Changes In Alternative Splicing Patterns Research Articles

Bulk and single-cell alternative splicing analyses reveal roles of TRA2B in myogenic differentiation.

Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that occur during myogenesis are not well understood. Here, we employed isoform long-reads RNA-seq (Iso-seq) and single-cell RNA-seq (scRNA-seq) to investigate the AS landscape during myogenesis. Our Iso-seq data identified 61,146 full-length isoforms representing 11,682 expressed genes, of which over 52% were novel. We identified 38,022 AS events, with most of these events altering coding sequences and exhibiting stage-specific splicing patterns. We identified AS dynamics in different types of muscle cells through scRNA-seq analysis, revealing genes essential for the contractile muscle system and cytoskeleton that undergo differential splicing across cell types. Gene-splicing analysis demonstrated that AS acts as a regulator, independent of changes in overall gene expression. Two isoforms of splicing factor TRA2B play distinct roles in myogenic differentiation by triggering AS of TGFBR2 to regulate canonical TGF-β signalling cascades differently. Our study provides a valuable transcriptome resource for myogenesis and reveals the complexity of AS and its regulation during myogenesis.

CELF1 Selectively Regulates Alternative Splicing of DNA Repair Genes Associated With Cataract in Human Lens Cell Line.

Cataract is a global eye disease caused by the opacification of lens, while its underlying molecular pathogenesis is not clear, making it difficult for prevention. CELF1, an RNA binding protein, mediates Alternative Splicing (AS) of genes involved in diverse diseases and regulates development or defects of lens. Utilizing transcriptome-wide approaches, we analyzed and compared AS patterns between human lens epithelial cells (SRA01/04) with CELF1 overexpression (CELF1-OE) and control cells. Extensive changes in AS patterns upon CELF1-OE were identified in SRA01/04 cells. We finally identified 840 CELF1-regulated AS events (RASEs) and found that CELF1-OE preferred to repress exon skipping events in SRA01/04 cells. CELF1-regulated AS genes were enriched in the regulation of DNA repair, cellular response to DNA damage stimulus, and apoptosis pathways (including HMGA2, CSNK1E, and YAP1). These biological functions and pathways have been reported to be associated with lens development or other eye diseases. To further explore the mechanisms of CELF1 in regulating AS genes, we downloaded and re-analyzed a set of CELF1-RNA interactome data. We found that 194 genes were bound and regulated by CELF1 at the AS level. 10 genes involved in DNA repair-related pathways were also bound by CELF1. Motif analysis for CELF1-bound peaks and splicing sites of RASEs showed that CELF1 regulates AS by binding to the AGGU[AG]AG motif in SRA01/04 cells. CELF1 could mediate AS of DNA repair-related genes through directly binding to their transcripts with distinct motif bias. The functional mechanism of CELF1 may ultimately participate in cataract formation and lens development.

Alternative splicing landscape of the neural transcriptome in a cytoplasmic-predominant Pten expression murine model of autism-like Behavior

Alternative splicing (AS) is a posttranscriptional mechanism regulating gene expression that complex organisms utilize to expand proteome diversity from a comparatively limited set of genes. Recent research has increasingly associated AS with increased functional complexity in the central nervous systems in higher order mammals. This work has heavily implicated aberrant AS in several neurocognitive and neurodevelopmental disorders, including autism. Due to the strong genetic association between germline PTEN mutations and autism spectrum disorder (ASD), we hypothesized that germline PTEN mutations would alter AS patterns, contributing to the pathophysiology of ASD. In a murine model of constitutional mislocalization of Pten, recapitulating an autism-like phenotype, we found significant changes in AS patterns across the neural transcriptome by analyzing RNA-sequencing data with the program rMATS. A few hundred significant alternative splicing events (ASEs) that differentiate each m3m4 genotype were identified. These ASEs occur in genes enriched in PTEN signaling, inositol metabolism, and several other pathways relevant to the pathophysiology of ASD. In addition, we identified expression changes in several splicing factors known to be enriched in the nervous system. For instance, the master regulator of microexons, Srrm4, has decreased expression, and consequently, we found decreased inclusion of microexons in the Ptenm3m4/m3m4 cortex (~10% decrease). We also demonstrated that the m3m4 mutation disrupts the interaction between Pten and U2af2, a member of the spliceosome. In sum, our observations point to germline Pten disruption changing the landscape of alternative splicing in the brain, and these changes may be relevant to the pathogenesis and/or maintenance of PTEN-ASD phenotypes.

