Tissue-specific Splicing Research Articles

SpliceTransformer predicts tissue-specific splicing linked to human diseases

We present SpliceTransformer (SpTransformer), a deep-learning framework that predicts tissue-specific RNA splicing alterations linked to human diseases based on genomic sequence. SpTransformer outperforms all previous methods on splicing prediction. Application to approximately 1.3 million genetic variants in the ClinVar database reveals that splicing alterations account for 60% of intronic and synonymous pathogenic mutations, and occur at different frequencies across tissue types. Importantly, tissue-specific splicing alterations match their clinical manifestations independent of gene expression variation. We validate the enrichment in three brain disease datasets involving over 164,000 individuals. Additionally, we identify single nucleotide variations that cause brain-specific splicing alterations, and find disease-associated genes harboring these single nucleotide variations with distinct expression patterns involved in diverse biological processes. Finally, SpTransformer analysis of whole exon sequencing data from blood samples of patients with diabetic nephropathy predicts kidney-specific RNA splicing alterations with 83% accuracy, demonstrating the potential to infer disease-causing tissue-specific splicing events. SpTransformer provides a powerful tool to guide biological and clinical interpretations of human diseases.

Machine learning-optimized targeted detection of alternative splicing.

RNA-sequencing (RNA-seq) is widely adopted for transcriptome analysis but has inherent biases which hinder the comprehensive detection and quantification of alternative splicing. To address this, we present an efficient targeted RNA-seq method that greatly enriches for splicing-informative junction-spanning reads. Local Splicing Variation sequencing (LSV-seq) utilizes multiplexed reverse transcription from highly scalable pools of primers anchored near splicing events of interest. Primers are designed using Optimal Prime, a novel machine learning algorithm trained on the performance of thousands of primer sequences. In experimental benchmarks, LSV-seq achieves high on-target capture rates and concordance with RNA-seq, while requiring significantly lower sequencing depth. Leveraging deep learning splicing code predictions, we used LSV-seq to target events with low coverage in GTEx RNA-seq data and newly discover hundreds of tissue-specific splicing events. Our results demonstrate the ability of LSV-seq to quantify splicing of events of interest at high-throughput and with exceptional sensitivity.

Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response

Mitochondrial morphology and function change dynamically in response to intracellular signaling and the surrounding environment. The mitochondrial fission factor Mff, which localizes to the outer mitochondrial membrane, mediates not only mitochondrial fission by recruiting the dynamin-related GTPase Drp1 to mitochondrial fission sites but also the double-stranded RNA-induced antiviral response on mitochondria through mitochondrial antiviral signaling (MAVS). Mff is reported to be regulated by AMP-activated protein kinase (AMPK)-mediated protein phosphorylation and alternative pre-mRNA splicing; however, the relationships among RNA splicing, phosphorylation, and multiple functions of Mff have not been fully understood. Here, we showed that mouse Mff has a tissue-specific splicing pattern, and at least eight Mff splice isoforms were expressed in mouse embryonic fibroblasts (MEFs). We introduced single Mff isoforms into Mff knockout MEFs and found that insertion of exon 6 just after the phosphorylation site, by the alternative splicing, reduced its phosphorylation by AMPK and its functions in mitochondrial fission and the antiviral response. In addition, the underlying mechanism repressing these functions was independent of phosphorylation. These results indicate that multiple functions of Mff on mitochondria are regulated by AMPK-mediated phosphorylation and alternative splicing, under the control of energy metabolism and cellular differentiation.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTβR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTβR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTβR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Comparative RNA-Seq Analysis Revealed Tissue-Specific Splicing Variations during the Generation of the PDX Model

