Decoding Cardiac Maturation Program: Insights from RNA Splicing Regulation.

Introduction: Cardiomyocyte maturation is a postnatal heart development that requires precise molecular, metabolic, structural, and electrophysiological changes to sustain the increased workload of the adult heart. Previous studies demonstrate that alternative RNA splicing is a key regulatory mechanism during this process. However, the full landscape of isoform switch and its role in functional specialization in adult cardiomyocyte remain largely unexplored. This study aims to provide a comprehensive landscape of isoform utilization during heart maturation. Methods: Using PacBio Iso-Seq, we profiled full-length transcriptomic changes in mouse left ventricles across neonatal to postnatal developmental stages. Isoform expression and usage ratios were analyzed to identify the genes with significant isoform switch. Functional validation of selected isoforms was performed in hESC-CMs. Results: While the total number of genes expressed remains relatively stable, the total number of full-length transcripts decreased from E18 to P49, indicating a progressive reduction in transcriptomic complexity at the isoform level. PCA analysis of isoform-specific expression and ratios shows stage-dependent clustering, highlighting that cardiac isoform dynamics is a defining feature for postnatal heart maturation. We identified at least 50 genes showing significant isoform switches across the maturation stages. In addition, an isoform of PDLIM5 is found to have exon 5B inclusion (PDLIM5-Ex5B) and its expression is increased in adult mouse heart but reversed to neonatal level in the mouse failing heart (n=3, p<0.05 compared to P49 and Sham control). hESC-CMs expressing PDLIM5-Ex5B improved sarcomere organization, enhanced binucleation, and increased expression of maturation markers. In contrast, isoforms lacking exon 5B were less effective in promoting cardiomyocyte maturation features. These findings further confirm that alternative splicing-mediated isoform switch is critical for cardiomyocyte structural and functional maturation. Conclusions: This study provides a comprehensive landscape of RNA splicing and isoform switching events during heart maturation, highlighting their importance in defining postnatal heart maturation stages and achieving functional specialization. Our results serve as a valuable resource for exploring maturation-related RNA splicing regulators and may represent a potential splicing-targeted therapy for heart diseases.

productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. [13][1]; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is implicated in both development and disease. However, the change of alternative mRNA splicing during cardiomyocytes maturation is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart. Further, we identified a highly conserved splicing regulator-RBFox1 to be significantly induced during zebrafish, mouse and human cardiac maturation. RBFox1 expression was also detected in cardiomyocytes derived from both mouse and human embryonic stem cells but at much lower levels comparing to adult heart. In zebrafish embryos, inactivation of RBFox1 caused cardiomyocyte maturation defects. Expression of RBFox1 in cultured neonatal cardiomyocytes was sufficient to promote maturation by reducing fetal marker gene expression while increasing calcium handling gene expression including RyR and promoting sarcomere organization. Deep RNA-Sequencing analysis showed that RBFox1 expression promoted alternative splicing in genes involved in calcium cycling, blood vessel development and muscle contraction. Finally, we identified a highly conserved mutually exclusive alternative splicing event of transcription factor MEF2 to be a direct downstream target of RBFox1. Expression of individual MEF2 splicing variants led to different cardiac developmental phenotypes in zebrafish, indicating their different transcriptional activities. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified RBFox1 as a key regulator for alternative RNA splicing during cardiomyocytes maturation. Further exploration of RBFox1 mediated RNA splicing regulation in heart may yield novel insight to the underlying mechanisms of cardiac maturation and new approach to improve cell based therapy for heart diseases.

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, the global pattern of alternative mRNA splicing during cardiac development and diseases is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we have identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart following pressure-overload induced heart failure. The alternative RNA splicing events observed in failing hearts mimics the profile in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. Using RNA-Seq database and real-time PCR analysis, we examined the expression profile of a large number of known alternative splicing regulators. Among them, we identified Fox1 as a significantly induced regulator during cardiac development in zebrafish, mouse and human, and down-regulated in both mouse and human failing hearts. Morpholino mediated Fox1 knockdown in zebrafish embryos led to lethal phenotype associated with reduced cardiac function and defects in chamber specificity. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene, suggesting a highly conserved cardiac function of Fox1 for normal cardiac development and function in vertebrates. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a key regulator for alternative RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome, and reveals the functional importance of RNA-splicing in cardiac development and diseases.

