RNA-Binding Proteins in Cardiomyopathies.

The post-transcriptional regulation of gene expression plays an important role in heart development and disease. Cardiac-specific alternative splicing, mediated by RNA-binding proteins, orchestrates the isoform switching of proteins that are essential for cardiomyocyte organization and contraction. Dysfunctions of RNA-binding proteins impair heart development and cause the main types of cardiomyopathies, which represent a heterogenous group of abnormalities that severely affect heart structure and function. In particular, mutations of RBM20 and RBFOX2 are associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, or hypoplastic left heart syndrome. Functional analyses in different animal models also suggest possible roles for other RNA-binding proteins in cardiomyopathies because of their involvement in organizing cardiac gene programming. Recent studies have provided significant insights into the causal relationship between RNA-binding proteins and cardiovascular diseases. They also show the potential of correcting pathogenic mutations in RNA-binding proteins to rescue cardiomyopathy or promote cardiac regeneration. Therefore, RNA-binding proteins have emerged as promising targets for therapeutic interventions for cardiovascular dysfunction. The challenge remains to decipher how they coordinately regulate the temporal and spatial expression of target genes to ensure heart function and homeostasis. This review discusses recent advances in understanding the implications of several well-characterized RNA-binding proteins in cardiomyopathies, with the aim of identifying research gaps to promote further investigation in this field.

RNA-Binding Proteins as Critical Post-Transcriptional Regulators of Cardiac Regeneration.

Myocardial injury causes death to cardiomyocytes and leads to heart failure. The adult mammalian heart has very limited regenerative capacity. However, the heart from early postnatal mammals and from adult lower vertebrates can fully regenerate after apical resection or myocardial infarction. Thus, it is of particular interest to decipher the mechanism underlying cardiac regeneration that preserves heart structure and function. RNA-binding proteins, as key regulators of post-transcriptional gene expression to coordinate cell differentiation and maintain tissue homeostasis, display dynamic expression in fetal and adult hearts. Accumulating evidence has demonstrated their importance for the survival and proliferation of cardiomyocytes following neonatal and postnatal cardiac injury. Functional studies suggest that RNA-binding proteins relay damage-stimulated cell extrinsic or intrinsic signals to regulate heart regenerative capacity by reprogramming multiple molecular and cellular processes, such as global protein synthesis, metabolic changes, hypertrophic growth, and cellular plasticity. Since manipulating the activity of RNA-binding proteins can improve the formation of new cardiomyocytes and extend the window of the cardiac regenerative capacity in mammals, they are potential targets of therapeutic interventions for cardiovascular disease. This review discusses our evolving understanding of RNA-binding proteins in regulating cardiac repair and regeneration, with the aim to identify important open questions that merit further investigations.

IGF2BP3 promotes adult myocardial regeneration by stabilizing MMP3 mRNA through interaction with m6A modification.

Myocardial infarction that causes damage to heart muscle can lead to heart failure. The identification of molecular mechanisms promoting myocardial regeneration represents a promising strategy to improve cardiac function. Here we show that IGF2BP3 plays an important role in regulating adult cardiomyocyte proliferation and regeneration in a mouse model of myocardial infarction. IGF2BP3 expression progressively decreases during postnatal development and becomes undetectable in the adult heart. However, it becomes upregulated after cardiac injury. Both gain- and loss-of-function analyses indicate that IGF2BP3 regulates cardiomyocyte proliferation in vitro and in vivo. In particular, IGF2BP3 promotes cardiac regeneration and improves cardiac function after myocardial infarction. Mechanistically, we demonstrate that IGF2BP3 binds to and stabilizes MMP3 mRNA through interaction with N6-methyladenosine modification. The expression of MMP3 protein is also progressively downregulated during postnatal development. Functional analyses indicate that MMP3 acts downstream of IGF2BP3 to regulate cardiomyocyte proliferation. These results suggest that IGF2BP3-mediated post-transcriptional regulation of extracellular matrix and tissue remodeling contributes to cardiomyocyte regeneration. They should help to define therapeutic strategy for ameliorating myocardial infarction by inducing cell proliferation and heart repair.

Planar cell polarity regulators in asymmetric organogenesis during development and disease.

The phenomenon of planar cell polarity is critically required for a myriad of morphogenetic processes in metazoan and is accurately controlled by several conserved modules. Six "core" proteins, including Frizzled, Flamingo (Celsr), Van Gogh (Vangl), Dishevelled, Prickle, and Diego (Ankrd6), are major components of the Wnt/planar cell polarity pathway. The Fat/Dchs protocadherins and the Scrib polarity complex also function to instruct cellular polarization. In vertebrates, all these pathways are essential for tissue and organ morphogenesis, such as neural tube closure, left-right symmetry breaking, heart and gut morphogenesis, lung and kidney branching, stereociliary bundle orientation, and proximal-distal limb elongation. Mutations in planar polarity genes are closely linked to various congenital diseases. Striking advances have been made in deciphering their contribution to the establishment of spatially oriented pattern in developing organs and the maintenance of tissue homeostasis. The challenge remains to clarify the complex interplay of different polarity pathways in organogenesis and the link of cell polarity to cell fate specification. Interdisciplinary approaches are also important to understand the roles of mechanical forces in coupling cellular polarization and differentiation. This review outlines current advances on planar polarity regulators in asymmetric organ formation, with the aim to identify questions that deserve further investigation.

Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure.

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration.

RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.

RBM24 in the Post-Transcriptional Regulation of Cancer Progression: Anti-Tumor or Pro-Tumor Activity?