Tissue-specific gene expression generates fundamental differences in the function of each tissue and affects the characteristics of the tumors that are created as a result. However, it is unclear how much the tissue specificity is conserved during grafting of the primary tumor into an immune-compromised mouse model. Here, we performed a comparative RNA-seq analysis of four different primary-patient derived xenograft (PDX) tumors. The analysis revealed a conserved RNA biotype distribution of primary−PDX pairs, as revealed by previous works. Interestingly, we detected significant changes in the splicing pattern of PDX, which was mainly comprised of skipped exons. This was confirmed by splicing variant-specific RT-PCR analysis. On the other hand, the correlation analysis for the tissue-specific genes indicated overall strong positive correlations between the primary and PDX tumor pairs, with the exception of gastric cancer cases, which showed an inverse correlation. These data propose a tissue-specific change in splicing events during PDX formation as a variable factor that affects primary−PDX integrity.

Neuronal and astrocyte NCX isoform/splice variants: How do they participate in Na+ and Ca2+ signalling?

NCX1, NCX2, and NCX3 gene isoforms and their splice variants are characteristically expressed in different regions of the brain. The tissue-specific splice variants of NCX1–3 isoforms show specific expression profiles in neurons and astrocytes, whereas the relevant NCX isoform/splice variants exhibit diverse allosteric modes of Na+- and Ca2+-dependent regulation. In general, overexpression of NCX1–3 genes leads to neuroprotective effects, whereas their ablation gains the opposite results. At this end, the partial contributions of NCX isoform/splice variants to neuroprotective effects remain unresolved. The glutamate-dependent Na+ entry generates Na+ transients (in response to neuronal cell activities), whereas the Na+-driven Ca2+ entry (through the reverse NCX mode) raises Ca2+ transients. This special mode of signal coupling translates Na+ transients into the Ca2+ signals while being a part of synaptic neurotransmission. This mechanism is of general interest since disease-related conditions (ischemia, metabolic stress, and stroke among many others) trigger Na+ and Ca2+ overload with deadly outcomes of downstream apoptosis and excitotoxicity. The recently discovered mechanisms of NCX allosteric regulation indicate that some NCX variants might play a critical role in the dynamic coupling of Na+-driven Ca2+ entry. In contrast, the others are less important or even could be dangerous under altered conditions (e.g., metabolic stress). This working hypothesis can be tested by applying advanced experimental approaches and highly focused computational simulations. This may allow the development of structure-based blockers/activators that can selectively modulate predefined NCX variants to lessen the life-threatening outcomes of excitotoxicity, ischemia, apoptosis, metabolic deprivation, brain injury, and stroke.

Counting pseudoalignments to novel splicing events.

Alternative splicing (AS) of introns from pre-mRNA produces diverse sets of transcripts across cell types and tissues, but is also dysregulated in many diseases. Alignment-free computational methods have greatly accelerated the quantification of mRNA transcripts from short RNA-seq reads, but they inherently rely on a catalog of known transcripts and might miss novel, disease-specific splicing events. By contrast, alignment of reads to the genome can effectively identify novel exonic segments and introns. Event-based methods then count how many reads align to predefined features. However, an alignment is more expensive to compute and constitutes a bottleneck in many AS analysis methods. Here, we propose fortuna, a method that guesses novel combinations of annotated splice sites to create transcript fragments. It then pseudoaligns reads to fragments using kallisto and efficiently derives counts of the most elementary splicing units from kallisto's equivalence classes. These counts can be directly used for AS analysis or summarized to larger units as used by other widely applied methods. In experiments on synthetic and real data, fortuna was around 7× faster than traditional align and count approaches, and was able to analyze almost 300 million reads in just 15 min when using four threads. It mapped reads containing mismatches more accurately across novel junctions and found more reads supporting aberrant splicing events in patients with autism spectrum disorder than existing methods. We further used fortuna to identify novel, tissue-specific splicing events in Drosophila. fortuna source code is available at https://github.com/canzarlab/fortuna.

HSF4 Transcriptionally Activates Autophagy by Regulating ATG9a During Lens Terminal Differentiation.