Our previous studies have shown that IWS1 (Interacts with Spt6) is a phosphorylation target of AKT and regulates the alternative RNA splicing of FGFR2, linking IWS1 with human Non-Small Cell Lung Cancer. To further address the role of IWS1 in alternative RNA splicing in lung cancer, we performed an RNA-seq study using lung adenocarcinoma cells in which IWS1 was knocked down or replaced by its phosphorylation site mutant. The results identified a novel, exon 2 deficient splice variant of the splicing factor U2 Associated-Factor 2 (U2AF2), whose abundance increases, upon the loss of phosphorylated IWS1. This exon encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). Here, we show that U2AF2 exon 2 inclusion depends on phosphorylated IWS1, by promoting histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes, in a cell-cycle specific manner. Inhibition of the pathway results in the downregulation of cell cycle division associated 5 (CDCA5), and its protein product, Sororin, a phosphorylation target of ERK and member of the cohesin complex, essential of G2/M phase progression. We also reveal the existence of a novel Sororin/ERK feedback loop controlled by the epigenetic regulation of U2AF2 RNA splicing, downstream of IWS1 phosphorylation. Given that the U2AF2 RNA splicing is regulated through the cell cycle and controls Sororin, our data unravel a novel RNA splicing pattern which is regulated through the cell cycle and feedbacks towards its regulation. Impairment of this signaling pathway leads to leading to G2/M phase arrest, impaired cell proliferation and tumor growth in mouse xenografts models, an effect more pronounced in EGFR mutant cells. Analysis of lung adenocarcinoma samples revealed strong correlations between IWS1 phosphorylation, U2AF2 RNA splicing, and Sororin/p-ERK levels, especially in EGFR, as opposed to K-RAS mutant patients. More importantly, IWS1 phosphorylation and U2AF2 RNA splicing pattern are positively correlated with tumor stage, grade and metastasis, and associated with poor survival in the same patients. This work highlights the instrumental role of the AKT/p-IWS1 axis to alternative RNA splicing in governing cell cycle progression and tumorigenesis and proposes this axis as a novel drug target in EGFR mutant lung adenocarcinoma, by concomitantly affecting the epigenetic regulation of RNA processing and oncogenic signals. Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Maria D. Paraskevopoulou, Abdul Kaba, Alessandro La Ferlita, Vollter Anastas, Arturo Orlacchio, Vasiliki Taraslia, Ioannis Vlachos, Marina Capece, Artemis Hatzigeorgiou, Dario Palmieri, Salvatore Alaimo, Christos Tsatsanis, Lalit Sehgal, David P. Carbone, Vincenzo Coppola, Philip N. Tsichlis. IWS1 phosphorylation promotes tumor growth and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the epigenetic regulation of U2AF2 RNA splicing [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-011.

While cancer-associated SF3B1 mutations causes alternative RNA splicing, the molecular mechanism underlying the alternative RNA splicing is not fully elucidated. Here, we analysed the proteins that interacted with the wild-type and K700E-mutated SF3B1 and found that the interactions of two RNA helicases, DDX42 and DDX46, with the mutated SF3B1 were reduced. Overexpression of DDX42 restored the decreased interaction between DDX42 and the K700E-mutated SF3B1, and suppressed some alternative RNA splicing associated with the SF3B1 mutation. Mutation that decreased the ATP hydrolysis activities of DDX42 abolished the suppressive effects of DDX42 on the alternative RNA splicing, suggesting that the ATP hydrolysis activity of DDX42 is involved in the mechanism of the altered RNA splicing associated with the SF3B1 mutation. Our study demonstrates an important function of the interaction between DDX42 and SF3B1 on regulating RNA splicing and revealed a potential role of DDX42 in the altered RNA splicing associated with the SF3B1 mutation.

Alternative RNA splicing is an important regulatory process used by genes to increase their diversity. This process is mainly executed by specific classes of RNA binding proteins that act in a dosage-dependent manner to include or exclude selected exons in the final transcripts. While these processes are tightly regulated in cells and tissues, little is known on how the dosage of these factors is achieved and maintained. Several recent studies have suggested that alternative RNA splicing may be in part modulated by microRNAs (miRNAs), which are short, non-coding RNAs (~22 nt in length) that inhibit translation of specific mRNA transcripts. As evidenced in tissues and in diseases, such as cancer and neurological disorders, the dysregulation of miRNA pathways disrupts downstream alternative RNA splicing events by altering the dosage of splicing factors involved in RNA splicing. This attractive model suggests that miRNAs can not only influence the dosage of gene expression at the post-transcriptional level but also indirectly interfere in pre-mRNA splicing at the co-transcriptional level. The purpose of this review is to compile and analyze recent studies on miRNAs modulating alternative RNA splicing factors, and how these events contribute to transcript rearrangements in tissue development and disease.

