RBFOX2 is required for establishing RNA regulatory networks essential for heart development.

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development.

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA-sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full-length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin-binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full-length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full-length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected generating predominantly muscle-specific Tpm1 isoforms in adult hearts. We found that the RNA-binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA-binding protein PTBP1. In sum, we defined full-length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA-binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle-enriched genes.

A developmentally regulated spliced variant of PTBP1 is upregulated in type 1 diabetic hearts.

Alternative splicing (AS) is dysregulated in Type 1 diabetic (T1D) hearts but mechanisms responsible are unclear. Here, we provide evidence that the RNA binding protein (RBP) PTBP1 is modulated in adult T1D hearts contributing to AS changes. We show that a spliced variant of PTBP1 that is highly expressed in normal newborn mouse hearts is aberrantly expressed in adult T1D mouse hearts. Comparing known PTBP1-target datasets to our T1D mouse transcriptome datasets, we discovered a group of genes with PTBP1 binding sites in their pre-mRNAs that are differentially spliced in T1D mouse hearts. We demonstrated that inducible expression of diabetes-induced PTBP1 spliced variant has less repressive splicing function. Notably, PTBP1 regulates AS of some of its targets antagonistically to RBFOX2. In sum, our results indicate that diabetic conditions disrupt developmental regulation of PTBP1 leading to differential AS of PTBP1 target genes. Identification of PTBP1 and PTBP1-regulated RNA networks can provide RNA-based therapies for the treatment of diabetes cardiac complications.

Rebuilding a broken heart: lessons from developmental and regenerative biology.

In May 2016, the annual Weinstein Cardiovascular Development and Regeneration Conference was held in Durham, North Carolina, USA. The meeting assembled leading investigators, junior scientists and trainees from around the world to discuss developmental and regenerative biological approaches to understanding the etiology of congenital heart defects and the repair of diseased cardiac tissue. In this Meeting Review, we present several of the major themes that were discussed throughout the meeting and highlight the depth and range of research currently being performed to uncover the causes of human cardiac diseases and develop potential therapies.

CELF1 contributes to aberrant alternative splicing patterns in the type 1 diabetic heart.

Dysregulated alternative splicing (AS) that contributes to diabetes pathogenesis has been identified, but little is known about the RNA binding proteins (RBPs) involved. We have previously found that the RBP CELF1 is upregulated in the diabetic heart; however, it is unclear if CELF1 contributes to diabetes-induced AS changes. Utilizing genome wide approaches, we identified extensive changes in AS patterns in Type 1 diabetic (T1D) mouse hearts. We discovered that many aberrantly spliced genes in T1D hearts have CELF1 binding sites. CELF1-regulated AS affects key genes within signaling pathways relevant to diabetes pathogenesis. Disruption of CELF1 binding sites impairs AS regulation by CELF1. In sum, our results indicate that CELF1 target RNAs are aberrantly spliced in the T1D heart leading to abnormal gene expression. These discoveries pave the way for targeting RBPs and their RNA networks as novel therapies for cardiac complications of diabetes.

Developmentally regulated alternative splicing is perturbed in type 1 diabetic skeletal muscle.

INTRODUCTION: Type 1 diabetic patients can develop skeletal muscle weakness and atrophy by molecular mechanisms that are not well understood. Alternative splicing (AS) is critical for gene expression in the skeletal muscle, and its dysregulation is implicated in muscle weakness and atrophy. Therefore, we investigated whether AS patterns are affected in type 1 diabetic skeletal muscle contributing to skeletal muscle defects. METHODS: AS patterns were determined by reverse transcription-polymerase chain reaction and levels of RNA binding proteins were assessed by Western blot in type 1 diabetic mouse skeletal muscle and during normal mouse skeletal muscle development. RESULTS: Five genes with critical functions in the skeletal muscle are misspliced in type 1 diabetic skeletal muscle, resembling their AS patterns at embryonic stages. AS of these genes undergoes dramatic transitions during skeletal muscle development, correlating with changes in specific RNA binding proteins. CONCLUSION: Embryonic spliced variants are inappropriately expressed in type 1 diabetic skeletal muscle. Muscle Nerve 56: 744-749, 2017.

Reactivation of fetal splicing programs in diabetic hearts is mediated by protein kinase C signaling.