HSF4 mutations are responsible for congenital cataract formation. Dysfunction of HSF4 leads to defects in lens terminal differentiation. We aimed to study the mechanism of how HSF4 promotes organelle degradation during lens differentiation. HSF4del42 mutant mice that developed congenital cataracts were employed. The organelle degradation and autophagic function in lens fibers were detected by immunofluorescence and Immunoblotting. Transcriptome analysis was performed to investigate the differentially expressed genes in HSF4del42 lenses, whereas luciferase report assay and ChIP assay were used to confirm the directly transcriptional regulation of ATG9a by HSF4. HSF4del42 mice displayed delayed organelle clearance and impaired autophagic degradation function in lens fibers. Activation of autophagy by rapamycin ameliorated the defects in organelle clearance in HSF4del42 lenses ex vivo and in vivo. Depletion of HSF4 attenuated autophagic flux by disrupting autophagosome biogenesis and maturation in lens epithelial cells. HSF4 directly transcriptionally activated the core autophagy protein ATG9a. Instead of the canonical ATG9a isoform, the ATG9a-X2 isoform was predominantly expressed in the lens and alleviated autophagic defects in HSF4 KO lens epithelial cells. The ATG9a-X2 protein displayed a short half-life, and rapamycin treatment restored its levels in HSF4 KO lens epithelial cells and HSF4del42 lenses. Our findings demonstrate that HSF4 facilitates organelle degradation probably by transcriptionally activating autophagy during lens terminal differentiation. We first report the involvement of HSF4 in autophagy and the tissue specific splicing of ATG9a. Our study indicates that autophagy activation is a possible therapeutic strategy for HSF4-related congenital cataracts.

Cause or consequence? The role of IL-1 family cytokines and receptors in neuroinflammatory and neurodegenerative diseases.

Cytokines and receptors of the IL-1 family are key mediators in innate immune and inflammatory reactions in physiological defensive conditions, but are also significantly involved in immune-mediated inflammatory diseases. Here, we will address the role of cytokines of the IL-1 superfamily and their receptors in neuroinflammatory and neurodegenerative diseases, in particular Multiple Sclerosis and Alzheimer's disease. Notably, several members of the IL-1 family are present in the brain as tissue-specific splice variants. Attention will be devoted to understanding whether these molecules are involved in the disease onset or are effectors of the downstream degenerative events. We will focus on the balance between the inflammatory cytokines IL-1β and IL-18 and inhibitory cytokines and receptors, in view of future therapeutic approaches.

Aberrant splicing prediction across human tissues.

Aberrant splicing is a major cause of genetic disorders but its direct detection in transcriptomes is limited to clinically accessible tissues such as skin or body fluids. While DNA-based machine learning models can prioritize rare variants for affecting splicing, their performance in predicting tissue-specific aberrant splicing remains unassessed. Here we generated an aberrant splicing benchmark dataset, spanning over 8.8 million rare variants in 49 human tissues from the Genotype-Tissue Expression (GTEx) dataset. At 20% recall, state-of-the-art DNA-based models achieve maximum 12% precision. By mapping and quantifying tissue-specific splice site usage transcriptome-wide and modeling isoform competition, we increased precision by threefold at the same recall. Integrating RNA-sequencing data of clinically accessible tissues into our model, AbSplice, brought precision to 60%. These results, replicated in two independent cohorts, substantially contribute to noncoding loss-of-function variant identification and to genetic diagnostics design and analytics.

