Tissue-specific Splicing Research Articles

The contribution of Alu exons to the human proteome.

BackgroundAlu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins.ResultsWe adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products.ConclusionsOur data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0876-5) contains supplementary material, which is available to authorized users.

Tissue Restricted Splice Junctions Originate Not Only from Tissue-Specific Gene Loci, but Gene Loci with a Broad Pattern of Expression.

Cellular mechanisms that achieve protein diversity in eukaryotes are multifaceted, including transcriptional components such as RNA splicing. Through alternative splicing, a single protein-coding gene can generate multiple mRNA transcripts and protein isoforms, some of which are tissue-specific. We have conducted qualitative and quantitative analyses of the Bodymap 2.0 messenger RNA-sequencing data from 16 human tissue samples and identified 209,363 splice junctions. Of these, 22,231 (10.6%) were not previously annotated and 21,650 (10.3%) were expressed in a tissue-restricted pattern. Tissue-restricted alternative splicing was found to be widespread, with approximately 65% of expressed multi-exon genes containing at least one tissue-specific splice junction. Interestingly, we observed many tissue-specific splice junctions not only in genes expressed in one or a few tissues, but also from gene loci with a broad pattern of expression.

Exome Sequencing RevealsAGBL5as Novel Candidate Gene and Additional Variants for Retinitis Pigmentosa in Five Turkish Families

Retinitis pigmentosa (RP) is the most common inherited retinal disease with high genetic heterogeneity and variable phenotypes. Characteristic symptoms include night blindness and progressive loss of visual field, leading to blindness. Mutations in >60 genes have been identified to date as causative for RP, and additional candidate genes are assumed. To find the disease-causing mutations in the affected members of five Turkish families, we sequenced whole exomes using an Illumina platform. Among all candidate genes for retinal degeneration we found two previously known sequence variations: a 4 bp deletion in the RPGR gene (c.1662_1665delAGAA; p.Glu555Glyfs*14) and a recently described USH1-causing missense mutation in MYO7A (c.472G>A, p.Gly158Arg). Furthermore, a novel 1 bp deletion in the VCAN gene (c.5118delA; p.Ser1707Valfs*44) was detected as well as a large deletion in EYS, spanning ∼ 400kb and comprising exons 16-26 (p.fs*). In one family, exome analyses of two affected individuals revealed a homozygous missense mutation (c.883G>A; p.Asp295Asn) in the AGBL5 (Agbl5; CCP5) gene, previously not reported to be associated with RP. RNA and protein analyses showed expression in human retina, as well as in mouse retina, brain and testis. Furthermore, cDNA analyses indicate the existence of tissue-specific AGBL5 splice variations in humans. AGBL5/CCP5 immunoreactivity was also visualized in human and mouse retinae. Due to the characteristic RP phenotype in patients carrying the AGBL5 missense mutation we suggest this gene as a candidate for a new form of autosomal recessively inherited RP and recommend further investigation to confirm this hypothesis.

Structure‐dynamic basis of splicing‐dependent regulation in tissue‐specific variants of the sodium‐calcium exchanger

Tissue-specific splice variants of Na(+)/Ca(2+) exchangers contain 2 Ca(2+)-binding regulatory domains (CBDs), CBD1 and CBD2. Ca(2+) interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca(2+) binding to CBD2 alleviates Na(+)-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca(2+) binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca(2+) salt bridge at the 2-domain interface, which secondarily modulates the Ca(2+)-bound states. In matching splice variants, the extent of Ca(2+)-induced rigidification correlates with decreased (up to 10-fold) Ca(2+) off rates, where the cardiac CBD12 exhibits the slowest Ca(2+) off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca(2+) binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.

Secretory Gene Recruitments in Vampire Bat Salivary Adaptation and Potential Convergences With Sanguivorous Leeches

Regulatory evolution is thought to be fundamental to adaptive evolution. However, the identification of specific regulatory changes responsible for adaptation are sparse. Bats of the family Phyllostomidae, owing to their unparalleled rate of ecological and morphological evolution, represent an ideal system to identify regulatory evolution of adaptive significance. Among ecological niche leaps observed in this family, the most dramatic is the evolution of obligate sanguivory by vampire bats, which due to their highly derived phenotype, the sporadic phylogenetic occurrence of blood-feeding, and the adaptive potential of salivary glands, has enabled the development of hypotheses about adaptive molecular phenotypes. Using comparative transcriptomics of vampire bat, outgroup insectivorous bats, and sanguivorous leeches we identify genes that have been convergently recruited as secretory products of salivary glands of vampire bats and leeches. Comparisons of vampire bat to lineages maintaining the primitive chiropteran condition of insectivory indicated gene recruitment of alternative splice variants, and 5’ exon evolution, as the mechanisms producing secretory expression in vampire bats, but not in the insectivorous bats Macrotus and Myotis. Biochemical functions of hypothesized recruited genes explain adaptive benefits to sanguivory by modulating host hemostasis and neural signaling. It is difficult to identify how complex phenotypic change and rapid ecological transition, such as that observed in vampire bats, evolved over a short evolutionary timescale. Results indicate that regulatory evolution controlling tissue-specific splicing patterns has been important to successful adaptation of this lineage. Future studies that leverage emerging long sequence-read technologies, increased sample sizes, and expression and sequence comparisons across other sanguivore lineages will further elucidate roles of alternative splicing and gene recruitment in the remarkable evolution of sanguivory.

Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

Targeting PTBP1 as a therapeutic strategy to reverse lineage-specific splicing of ANXA7 and ensuing EGFR activation in glioblastoma.

2075 Background: Tissue-specific splicing involves evolutionarily conserved, alternative exons many of which have functional features that influence cell signalling. The membrane-binding tumor supp...

Neural differentiation modulates the vertebrate brain specific splicing program.

Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation.

Analysis of pollen-specific alternative splicing inArabidopsis thalianavia semi-quantitative PCR

Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results.

Incomplete splicing of neutrophil-specific genes affects neutrophil development in a zebrafish model of poikiloderma with neutropenia

Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.

Two decades of RNA as I see it.

Two decades of RNA as I see it.

Cloning and expression of hepatic synaptotagmin 1 in mouse

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5′-and 3′-untranslated regions were 66 and 476bp, respectively; the open reading frame of 1266bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ.

Phosphoprotein phosphatase 1 complexes in spermatogenesis.

The major post-translational modification in eukaryotes is protein phosphorylation which mediates responses to signals in a myriad of cellular processes. Not surprisingly, many steps in spermatogenesis involve the concerted action of the protein (de)phosphorylation key players, kinases and phosphatases. Phosphoprotein phosphatase 1 catalytic subunit (PPP1C), an evolutionarily conserved Ser/Thr-protein phosphatase, catalyzes the majority of eukaryotic protein dephosphorylation reactions. Three genes, PPP1CA, PPP1CB and PPP1CC, encode four PPP1C isoforms, PPP1CA, PPP1CB, PPP1CC1, and PPP1CC2. After transcription, PPP1CC undergoes tissue-specific splicing, originating a ubiquitously expressed isoform, PPP1CC1 and a testis-enriched and sperm-specific isoform, PPP1CC2 which is essential for completion of spermatogenesis. Highly similar PPP1C isoforms - PPP1CA and PPP1CB - are capable of compensating the loss of Ppp1cc in every tissue except in testis. PPP1C cellular functions depend on the complexes it forms with PPP1C Interacting Proteins (PIPs), which together with the different catalytic subunits, account for PPP1C specificity. This review will focus on the role of the major serine/threonine phosphatase - PPP1C and its holoenzymes in spermatogenesis. Furthermore, current challenges on the protein phosphatases field as targets to male contraception will be addressed.

Transcriptome-wide identification and study of cancer-specific splicing events across multiple tumors.

Dysregulation of alternative splicing (AS) is one of the molecular hallmarks of cancer, with splicing alteration of numerous genes in cancer patients. However, studying splicing mis-regulation in cancer is complicated by the large noise generated from tissue-specific splicing. To obtain a global picture of cancer-specific splicing, we analyzed transcriptome sequencing data from 1149 patients in The Cancer Genome Atlas project, producing a core set of AS events significantly altered across multiple cancer types. These cancer-specific AS events are highly conserved, are more likely to maintain protein reading frame, and mainly function in cell cycle, cell adhesion/migration, and insulin signaling pathways. Furthermore, these events can serve as new molecular biomarkers to distinguish cancer from normal tissues, to separate cancer subtypes, and to predict patient survival. We also found that most genes whose expression is closely associated with cancer-specific splicing are key regulators of the cell cycle. This study uncovers a common set of cancer-specific AS events altered across multiple cancers, providing mechanistic insight into how splicing is mis-regulated in cancers.

Functional roles of alternative splicing factors in human disease.

Alternative splicing (AS) is an important mechanism used to generate greater transcriptomic and proteomic diversity from a finite genome. Nearly all human gene transcripts are alternatively spliced and can produce protein isoforms with divergent and even antagonistic properties that impact cell functions. Many AS events are tightly regulated in a cell-type or tissue-specific manner, and at different developmental stages. AS is regulated by RNA-binding proteins, including cell- or tissue-specific splicing factors. In the past few years, technological advances have defined genome-wide programs of AS regulated by increasing numbers of splicing factors. These splicing regulatory networks (SRNs) consist of transcripts that encode proteins that function in coordinated and related processes that impact the development and phenotypes of different cell types. As such, it is increasingly recognized that disruption of normal programs of splicing regulated by different splicing factors can lead to human diseases. We will summarize examples of diseases in which altered expression or function of splicing regulatory proteins has been implicated in human disease pathophysiology. As the role of AS continues to be unveiled in human disease and disease risk, it is hoped that further investigations into the functions of numerous splicing factors and their regulated targets will enable the development of novel therapies that are directed at specific AS events as well as the biological pathways they impact. WIREs RNA 2015, 6:311–326. doi: 10.1002/wrna.1276For further resources related to this article, please visit the http://wires.wiley.com/remdoi.cgi?doi=10.1002/wrna.1276WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.

Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation.

Among the Mef2 family of transcription factors, Mef2D is unique in that it undergoes tissue-specific splicing to generate an isoform that is essential for muscle differentiation. However, the mechanisms mediating this muscle-specific processing of Mef2D remain unknown. Using bioinformatics, we identified Rbfox proteins as putative modulators of Mef2D muscle-specific splicing. Accordingly, we found direct and specific Rbfox1 and Rbfox2 binding to Mef2D pre-mRNA in vivo. Gain- and loss-of-function experiments demonstrated that Rbfox1 and Rbfox2 cooperate in promoting Mef2D splicing and subsequent myogenesis. Thus, our findings reveal a new role for Rbfox proteins in regulating myogenesis through activation of essential muscle-specific splicing events.

Tissue-specific splice variants of HARE/Stabilin-2 are expressed in bone marrow, lymph node, and spleen

The hyaluronan receptor for endocytosis (HARE), or Stabilin-2, is the mammalian endocytic clearance receptor for HA, heparin, advanced glycation end-products, acetylated and oxidized low-density lipoproteins and collagen N-terminal propeptides. This large 2551 amino acid receptor is encoded by a gene that covers over 180kbp on human chromosome 12 and is predicted to be composed of 69 exons. Due to the expression profile of this gene and the number of exons it contains, we hypothesized that splice variants of stab2 are encoded in these tissues. In addition, a correlation between alternative splice variants and cancer progression has been shown in other HA receptors such as RHAMM and CD42. In this study, two methods were utilized in identifying and/or isolating the HARE splice variants. The first method used primer sets to amplify the 190-HARE encoding region that could contain splice junctions; therefore, they were purified from agarose gels and sequenced. Five splice variants were detected in that manner. In the second approach, the entire open reading frame of HARE was amplified. This allowed four splice variants with extensive exon splicing to be isolated. After the splice variants were sequenced, three were cloned into a mammalian expression vector. Next, stable cell lines expressing the variants were created in order to determine stable protein expression. In this study, the splice variants were found to be tissue specific in most cases. This suggests that tissue specific regulatory splicing mechanisms may lead to differences in functionality between the splice variants.

RBM24 Is a Major Regulator of Muscle-Specific Alternative Splicing

Cell-type-specific splicing generates numerous alternatively spliced transcripts playing important roles for organ development and homeostasis, but only a few tissue-specific splicing factors have been identified. We found that RBM24 governs a large number of muscle-specific splicing events that are critically involved in cardiac and skeletal muscle development and disease. Targeted inactivation of RBM24 in mice disrupted cardiac development and impaired sarcomerogenesis in striated muscles. In vitro splicing assays revealed that recombinant RBM24 is sufficient to promote muscle-specific exon inclusion in nuclear extracts of nonmuscle cells. Furthermore, we demonstrate that binding of RBM24 to an intronic splicing enhancer (ISE) is essential and sufficient to overcome repression of exon inclusion by an exonic splicing silencer (ESS) containing PTB and hnRNP A1/A2 binding sites. Introduction of ESS and ISE converted a constitutive exon into an RMB24-dependent alternative exon. We reason that RBM24 is a major regulator of alternative splicing in striated muscles.

P01.08 * LINEAGE-SPECIFIC SPLICING OF AN ALTERNATIVE EXON OF ANXA7 PROMOTES EGFR SIGNALING ACTIVATION AND TUMOR PROGRESSION IN GLIOBLASTOMA

Tissue-specific alternative splicing is critical to the emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence signaling pathways to profound biological effect. In the brain, Annexin A7 isoform 1 (ANXA7-I1) is exclusively expressed in mature neurons, while isoform 2 (ANXA7-I2) in which exon 6 is skipped, is expressed in glial and progenitor cells. We show that lineage-specific splicing of the cassette exon 6 in the membrane-binding tumor suppressor ANXA7diminishes endosomal targeting and consequent signal termination of the EGFR oncoprotein during brain tumor progression. Splicing of this exon is mediated by Polypyrimidine Tract-Binding Protein 1 (PTBP1), a ribonucleoprotein normally repressed during neuronal development but which we found to be highly expressed also in glioblastomas through loss of a brain-enriched microRNA, miR-124, and gene amplification. Here, we show that the PTBP1-ANXA7 splicing-EGFR signal activation axis promotes in vitro cell migration and invasion, and tumor angiogenesis in vivo. In glioblastoma, ANXA7 splicing is likely inherited from a potential tumor-initiating ancestor but this trait is further exploited through accumulation of mutations that enhance EGFR signaling. Our data illustrate how lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates its tumor suppressor function and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can contribute to reprogramming normal development to oncogenesis.

RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing.

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.