Genomic profiling of human tumors has had a major impact on translational and clinical research and clinical practice by providing important molecular information to researchers, health care providers and patients. Most studies have focused on mutations and aggregate gene expression in patients of European ancestry, resulting in potential missed drivers of cancer heterogeneity among patients of different ancestries. This underscores the importance of conducting further translational and clinical research on additional oncogenic molecular mechanisms in diverse populations to aid in development of new diagnostic and therapeutic interventions. Cancer disparities are the result of a complex interplay among social, structural (health system), lifestyle and biological determinants of health. Biological determinants of health vary between populations as a function of the human diaspora and the dynamic interplay between genome and environment over time, resulting in both genotypic and phenotypic diversity. An important driver of both organismal and cellular biological heterogeneity is Alternative RNA Splicing (ARS), a key step in gene expression and protein diversification in higher eukaryotes. Consistent with its role in driving evolutionary biological diversity, ARS is increasingly implicated as a major driver of tumor-related biological diversity, yet it is often overlooked in translational and clinical cancer research. Genetic variation in cis-acting splicing elements, differential expression of trans-acting splicing factors, mutation in genes encoding components of the RNA splicing machinery and aberrant and ARS events can all contribute to cancer. Work from our laboratory and others highlights the importance of ancestry-related ARS in cancer biology and cancer disparities, and demonstrates that dysregulation of ARS is a principal feature differentiating cancers from their host tissues of origin. Extensive race-related differences in expression of RNA splice variants between prostate cancer (PCa) in self-identified African American (AA) and white (W) patients were observed, and approximately one-third of the variants enriched in PCa in AA patients were also present in patient-matched normal prostate specimens, indicating potential germline origin and clinical significance as biomarkers. Ancestry-specific PCa cell lines and xenografts were used to demonstrate the functional significance of these AA-enriched RNA splice variants in driving ancestry-related PCa aggressiveness and influencing drug response to corresponding targeted therapeutics. This approach has revealed novel molecular targets that are being studied further pre-clinically in population-specific PCa cell lines and mouse trials, at the population-level using diverse human PCa specimens and controls and in first-ever clinical trials in PCa patients stratified by both biomarker and ancestry. We have also developed an algorithm to enable analysis of race-related ARS using annotated publicly available RNA Seq data. Using this algorithm and data from The Cancer Genome Atlas, we have identified additional race-related ARS events between PCa from AA and W patients. The majority of these events involve exon skipping. Genes that undergo race-related ARS do not overlap with genes that exhibit race-related aggregate gene expression in the same specimens; however, a significant number of the Gene Ontology terms corresponding to the genes exhibiting race-related aggregate gene expression or ARS do overlap, indicating that, despite these two distinct mechanisms of regulation (transcription and RNA splicing), both differentially regulate common pathways in PCa between AA and W patients. We have also identified 10 trans-acting splicing factors, whose aggregate gene expression significantly differed between PCa from AA and W patients, suggesting a potential mechanistic relationship between these factors and the identified race-related ARS events. In addition, analysis of race-related ARS in PCa tissue stratified by Gleason grade 6, 7, >8 from AA and white patients has revealed additional novel ARS events now under preclinical evaluation, and a subset of genes exhibiting race-related ARS that are common among prostate, lung, breast and liver cancer. Finally, we have computationally identified associations between single nucleotide polymorphisms predicted to regulate RNA splicing and disparities in PCa risk, PCa aggressiveness and PCa survival, and we are currently investigating the significance of such single nucleotide polymorphisms in mechanistic and functional biology. Our laboratory is also engaged in developing therapeutic approaches to manipulate ARS, correct aberrant RNA splicing or produce novel RNA splice variants. These include splice-switching oligonucleotides (SSOs) that can modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the RNA splicing machinery to a particular splice site, thereby simultaneously limiting production of pathogenic RNA splice variants and maintaining/inducing expression of RNA splice variants with therapeutic value. In addition, we are using phenotypic readouts and RNA splicing-specific reporters to conduct high-throughput screens of small molecules and have identified novel potential modulators of RNA splicing. Despite the significance of ARS to cancer, most profiling of tumor heterogeneity and clinically-oriented studies of cancer biomarkers and therapeutics overlook its importance. We are only now starting to appreciate the translational importance of ARS in cancer, including its emerging role in generating novel immunogenic neoantigens, predicting clinical responses to therapy and in dysregulating major tumor suppressor genes and oncogenes affecting virtually every hallmark of cancer. Also under-appreciated are its contributions to ancestry-related tumor aggressiveness, heterogeneity of tumor and host cell stress responses, and therapeutic target potential. Increased collaborations among bioinformaticians, cancer biologists and clinicians are needed to identify, analyze, and develop ARS into precision oncology. This work is supported by a DoD Prostate Cancer Research Program Health Disparity Research Award PC131972, a NIH Feasibility Studies to Build Collaborative Partnerships in Cancer Research P20 Award 1P20-CA202925-01A1 to, a NIH Basic Research in Cancer Health Disparities R01 Award R01CA220314 and a Prostate Cancer Foundation 2018 Challenge Award. Citation Format: Steven R. Patierno. Spliceomics: Alternative RNA splicing as a source of ancestry-related molecular targets in precision oncology and cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr PL02-02.