P53-induced ARVCF modulates the splicing landscape and supports the tumor suppressive function of p53

p53 is one of the most important tumor suppressor genes, and the exploration of p53-target genes is important for elucidation of its functional mechanisms. In this study, we identified Armadillo Repeat gene deleted in Velo-Cardio-Facial syndrome (ARVCF) as a direct target of p53 through ChIP-sequencing analysis. Activated p53 protein was found to bind to two distinct sites in the ARVCF gene, resulting in induction of ARVCF expression at both the mRNA and protein levels. We revealed that the knockdown of ARVCF inhibited p53-induced apoptosis. Interestingly, ARVCF interacted with hnRNPH2, which is involved in pre-mRNA splicing, and ARVCF knockdown induced dynamic changes in alternative splicing patterns. These results suggest that p53-induced ARVCF indirectly, but not directly, regulates p53 target selectivity through splicing alterations of specific genes. Thus, we demonstrated that the induction of ARVCF expression contributed to the tumor suppressive function of p53. Recently, it has been reported that many tumors have thousands of alternative splicing events that are not detectable in normal samples. ARVCF may play a role in alternative splicing events in cancer and may provide clues to explore novel approaches for cancer diagnosis and therapy.

Translocation of a Cell Surface Spliceosomal Complex Induces Alternative Splicing Events and Lymphoma Cell Necrosis

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.

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.

Conservation of mRNA quality control factor Ski7 and its diversification through changes in alternative splicing and gene duplication

Eukaryotes maintain fidelity of gene expression by preferential degradation of aberrant mRNAs that arise by errors in RNA processing reactions. In Saccharomyces cerevisiae, Ski7 plays an important role in this mRNA quality control by mediating mRNA degradation by the RNA exosome. Ski7 was initially thought to be restricted to Saccharomyces cerevisiae and close relatives because the SKI7 gene and its paralog HBS1 arose by whole genome duplication (WGD) in a recent ancestor. We have recently shown that the preduplication gene was alternatively spliced and that Ski7 function predates WGD. Here, we use transcriptome analysis of diverse eukaryotes to show that diverse eukaryotes use alternative splicing of SKI7/HBS1 to encode two proteins. Although alternative splicing affects the same intrinsically disordered region of the protein, the pattern of splice site usage varies. This alternative splicing event arose in an early eukaryote that is a common ancestor of plants, animals, and fungi. Remarkably, through changes in alternative splicing and gene duplication, the Ski7 protein has diversified such that different species express one of four distinct Ski7-like proteins. We also show experimentally that the Saccharomyces cerevisiae SKI7 gene has undergone multiple changes that are incompatible with the Hbs1 function and may also have undergone additional changes to optimize mRNA quality control. The combination of transcriptome analysis in diverse eukaryotes and genetic analysis in yeast clarifies the mechanism by which a Ski7-like protein is expressed across eukaryotes and provides a unique view of changes in alternative splicing patterns of one gene over long evolutionary time.

LOX-1 and Its Splice Variants: A New Challenge for Atherosclerosis and Cancer-Targeted Therapies.

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both “risk” and “protective” splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target.

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology

SMN deficiency alters Nrxn2 expression and splicing in zebrafish and mouse models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.

Alternative Splicing: Role in Cancer Development and Progression

Alternative Splicing: Role in Cancer Development and Progression

Polypyrimidine tract binding protein homologs from Arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes.

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5' splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner.

