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Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale data sets of the Genotype-Tissue Expression (GTEx) project to study genetically modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues versus individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMAS-associated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.

Transcriptomic analyses of postmortem brains have begun to elucidate molecular abnormalities in autism spectrum disorder (ASD). However, a crucial pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here we profiled global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of postmortem brains of people with ASD. We observed a global bias for hypoediting in ASD brains, which was shared across brain regions and involved many synaptic genes. We show that the Fragile X proteins FMRP and FXR1P interact with RNA-editing enzymes (ADAR proteins) and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA-editing alterations in ASD and Fragile X syndrome, establishing this as a molecular link between these related diseases. Our findings, which are corroborated across multiple data sets, including dup15q (genomic duplication of 15q11.2-13.1) cases associated with intellectual disability, highlight RNA-editing dysregulation in ASD and reveal new mechanisms underlying this disorder.

Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale datasets of the Genotype-Tissue Expression (GTEx) project to study genetically-modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues vs. individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMAS-associated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.

Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting that cis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon.

Autism spectrum disorder (ASD) is a genetically complex, clinically heterogeneous neurodevelopmental disease. Recently, our understanding of the molecular abnormalities in ASD has been expanded through transcriptomic analyses of postmortem brains. However, a crucial molecular pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here, we profiled the global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of post-mortem ASD brains. Strikingly, we observed a global bias of hypo-editing in ASD brains, common to different brain regions and involving many genes with known neurobiological functions. Through genome-wide protein-RNA binding analyses and detailed molecular assays, we show that the Fragile X proteins, FMRP and FXR1P, interact with ADAR proteins and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA editing alterations in ASD and Fragile X syndrome, thus establishing RNA editing as a molecular link underlying these two highly related diseases. Our findings support a role for RNA editing dysregulation in ASD and highlight novel mechanisms for RNA editing regulation.

Background: RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with heart failure in mouse and human. Genetic manipulation of RBFox1 reveals a major function of RBFox1 in the pathogenesis of cardiac hypertrophy, fibrosis and dysfunction under pathological stresses. However, much of our current knowledge about RBFox1 focuses on the nuclear RBFox1 with a major impact on global alternative splicing changes. Yet, RBFox1 gene also generates a cytosolic isoform-RBFox1c through alternative splicing, and the specific function of RBFox1c has not been characterized. Goal and Methods: This study investigated the functional impact and the underlying mechanism of the RBFox1c in cardiac pathological remodeling and targeted gene regulation. Results: RBFox1c expression is significantly repressed in stressed cardiomyocytes in vivo and in vitro. RNA-Seq combined with IPA analyses from cardiomyocytes revealed RBFox1c but not the nucleus RBFox1 specifically suppressed pro-inflammatory genes. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis is observed following I/R injury associated with elevated expression of pro-inflammatory genes. In contrast, cardiac specific expression of RBFox1c reduced cardiac fibrosis and inflammatory gene expression following pressure-overload and myocardial infarct injury associated with improved ejection fraction. Motif enrichment analysis identified significant enrichment of the RBFox1 binding motif in the 3’UTR of the RBFox1c regulated genes. We performed CLIP analysis followed by RT-PCR and observed RBFox1c interacted with inflammatory gene 3’UTR. Lastly, we explored the interactome of RBFox1c and found RBFox1c specifically interacted with a component of RNA decay machinery-Upf1. Indeed, while expression of RBFox1c repressed inflammatory gene expression, inactivation of Upf1 abolished this effect in cardiomyocytes. Conclusion: RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates RNA splicing reprogramming, and the RBFox1c represses proinflammatory genes via recruitment of Upf1 mediated RNA degradation.

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.

RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with pathological hypertrophy and heart failure. However, much of our current knowledge about RBFox1 focuses on nuclear RBFox1 with a major impact on global alternative splicing changes in the diseased heart. Yet, RBFox1 gene also generates a cytosolic isoform through alternative splicing (RBFox1c), but the specific function of RBFox1c in heart has not been characterized. RBFox1c expression is significantly repressed in the mouse failing heart and hypertrophic cardiomyocytes. We performed RNA-seq combined with GO and IPA analysis to determine the impact of RBFox1c expression in culture. Among the genes suppressed specifically by RBFox1c but not the nucleus RBFox1 are groups of pro-inflammatory genes. Both Motif enrichment analysis and de novo motif discovery identified significant enrichment of RBFox1 binding motif in the 3’UTRs of the RBFox1c regulated genes. Using CLIP analysis followed by RT-PCR, we observed RBFox1c, but not nuclear RBFox1 specifically interacted with targeted inflammatory gene 3’UTR. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis was observed following TAC, associated with elevated expression of RBFox1c dependent inflammatory genes. In contrast, cardiac specific expression of RBFox1c significantly reduced cardiac fibrosis and inflammatory gene expression following TAC, associated with improved ejection fraction and reduced hypertrophic marker gene expression. Further, we tested the effect of RBFox1c expression on cardiac fibrosis response using NRVM conditioned media. We showed the conditioned media from the hypertrophic cardiomyocytes potently induce fibroblast proliferation. However, RBFox1c expression can suppress phenylephrine and isoproterenol induced fibroblasts proliferation. RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates global RNA splicing reprogramming in heart, while the RBFox1c regulates inflammatory gene expression and fibrotic remodeling potentially through potential interaction with their 3’UTR and targeted RNA degradation.

Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.

In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation.

Pre-mRNA splicing is an essential RNA processing step in eukaryotes. Alternative splicing generates distinct spliced isoforms of the same gene, thereby dramatically increasing transcriptome diversity. Since most human genes undergo alternative splicing, this process contributes to a wide spectrum of biological functions in healthy and disease states. Splicing is closely regulated by various cis-regulatory elements and trans-factors. With the advent of high-throughput experimental technologies and bioinformatic algorithms, we now have powerful means to study alternative splicing globally and uncover its functional impact and regulatory mechanisms. As more RNA sequencing (RNA-Seq) data from normal and disease conditions are becoming available, many studies are underway to dissect global misregulation of splicing in diseases and develop novel splicing-targeted therapeutics. In this chapter, we first discuss the experimental and bioinformatic approaches for identification of alternative splicing, followed by a comprehensive review on the state-of-the-art methodologies to study splicing regulation. In addition, we discuss the current challenges and open questions in the RNA splicing field including gene expression kinetics, co-transcriptional splicing, and therapeutic approaches targeting splicing.

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.