Abstract 3845: Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain

Abstract PTEN negatively regulates PI3K/AKT/mTOR signaling through dephosphorylation of PIP3 to PIP2. PTEN dysfunction triggers disease phenotypes, ranging from cancer and PHTS to NDDs such as ASD. The most common PTEN mutations, nonsense, frameshift, and deletion/insertion, may cause premature termination of translation. PTEN has a considerable number of missense mutations that may result in loss of protein function through reduced catalytic activity and protein stability. To decipher mechanistic details of the mutations and structural features of the mutant proteins, comprehensive computational studies were performed for PTEN mutants at an anionic lipid bilayer containing PC, PS, PIP2, and PIP3. Six PTP mutations (Y68H, H93R, A126T, R130Q, G132D, R173C) and two C2 mutations (F241S, D252G) were considered. The PTP mutations are known to associate with cancer, PHTS, macrocephaly, and ASD, while the C2 mutations are exclusively related to macrocephaly and ASD. Our studies indicate that the PTEN mutants can effectively absorb the anionic lipid bilayer, similar to wild-type PTEN. However, Y68H significantly disrupts the core of PTP domain, reducing protein stability. H93R hijacks the substate PIP3, interrupting the catalytic site residues recruitment of the substate for catalysis. A126T, R130Q, and G132D in the P loop directly influence the dynamics of the loop, yielding a collapsed loop conformation. The collapsed P loop loses the interaction with the WPD loop, leading to dehydration in the catalytic site. R173C and D252G at the interface between two major domains disrupt the domain-domain interaction, allosterically biasing the P loop dynamics. However, F241S in C2 exhibits less effective allosteric connection to the catalytic site than that observed in the wild type. PCA of the sampled conformations found that the macrocephaly and ASD-related H93R and F241S are likely to sample the conformations present in the wild type. In contrast, the sampled conformations for the cancer and PHTS-related Y68H, A126T, and G132D are different from those for the wild type. These distinct structural features seem to correlate with mutation strength and timing of the expression of the genes that determine the cancer and NDD outcomes. The isoform expression data extracted from the BrainSpan indicate that exon 5 is impacted by NDD or non-NDD mutations. Interestingly, PTEN splicing isoforms that do not carry exon 5 are exclusively impacted by the NDD mutations, F241S and D252G. The increased life expectancy of PTEN variants carriers carrying exons 5 and 7 is highly correlated to increased lifetime risk for certain cancers. Our results underscore the detailed structural and functional mechanisms of PTEN with mutations at the membrane, and suggest that tissue-specific splicing isoform expression should be taken into consideration while predicting the effect of mutations in various diseases. Citation Format: Hyunbum Jang, Jiaye Chen, Lilia M. Iakoucheva, Ruth Nussinov. Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3845.

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation.

Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3'-ends have uncovered widespread intronic polyadenylation (IPA) in normal and disease conditions, where it is currently attributed to stochastic variations in the pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific expression of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines revealed that APA events are more abundant in introns than in exons. While the rate of IPA in composite terminal exons and skipped terminal exons expectedly correlates with splicing, we observed a considerable fraction of IPA events that lack AS support and attributed them to spliced polyadenylated introns (SPI). We hypothesize that SPIs represent transient byproducts of a dynamic coupling between APA and AS, in which the spliceosome removes the intron while it is being cleaved and polyadenylated. These findings indicate that cotranscriptional pre-mRNA splicing could serve as a rescue mechanism to suppress premature transcription termination at intronic polyadenylation sites.

Major histocompatibility class I antigenic peptides derived from translation of pre-mRNAs generate immune tolerance

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the β-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The β-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.

Isoform changes of action potential regulators in the ventricles of arrhythmogenic phospholamban-R14del humanized mouse hearts

Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN-R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with “cardiac cell action potential”, specifically in the RV. We found that splicing of 2 key genes, Trpm4 and Camk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+-homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN-R14del ACM.