RNA-binding proteins are critical post-transcriptional regulators of gene expression. They are implicated in a wide range of physiological and pathological processes by modulating nearly every aspect of RNA metabolisms. Alterations in their expression and function disrupt tissue homeostasis and lead to the occurrence of various cancers. RBM24 is a highly conserved protein that binds to a large spectrum of target mRNAs and regulates many post-transcriptional events ranging from pre-mRNA splicing to mRNA stability, polyadenylation and translation. Studies using different animal models indicate that it plays an essential role in promoting cellular differentiation during organogenesis and tissue regeneration. Evidence is also accumulating that its dysregulation frequently occurs across human cancers. In several tissues, RBM24 clearly functions as a tumor suppressor, which is consistent with its inhibitory potential on cell proliferation. However, upregulation of RBM24 in other cancers appears to promote tumor growth. There is a possibility that RBM24 displays both anti-tumor and pro-tumor activities, which may be regulated in part through differential interactions with its protein partners and by its post-translational modifications. This makes it a potential biomarker for diagnosis and prognosis, as well as a therapeutic target for cancer treatment. The challenge remains to determine the post-transcriptional mechanisms by which RBM24 modulates gene expression and tumor progression in a context- or background-dependent manner. This review discusses recent findings on the potential function of RBM24 in tumorigenesis and provides future directions for better understanding its regulatory role in cancer cells.

Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish.

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish.

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease.

Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.

Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish.

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site-spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.

Syne2b/Nesprin-2 Is Required for Actin Organization and Epithelial Integrity During Epiboly Movement in Zebrafish.

Syne2b/nesprin-2 is a giant protein implicated in tethering the nucleus to the cytoskeleton and plays an important role in maintaining cellular architecture. Epiboly is a conserved morphogenetic movement that involves extensive spreading and thinning of the epithelial blastoderm to shape the embryo and organize the three germ layers. Dynamic cytoskeletal organization is critical for this process, but how it is regulated remains elusive. Here we generated a zebrafish syne2b mutant line and analyzed the effects of impaired Syne2b function during early development. By CRISPR/Cas9-mediated genome editing, we obtained a large deletion in the syne2b locus, predicted to cause truncation of the nuclear localization KASH domain in the translated protein. Maternal and zygotic syne2b embryos showed delayed epiboly initiation and progression without defects in embryonic patterning. Remarkably, disruption of Syne2b function severely impaired cytoskeletal organization across the embryo, leading to aberrant clustering of F-actin at multiple cell contact regions and abnormal cell shape changes. These caused disintegration of the epithelial blastoderm before the end of gastrulation in most severely affected embryos. Moreover, the migration of yolk nuclear syncytium also became defective, likely due to disorganized cytoskeletal networks at the blastoderm margin and in the yolk cell. These findings demonstrate an essential function of Syne2b in maintaining cytoskeletal architecture and epithelial integrity during epiboly movement.

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration.

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

Decoding Dishevelled-Mediated Wnt Signaling in Vertebrate Early Development.

Dishevelled proteins are key players of Wnt signaling pathways. They transduce Wnt signals and perform cellular functions through distinct conserved domains. Due to the presence of multiple paralogs, the abundant accumulation of maternal transcripts, and the activation of distinct Wnt pathways, their regulatory roles during vertebrate early development and the mechanism by which they dictate the pathway specificity have been enigmatic and attracted much attention in the past decades. Extensive studies in different animal models have provided significant insights into the structure-function relationship of conserved Dishevelled domains in Wnt signaling and the implications of Dishevelled isoforms in early developmental processes. Notably, intra- and inter-molecular interactions and Dishevelled dosage may be important in modulating the specificity of Wnt signaling. There are also distinct and redundant functions among Dishevelled isoforms in development and disease, which may result from differential spatiotemporal expression patterns and biochemical properties and post-translational modifications. This review presents the advances and perspectives in understanding Dishevelled-mediated Wnt signaling during gastrulation and neurulation in vertebrate early embryos.

RNA-Binding Protein Rbm24 as a Multifaceted Post-Transcriptional Regulator of Embryonic Lineage Differentiation and Cellular Homeostasis.

RNA-binding proteins control the metabolism of RNAs at all stages of their lifetime. They are critically required for the post-transcriptional regulation of gene expression in a wide variety of physiological and pathological processes. Rbm24 is a highly conserved RNA-binding protein that displays strongly regionalized expression patterns and exhibits dynamic changes in subcellular localization during early development. There is increasing evidence that it acts as a multifunctional regulator to switch cell fate determination and to maintain tissue homeostasis. Dysfunction of Rbm24 disrupts cell differentiation in nearly every tissue where it is expressed, such as skeletal and cardiac muscles, and different head sensory organs, but the molecular events that are affected may vary in a tissue-specific, or even a stage-specific manner. Recent works using different animal models have uncovered multiple post-transcriptional regulatory mechanisms by which Rbm24 functions in key developmental processes. In particular, it represents a major splicing factor in muscle cell development, and plays an essential role in cytoplasmic polyadenylation during lens fiber cell terminal differentiation. Here we review the advances in understanding the implication of Rbm24 during development and disease, by focusing on its regulatory roles in physiological and pathological conditions.

Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation.

Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of crystallin mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.

The regulatory proteins DSCR6 and Ezh2 oppositely regulate Stat3 transcriptional activity in mesoderm patterning during Xenopus development.

Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventral mesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycomb-repressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventral mesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and post-translationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventral mesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.