifications are critical
   to mammalian gene expression^[68]1. RNA splicing is a
   posttranscriptional process by which introns are removed from the newly
   transcribed sequences of immature pre-mRNAs and this process is
   required for generating mature protein-coding mRNAs^[69]2. In contrast
   to constitutive splicing, alternative splicing is a dynamic process
   that is highly regulated upon cellular differentiation or in response
   to distinct physiological states, resulting in specific exons being
   either included or excluded in unique combinations to generate multiple
   mRNA isoforms from a single gene^[70]3. Genome-wide studies estimated
   that up to 95% of human genes undergo some level of alternative
   splicing^[71]4, and these splicing events are associated with almost
   all normal cellular and physiological functions as well as
   pathophysiological conditions.

   Alternative splicing is regulated by various cis-regulatory sequences
   and trans-acting factors^[72]5–[73]8. RNA-binding proteins (RBPs) can
   recognize and bind to specific RNA sequences to form ribonucleoprotein
   complexes and act as regulators of diverse biological functions, such
   as modifying RNA posttranscriptionally and transporting RNA^[74]9. Many
   RBPs play particularly important roles in pre-mRNA
   splicing^[75]10,[76]11. In general, upon receiving upstream biological
   signals, RBPs coordinate alternative splicing events by recognizing
   specific cis-elements (e.g., enhancers or silencers) to either further
   promote or inhibit specific splicing events. The most well-known RBP
   families involved in RNA splicing are the serine/arginine-rich (SR)
   protein family, with members such as SF2/ASF, SRp20, SRp40, SRp55, and
   SRp75, and the heterogeneous nuclear ribonucleoprotein (hnRNP) protein
   family, with members such as PTB, hnRNP A1, hnRNP C, hnRNP D, hnRNP E,
   hnRNP F/H, hnRNP G, and hnRNP H^[77]12–[78]14. The heart is the first
   functional organ to be formed during embryonic development.
   Interestingly, among ~1148 RBPs in the heart, there are ~390
   cardiac-specific RBPs and many of them are present in the developing
   hearts^[79]15, suggesting that alternative splicing is one of the major
   regulatory events for cardiogenesis and cardiac physiology. Indeed, it
   has been shown that mutations of several RBPs and cis-regulatory
   sequences are associated with cardiomyopathies, muscular atrophy,
   hypercholesterolemia, and other cardiac disorders^[80]10,[81]16–[82]22.

   Previously, several studies have suggested that alternative splicing
   involved in sarcomerogenesis impacts heart development and
   function^[83]10. These prior investigations strongly indicated a
   critical role for alternative splicing and the associated RBPs in
   cardiac development and cardiac physiology, as well as the pathogenesis
   of heart diseases. RBM24 and RBM20 are two of the most well-known RBPs
   that have been implicated in the pathogenesis of
   cardiomyopathies^[84]23–[85]25. The loss of RBM20 function resulted in
   aberrant splicing events that led to abnormal sarcomerogenesis in
   embryonic development^[86]24. Genetic ablation of SRp38 in mice
   resulted in mis-splicing of triadin, a cardiac protein that regulates
   calcium release from the sarcoplasmic reticulum during
   excitation–contraction (E–C) coupling^[87]26. SF2/ASF
   cardiomyocyte-specific ablation results in the development of dilated
   cardiomyopathy and rapid progression of heart failure^[88]27. RBFox1
   acts as a vital regulator for the conserved splicing process of
   transcription factor MEF2 family members and is involved in the heart
   failure progression^[89]28.

   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^[90]29–[91]32. QKI contains an RNA-binding motif (KH domain),
   which is flanked by QUA1 and QUA2 domains. The QUA domain is involved
   in forming homo- or heterodimers and is required for RNA
   binding^[92]33–[93]36. In addition to these functional domains, there
   is a tyrosin cluster located within the proline-rich PXXP motif that
   can be phosphorylated by Src kinases^[94]37, suggesting that QKI can be
   regulated by intracellular signaling. At least three major
   alternatively spliced mRNA isoforms are generated from the QKI gene,
   QKI−5, QKI-6, and QKI-7, in which exons 1–6 encode identical structures
   in these isoforms but differ in their C-terminal end encoded by exons 7
   and 8^[95]38. QKI-5 has a nuclear localization signal
   (NLS)^[96]39,[97]40 and has been shown to play a major function in
   pre-mRNA splicing regulators^[98]41–[99]46. QKI-6 and QKI-7 lack NLS
   and apparently have different biological
   functions^[100]38,[101]40,[102]47,[103]48. They seem to play more
   important roles in regulating mRNA stability and other
   posttranscriptional mRNA processing and intracellular
   transportation^[104]49–[105]52. These functional differences reflect
   the complexity of QKI biological functions. Based on the early
   embryonic lethal phenotype of Qki-deficient mice, Qki is considered
   essential for early embryonic development^[106]53. Interestingly, a
   spontaneous mutant mouse line, known as the Qk^v mouse line, in which a
   1 Mb promoter/enhancer deletion upstream of the Qki transcription start
   site resulted in deficient myelination in the central nervous
   system^[107]34,[108]54. Over the past decades, the use of these Qki
   mutant animal models has suggested important functions in neural
   progenitors, myelin formation, smooth muscle differentiation, and
   monocyte to macrophage differentiation^[109]45,[110]55,[111]56. 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.

   In this work, we analyze the biological function of QKI-mediated
   post-transcriptional regulation in cardiogenesis and cardiac
   physiology. By taking advantage of a defined in vitro system for
   differentiating human embryonic stem cells (hESCs) into cardiomyocytes
   and the Qki^βGeo mouse model, we are able to reveal that QKI is a
   critical pre-mRNA alternative splicing regulator in cardiomyocytes and
   is essential for myofibrillogenesis and contractile physiology during
   cardiomyocyte differentiation and maturation. Our finding shows that
   QKI is indispensable to normal cardiogenesis and cardiac function.

Results

The expression of QKI in cardiomyocytes and the generation of hESC-QKI^del

   QKI expression was previously found in the hearts^[112]29. To determine
   the temporal expression pattern of QKI during hESC-to-cardiomyocyte
   differentiation (Supplementary Fig. [113]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 (qRT-PCR) (Fig. [114]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. [115]1B), we were able to demonstrate that QKI-5