Embracing the complexity of pre-mRNA splicing

NIH Public Access Author Manuscript Cell Res. Author manuscript; available in PMC 2014 February 03. NIH-PA Author Manuscript Published in final edited form as: Cell Res. 2010 August ; 20(8): 866–868. doi:10.1038/cr.2010.98. Embracing the Complexity of Pre-mRNA Splicing Peter J. Shepard and Klemens J. Hertel * Department of Microbiology & Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA Keywords alternative pre-mRNA splicing; exon; intron; computation NIH-PA Author Manuscript Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of non-coding intronic sequences to assemble exons into mature mRNAs prior to export and translation. Defects in splicing lead to many human genetic diseases 1 , and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer 2 . Given the complexity of higher eukaryotic genes and the relatively low level of splice-site conservation, the precision of the splicing machinery in recognizing and pairing splice sites is remarkable. Introns ranging in size from less than 100 up to 100,000 bases are removed efficiently. At the same time, a large number of alternative splicing events are observed between different cell-types, developmental stages, and during other biological processes. Of the approximately 25,000 genes encoded by the human genome 3 , more than 90% are believed to produce transcripts that are alternatively spliced 4 . Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, significantly enriching the proteomic diversity of higher eukaryotic organisms 5 . Because regulation of this process can determine the timing and location in which a particular protein isoform is produced, changes in alternative splicing patterns modulate many cellular activities. This extensive alternative splicing implies a significant flexibility of the spliceosome to identify and process exons within a given pre-mRNA. NIH-PA Author Manuscript Over the last few years, research from different laboratories has demonstrated that the regulation of alternative splicing depends on many different cis-acting RNA elements 6 . While most of these RNA elements appeared to have a common mechanistic goal, the initial recruitment of spliceosomal components, the sheer number of RNA sequences involved in regulating alternative splicing became more and more daunting. As a consequence, the enthusiasm to derive alternative splicing predictions based only on pre-mRNA sequence, appropriately referred to as the “splicing code”, gave way to the growing realization that alternative splicing is much more complex than initially anticipated. Computational analyses of the identified RNA elements proved to be the first efficient tools to classify and group these RNA elements, while at the same time expanding the repertoire of additional RNA sequence elements likely to be important for splicing regulation 7 . Sequence motifs that define the exon/intron boundary, the presence or absence of splicing enhancer or silencer sequences, RNA secondary structures, and the length of exons and their flanking introns are the most prominent classes of RNA elements involved in mediating efficient exon splicing 6 . While it was appreciated that the relative contribution of each of these RNA sequence elements controls how efficient splice sites are recognized and flanking introns are removed, To whom correspondence should be addressed: khertel@uci.edu.

Regulation of plant gene expression by alternative splicing

AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed 'combinatorial' control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)-PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT-PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process.

The search for alternative splicing regulators: new approaches offer a path to a splicing code

Complex multicellular organisms require a diverse set of proteins to set the form and function of specialized cell types. The availability of complete genomic sequences has revealed that instead of a large increase in the number of protein coding genes compared with unicellular organisms, more complex eukaryotes instead obtain more diversity out of a relatively limited number of genes through the process of alternative splicing (AS). AS results in the cell type-, developmental stage-, sex-, or signal-regulated changes in composition of an mRNA produced from a given gene, brought about by changes in splice site choice (Black 2003; Matlin et al. 2005). There are many different types of AS events, ranging from the tissue-specific inclusion of a cassette exon to the Dscam gene in Drosophila, which contains four clusters of exons containing 12, 48, 33, and 2 mutually exclusive variants, an extreme example of AS complexity (Bharadwaj and Kolodkin 2006). Additionally, it is clear from the relatively small number of AS events that have been studied extensively at a mechanistic level that regulation of AS takes many forms, as will be discussed in more detail later in this article. Lastly, the functional outcomes of AS vary greatly, from effectively turning off a gene (the result of including an exon containing a premature stop codon, for example), to a subtle change in a protein’s function. In spite of this complexity, the goal of understanding the changes in AS patterns in terms of changes in expression and regulation of factors that regulate AS across cell types appears achievable. This is in part because recent technical advances allow us, starting with an individual splicing factor, to determine its genome-wide role in AS regulation. However, the ambitious goal of determining a cellular code for AS will be impossible to realize without a complete list of AS regulators, and currently there is no reason to believe we are anywhere near completing such a list in any metazoan. Kuroyanagi et al. (2006) have developed recently a system in Caenorhabditis elegans, which allows for the straightforward visualization of individual AS events in vivo, which in turn allows for the identification of mutants defective in regulating the event. Using this approach, Kuroyanagi and colleagues (Kuroyanagi et al. 2006, 2007; Ohno et al. 2008) have identified four proteins that regulate two different AS events, a feat that would be laborious using in vitro approaches. Three of these proteins had not been shown previously to participate in regulation of AS, indicating that we may be far from a complete list of AS regulators. Their data also highlight the fact that regulators of AS are often highly conserved throughout metazoans, meaning that filling the list of splicing regulators in C. elegans will likely contribute to filling our own.

A double-reporter splicing assay for determining splicing efficiency in mammalian cells

Changes in alternative splicing patterns can result from both inherited and acquired defects, and they are increasingly recognized as causes of human diseases. Hence, improvements in the understanding of alternative splicing regulation may provide opportunities for restoring productive patterns of splicing. The identification of factors (such as proteins, nucleic acids or small molecules) that modulate the splicing pattern would be facilitated by systems with which many samples can be screened. The absence of reliable systems prompted us to develop an assay system based on dual enzymatic activities. Two distinct signals derived from spliced and unspliced RNA are measured, providing the basis for a robust, rapid and convenient assay for investigating splicing. This protocol describes how to use this system; the time required for lysing the cells and recording enzymatic activity is about 2 hours.