Diabetic cardiomyopathy is one of the complications of diabetes that eventually leads to heart failure and death. Aberrant activation of PKC signaling contributes to diabetic cardiomyopathy by mechanisms that are poorly understood. Previous reports indicate that PKC is implicated in alternative splicing regulation. Therefore, we wanted to test whether PKC activation in diabetic hearts induces alternative splicing abnormalities. Here, using RNA sequencing we identified a set of 22 alternative splicing events that undergo a developmental switch in splicing, and we confirmed that splicing reverts to an embryonic pattern in adult diabetic hearts. This network of genes has important functions in RNA metabolism and in developmental processes such as differentiation. Importantly, PKC isozymes α/ß control alternative splicing of these genes via phosphorylation and up-regulation of the RNA-binding proteins CELF1 and Rbfox2. Using a mutant of CELF1, we show that phosphorylation of CELF1 by PKC is necessary for regulation of splicing events altered in diabetes. In summary, our studies indicate that activation of PKCα/ß in diabetic hearts contributes to the genome-wide splicing changes through phosphorylation and up-regulation of CELF1/Rbfox2 proteins. These findings provide a basis for PKC-mediated cardiac pathogenesis under diabetic conditions.

RNA binding proteins in cardiovascular development and disease.

Congenital heart disease (CHD) is the most common birth defect affecting>1.35 million newborn babies worldwide. CHD can lead to prenatal, neonatal, postnatal lethality or life-long cardiac complications. RNA binding protein (RBP) mutations or variants are emerging as contributors to CHDs. RBPs are wizards of gene regulation and are major contributors to mRNA and protein landscape. However, not much is known about RBPs in the developing heart and their contributions to CHD. In this chapter, we will discuss our current knowledge about specific RBPs implicated in CHDs. We are in an exciting era to study RBPs using the currently available and highly successful RNA-based therapies and methodologies. Understanding how RBPs shape the developing heart will unveil their contributions to CHD. Identifying their target RNAs in the embryonic heart will ultimately lead to RNA-based treatments for congenital heart disease.

Alternative polyadenylation regulation in cardiac development and cardiovascular disease.

Cleavage and polyadenylation of pre-mRNAs is a necessary step for gene expression and function. Majority of human genes exhibit multiple polyadenylation sites, which can be alternatively used to generate different mRNA isoforms from a single gene. Alternative polyadenylation (APA) of pre-mRNAs is important for the proteome and transcriptome landscape. APA is tightly regulated during development and contributes to tissue-specific gene regulation. Mis-regulation of APA is linked to a wide range of pathological conditions. APA-mediated gene regulation in the heart is emerging as a new area of research. Here, we will discuss the impact of APA on gene regulation during heart development and in cardiovascular diseases. First, we will briefly review how APA impacts gene regulation and discuss molecular mechanisms that control APA. Then, we will address APA regulation during heart development and its dysregulation in cardiovascular diseases. Finally, we will discuss pre-mRNA targeting strategies to correct aberrant APA patterns of essential genes for the treatment or prevention of cardiovascular diseases. The RNA field is blooming due to advancements in RNA-based technologies. RNA-based vaccines and therapies are becoming the new line of effective and safe approaches for the treatment and prevention of human diseases. Overall, this review will be influential for understanding gene regulation at the RNA level via APA in the heart and will help design RNA-based tools for the treatment of cardiovascular diseases in the future.

A large-scale LC-MS dataset of murine liver proteome from time course of heavy water metabolic labeling.

Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies. Several algorithms and tools have been developed to determine the turnover rates of peptides and proteins from time-course stable isotope labeling experiments. The availability of benchmark mass spectrometry data is crucial to compare and validate the effectiveness of newly developed techniques and algorithms. In this work, we report a heavy water-labeled LC-MS dataset from the murine liver for protein turnover rate analysis. The dataset contains eighteen mass spectral data with their corresponding database search results from nine different labeling durations and quantification outputs from d2ome+ software. The dataset also contains eight mass spectral data from two-dimensional fractionation experiments on unlabeled samples.

Quantifying label enrichment from two mass isotopomers increases proteome coverage for in vivo protein turnover using heavy water metabolic labeling.

Heavy water metabolic labeling followed by liquid chromatography coupled with mass spectrometry is a powerful high throughput technique for measuring the turnover rates of individual proteins in vivo. The turnover rate is obtained from the exponential decay modeling of the depletion of the monoisotopic relative isotope abundance. We provide theoretical formulas for the time course dynamics of six mass isotopomers and use the formulas to introduce a method that utilizes partial isotope profiles, only two mass isotopomers, to compute protein turnover rate. The use of partial isotope profiles alleviates the interferences from co-eluting contaminants in complex proteome mixtures and improves the accuracy of the estimation of label enrichment. In five different datasets, the technique consistently doubles the number of peptides with high goodness-of-fit characteristics of the turnover rate model. We also introduce a software tool, d2ome+, which automates the protein turnover estimation from partial isotope profiles.