Human embryonic stem cells (hESCs) and neural progenitor (NP) cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation. Alternative RNA splicing (AS), a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function. Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays. We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol), to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC. Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%. REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events. Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes. In addition, we found that REAP predictions are enriched in genes encoding serine/threonine kinase and helicase activities. An example is a REAP-predicted alternative exon in the SLK (serine/threonine kinase 2) gene that is differentially included in hESC, but skipped in NP as well as in other differentiated tissues. Lastly, comparative sequence analysis revealed conserved intronic cis-regulatory elements such as the FOX1/2 binding site GCAUG as being proximal to candidate AS exons, suggesting that FOX1/2 may participate in the regulation of AS in NP and hESC. In summary, a new methodology for exon array analysis was introduced, leading to new insights into the complexity of AS in human embryonic stem cells and their transition to neural stem cells.

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.

Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases.

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

SUMMARYCardiac maturation is crucial for postnatal cardiac development and is increasingly known to be regulated by a series of transcription factors. However, post-translational mechanisms regulating this process remain unclear. Here we report the indispensable role of neddylation in cardiac maturation. Mosaic deletion of NAE1, an essential enzyme for neddylation, in neonatal hearts results in the rapid development of cardiomyopathy and heart failure. NAE1 deficiency disrupts transverse tubule formation, inhibits physiological hypertrophy, and represses fetal-to-adult isoform switching, thus culminating in cardiomyocyte immaturation. Mechanistically, we find that neddylation is needed for the perinatal metabolic transition from glycolytic to oxidative metabolism in cardiomyocytes. Further, we show that HIF1α is a putative neddylation target and that inhibition of neddylation accumulates HIF1α and impairs fatty acid utilization and bioenergetics in cardiomyocytes. Together, our data show neddylation is required for cardiomyocyte maturation through promoting oxidative metabolism in the developing heart.

Metabolic Imaging of Pain Matrix Using 18F Fluoro-deoxyglucose Positron Emission Tomography/Computed Tomography for Patients Undergoing L2 Dorsal Root Ganglion Stimulation for Low Back Pain

Retrospective diagnosis of a seizure type is pivotal for effective management and treatment of epilepsy. Previously, we demonstrated that RNA signatures could discriminate between non-epileptic spells and epileptic seizures. Here, we investigate the utility of alternative RNA splicing to distinguish generalized versus focal epileptic seizures. Blood samples were collected at baseline, 4–6 h post-seizure, and at discharge from 27 patients undergoing video-electroencephalogram (vEEG) monitoring at the Emory University Hospital. Epileptologists determined seizure classification through vEEG data review. RNA was extracted, sequenced, and analyzed for RNA expression and transcript usage. Classification models were generated to distinguish between patients who had a focal or generalized seizure. The study shows transcriptomic profile changes following EEG-verified focal and generalized seizures. Compared to baseline, focal seizure exhibits limited changes in transcriptomic expression 4–6 h post-seizure and discharge samples. In contrast, generalized seizures demonstrated a broader transcript response, with 74 differentially expressed transcripts at 4–6 h and 70 at discharge. The changes were also evident across different time points between focal and generalized seizure. The study for the first time described the landscape of isoform switching in seizure type. Notably, significant isoform switching without differences in gene expression was observed. We identified 2689 isoform switches linked to 1249 genes among which 742 genes were sensitive to nonsense-mediated mRNA decay (NMD). Significant switches were observed in genes such as CORO1C, ZBTB44, SNHG1, and RPS17. Notably, we also observed novel isoforms, including CD300 (MSTRG.26116.1), RNF216 (MSTRG.52862.7), and RN7SL1 (MSTRG.17010.3) which exhibited significant switching, revealing potential new regulators of gene expression. Differentially expressed transcripts were utilized as classifiers for machine learning (ML) modeling using random forest (rf) and radial support vector machine (rSVM) algorithms, achieving ~ 83% accuracy in classifying generalized seizures, and multivariate adaptive regression splines (mars) algorithm achieving 100% accuracy in identifying focal seizure events. Our findings of blood transcript expression changes, including isoform switch analysis, underscore the potential of blood-based transcriptome analysis for retrospectively distinguishing seizure types and identifying biomarkers for epilepsy management. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-025-05110-1.