Association between Polymorphisms in CFH, ARMS2, CFI, and C3 Genes and Response to Anti-VEGF Treatment in Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (nAMD) is the leading cause of vision loss in the elderly. The gold standard of nAMD treatment is intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors. Genetic factors may influence the response to anti-VEGF therapy and result in a high degree of response variability. The aim of the study was to evaluate the association of the polymorphisms in genes related to the complement system (rs2285714-CFI, rs10490924-ARMS2, rs2230199-C3, rs800292-CFH, and rs6677604-CFH) with nAMD its clinical features and optical coherent tomography (OCT) biomarkers of treatment response to anti-VEGF therapy. Genotyping by allele-specific PCR was performed in 193 AMD patients and 147 age-matched controls. A prospective study of the dynamics of changes in OCT biomarkers during aflibercept treatment included 110 treatment-naive patients. Allele T rs10490924 was associated with the increased risk of nAMD. For both rs800292 and rs6677604, carriage of the A allele was protective and decreased the nAMD risk. Associations of rs2230199 with central retinal thickness (CRT) and intraretinal cysts were revealed. The height of pigment epithelium detachment and the height of neuroretinal detachment were significantly higher in carriers of the minor allele of rs2285714, both at baseline and during treatment. The reduction of CRT was associated with higher CRT at baseline and the presence of the T allele of rs2285714. By the end of one-year follow-up the patients homozygous for the minor allele rs2285714 had significantly higher odds of the presence of anastomoses and loops and active neovascular membrane. Furthermore, minor allele carriers had decreased levels of complement factor I level in aqueous humor but not in the plasma, which may be due to the influence of rs2285714 on tissue-specific splicing. Our results suggest that the severity of AMD macular lesions is associated with rs2285714 and rs2230199 polymorphisms, which could be explained by their high regulatory potential. Patients with the minor allele of rs2285714 respond worse to antiangiogenic therapy.

Selective retinal ganglion cell loss and optic neuropathy in a humanized mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner.

Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

Alternative Splicing in Cardiovascular Disease-A Survey of Recent Findings.

Alternative splicing, a driver of posttranscriptional variance, differs from canonical splicing by arranging the introns and exons of an immature pre-mRNA transcript in a multitude of different ways. Although alternative splicing was discovered almost half a century ago, estimates of the proportion of genes that undergo alternative splicing have risen drastically over the last two decades. Deep sequencing methods and novel bioinformatic algorithms have led to new insights into the prevalence of spliced variants, tissue-specific splicing patterns and the significance of alternative splicing in development and disease. Thus far, the role of alternative splicing has been uncovered in areas ranging from heart development, the response to myocardial infarction to cardiac structural disease. Circular RNAs, a product of alternative back-splicing, were initially discovered in 1976, but landmark publications have only recently identified their regulatory role, tissue-specific expression, and transcriptomic abundance, spurring a renewed interest in the topic. The aim of this review is to provide a brief insight into some of the available findings on the role of alternative splicing in cardiovascular disease, with a focus on atherosclerosis, myocardial infarction, heart failure, dilated cardiomyopathy and circular RNAs in myocardial infarction.

FIVEx: an interactive eQTL browser across public datasets.

Expression quantitative trait loci (eQTLs) characterize the associations between genetic variation and gene expression to provide insights into tissue-specific gene regulation. Interactive visualization of tissue-specific eQTLs or splice QTLs (sQTLs) can facilitate our understanding of functional variants relevant to disease-related traits. However, combining the multi-dimensional nature of eQTLs/sQTLs into a concise and informative visualization is challenging. Existing QTL visualization tools provide useful ways to summarize the unprecedented scale of transcriptomic data but are not necessarily tailored to answer questions about the functional interpretations of trait-associated variants or other variants of interest. We developed FIVEx, an interactive eQTL/sQTL browser with an intuitive interface tailored to the functional interpretation of associated variants. It features the ability to navigate seamlessly between different data views while providing relevant tissue- and locus-specific information to offer users a better understanding of population-scale multi-tissue transcriptomic profiles. Our implementation of the FIVEx browser on the EBI eQTL catalogue, encompassing 16 publicly available RNA-seq studies, provides important insights for understanding potential tissue-specific regulatory mechanisms underlying trait-associated signals. A FIVEx instance visualizing EBI eQTL catalogue data can be found at https://fivex.sph.umich.edu. Its source code is open source under an MIT license at https://github.com/statgen/fivex. Supplementary data are available at Bioinformatics online.

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis.

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.