Alternative Splicing of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Is Regulated by RBFOX2 in Lymphoid Malignancies.

Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans-regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT-specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.

Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells.

Flaviviruses are small RNA viruses that are mainly transmitted via arthropod vectors and are found in tropic and sub-tropical regions. Most infections are asymptomatic (90-95%), but symptoms can be as severe as hemorrhagic fever and encephalitis. One recently emerged flavivirus is Zika virus (ZIKV), which was originally isolated from rhesus monkeys in Uganda roughly 70 years ago but has recently spread east, reaching S. America in 2015-2016. This outbreak was associated with the development of Guillain-Barré syndrome in adults and microcephaly in infants born to expectant mothers infected early in pregnancy. ZIKV must traverse the placenta to impact the development of the fetus, but the mechanisms responsible are unknown. While flaviviruses are known to disrupt splicing patterns in host cells, little is known about how flaviviruses such as ZIKV impact the alternative polyadenylation (APA) of host transcripts. This is important as APA is well-established as a mechanism in the regulation of mRNA metabolism and translation. Thus, we sought to characterize transcriptomic changes including APA in human placental (JEG3) cells in response to ZIKV infection using Poly(A)-ClickSeq (PAC-Seq). We used our differential Poly(A)-cluster (DPAC) analysis pipeline to characterize changes in differential gene expression, alternative poly-adenylation (APA) and the use of alternative terminal exons. We identified 98 upregulated genes and 28 downregulated genes. Pathway enrichment analysis indicated that many RNA processing and immune pathways were upregulated in ZIKV-infected JEG3 cells. We also updated DPAC to provide additional metrics of APA including the percentage-distal usage index (PDUI), which revealed that APA was extensive and the 3' UTRs of 229 genes were lengthened while 269 were shortened. We further found that there were 214 upregulated and 59 downregulated poly(A)-clusters (PACs). We extracted the nucleotide sequences surrounding these PACs and found that the canonical signals for poly-adenylation (binding site for poly-A binding protein (PABP) upstream and a GU-rich region down-stream of the PAC) were only enriched in the downregulated PACs. These results indicate that ZIKV infection makes JEG3 cells more permissive to non-canonical poly-adenylation signals.

RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3'-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

Genes of the cGMP-PKG-Ca2+ signaling pathway are alternatively spliced in cardiomyopathy: Role of RBFOX2.

Aberrations in the cGMP-PKG-Ca2+ pathway are implicated in cardiovascular complications of diverse etiologies, though involved molecular mechanisms are not understood. We performed RNA-Seq analysis to profile global changes in gene expression and exon splicing in Chagas disease (ChD) murine myocardium. Ingenuity-Pathway-Analysis of transcriptome dataset identified 26 differentially expressed genes associated with increased mobilization and cellular levels of Ca2+ in ChD hearts. Mixture-of-isoforms and Enrichr KEGG pathway analyses of the RNA-Seq datasets from ChD (this study) and diabetic (previous study) murine hearts identified alternative splicing (AS) in eleven genes (Arhgef10, Atp2b1, Atp2a3, Cacna1c, Itpr1, Mef2a, Mef2d, Pde2a, Plcb1, Plcb4, and Ppp1r12a) of the cGMP-PKG-Ca2+ pathway in diseased hearts. AS of these genes was validated by an exon exclusion-inclusion assay. Further, Arhgef10, Atp2b1, Mef2a, Mef2d, Plcb1, and Ppp1r12a genes consisted RBFOX2 (RNA-binding protein) binding-site clusters, determined by analyzing the RBFOX2 CLIP-Seq dataset. H9c2 rat heart cells transfected with Rbfox2 (vs. scrambled) siRNA confirmed that expression of Rbfox2 is essential for proper exon splicing of genes of the cGMP-PKG-Ca2+ pathway. We conclude that changes in gene expression may influence the Ca2+ mobilization pathway in ChD, and AS impacts the genes involved in cGMP/PKG/Ca2+ signaling pathway in ChD and diabetes. Our findings suggest that ChD patients with diabetes may be at increased risk of cardiomyopathy and heart failure and provide novel ways to restore cGMP-PKG regulated signaling networks via correcting splicing patterns of key factors using oligonucleotide-based therapies for the treatment of cardiovascular complications.

Emerging roles of RNA-binding proteins in diabetes and their therapeutic potential in diabetic complications.

Diabetes is a debilitating health care problem affecting 422 million people around the world. Diabetic patients suffer from multisystemic complications that can cause mortality and morbidity. Recent advancements in high-throughput next-generation RNA-sequencing and computational algorithms led to the discovery of aberrant posttranscriptional gene regulatory programs in diabetes. However, very little is known about how these regulatory programs are mis-regulated in diabetes. RNA-binding proteins (RBPs) are important regulators of posttranscriptional RNA networks, which are also dysregulated in diabetes. Human genetic studies provide new evidence that polymorphisms and mutations in RBPs are linked to diabetes. Therefore, we will discuss the emerging roles of RBPs in abnormal posttranscriptional gene expression in diabetes. Questions that will be addressed are: Which posttranscriptional mechanisms are disrupted in diabetes? Which RBPs are responsible for such changes under diabetic conditions? How are RBPs altered in diabetes? How does dysregulation of RBPs contribute to diabetes? Can we target RBPs using RNA-based methods to restore gene expression profiles in diabetic patients? Studying the evolving roles of RBPs in diabetes is critical not only for a comprehensive understanding of diabetes pathogenesis but also to design RNA-based therapeutic approaches for diabetic complications. WIREs RNA 2018, 9:e1459. doi: 10.1002/wrna.1459 This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing Translation > Translation Regulation.

Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice.

Fortilin, a pro-survival molecule, inhibits p53-induced apoptosis by binding to the sequence-specific DNA-binding domain of the tumor suppressor protein and preventing it from transcriptionally activating Bax. Intriguingly, fortilin protects cells against ROS-induced cell death, independent of p53. The signaling pathway through which fortilin protects cells against ROS-induced cell death, however, is unknown. Here we report that fortilin physically interacts with the antioxidant enzyme peroxiredoxin-1 (PRX1), protects it from proteasome-mediated degradation, and keeps it enzymatically active by blocking its deactivating phosphorylation by Mst1, a serine/threonine kinase. At the whole animal level, the liver-specific overexpression of fortilin reduced PRX1 phosphorylation in the liver, enhanced PRX1 activity, and protected the transgenic animals against alcohol-induced, ROS-mediated, liver damage. These data suggest the presence of a novel oxidative-stress-handling pathway where the anti-p53 molecule fortilin augments the peroxidase PRX1 by protecting it against degradation and inactivation of the enzyme. Fortilin-PRX1 interaction in the liver could be clinically exploited further to prevent acute alcohol-induced liver damage in humans.

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure.

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

Rbfox2 function in RNA metabolism is impaired in hypoplastic left heart syndrome patient hearts.

Hypoplastic left heart syndrome (HLHS) is a fatal congenital heart disease in which the left side of the heart is underdeveloped, impairing the systemic circulation. Underdeveloped left ventricle exerts biomechanical stress on the right ventricle that can progress into heart failure. Genome-wide transcriptome changes have been identified at early stages in the right ventricle (RV) of infants with HLHS, although the molecular mechanisms remain unknown. Here, we demonstrate that the RNA binding protein Rbfox2, which is mutated in HLHS patients, is a contributor to transcriptome changes in HLHS patient RVs. Our results indicate that majority of transcripts differentially expressed in HLHS patient hearts have validated Rbfox2 binding sites. We show that Rbfox2 regulates mRNA levels of targets with 3'UTR binding sites contributing to aberrant gene expression in HLHS patients. Strikingly, the Rbfox2 nonsense mutation identified in HLHS patients truncates the protein, impairs its subcellular distribution and adversely affects its function in RNA metabolism. Overall, our findings uncover a novel role for Rbfox2 in controlling transcriptome in HLHS.

Dysregulation of RBFOX2 Is an Early Event in Cardiac Pathogenesis of Diabetes.

Alternative splicing (AS) defects that adversely affect gene expression and function have been identified in diabetic hearts; however, the mechanisms responsible are largely unknown. Here, we show that the RNA-binding protein RBFOX2 contributes to transcriptome changes under diabetic conditions. RBFOX2 controls AS of genes with important roles in heart function relevant to diabetic cardiomyopathy. RBFOX2 protein levels are elevated in diabetic hearts despite low RBFOX2 AS activity. A dominant-negative (DN) isoform of RBFOX2 that blocks RBFOX2-mediated AS is generated in diabetic hearts. DN RBFOX2 interacts with wild-type (WT) RBFOX2, and ectopic expression of DN RBFOX2 inhibits AS of RBFOX2 targets. Notably, DN RBFOX2 expression is specific to diabetes and occurs at early stages before cardiomyopathy symptoms appear. Importantly, DN RBFOX2 expression impairs intracellular calcium release in cardiomyocytes. Our results demonstrate that RBFOX2 dysregulation by DN RBFOX2 is an early pathogenic event in diabetic hearts.