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Sample records for gene alternative splicing

  1. Evolution of alternative splicing after gene duplication.

    PubMed

    Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

    2006-02-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates. PMID:16365379

  2. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

  3. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  4. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  5. Vials: Visualizing Alternative Splicing of Genes.

    PubMed

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  6. Alternative splicing of DNA damage response genes and gastrointestinal cancers

    PubMed Central

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

    2014-01-01

    Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the potential relationship of alternative splicing, DNA damage, and gastrointestinal cancers. We will also discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies. PMID:25516641

  7. Insights into alternative splicing of sarcomeric genes in the heart.

    PubMed

    Weeland, Cornelis J; van den Hoogenhof, Maarten M; Beqqali, Abdelaziz; Creemers, Esther E

    2015-04-01

    Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical properties of the sarcomere are carefully orchestrated through alternative splicing to fit the varying demands on the heart. By the recent discovery of RBM20 and RBM24, two major heart and skeletal muscle-restricted splicing factors, it became evident that alternative splicing events in the heart occur in regulated networks rather than in isolated events. Analysis of knockout mice of these splice factors has shed light on the importance of these fundamental processes in the heart. In this review, we discuss recent advances in our understanding of the role and regulation of alternative splicing in the developing and diseased heart, specifically within the sarcomere. Through various examples (titin, myomesin, troponin T, tropomyosin and LDB3) we illustrate how alternative splicing regulates the functional properties of the sarcomere. Finally, we evaluate opportunities and obstacles to modulate alternative splicing in therapeutic approaches for cardiac disease. PMID:25683494

  8. Width of Gene Expression Profile Drives Alternative Splicing

    PubMed Central

    Wegmann, Daniel; Dupanloup, Isabelle; Excoffier, Laurent

    2008-01-01

    Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection. PMID:18974852

  9. Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes.

    PubMed

    Simpson, Craig G; Fuller, John; Calixto, Cristiane P G; McNicol, Jim; Booth, Clare; Brown, John W S; Staiger, Dorothee

    2016-01-01

    Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative splicing events. The use of the panel allows the quantification of changes in the proportion of splice isoforms between different samples, e.g., different time points, different tissues, genotypes, ecotypes, or treatments. PMID:26867620

  10. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases.

    PubMed

    Dlamini, Zodwa; Tshidino, Shonisani C; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  11. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  12. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    PubMed

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. PMID:26685868

  13. A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

    PubMed Central

    Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

    2015-01-01

    Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

  14. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  15. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon.

    PubMed

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ?5,655 intronless genes, ?13,302 constitutively spliced, and ?7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ?5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes. PMID:26156297

  16. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon

    PubMed Central

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ?5,655 intronless genes, ?13,302 constitutively spliced, and ?7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ?5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes. PMID:26156297

  17. Alternative Splicing of an Insect Sodium Channel Gene Generates Pharmacologically Distinct Sodium Channels

    PubMed Central

    Tan, Jianguo; Liu, Zhiqi; Nomura, Yoshiko; Goldin, Alan L.; Dong, Ke

    2011-01-01

    Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, paraCSMA. The splice site is conserved in the mouse, fish, and human Nav1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels. PMID:12097481

  18. Alternative splicing in C. elegans.

    PubMed Central

    Zahler, Alan M

    2005-01-01

    Alternative splicing is a common mechanism for the generation of multiple isoforms of proteins. It can function to expand the proteome of an organism and can serve as a way to turn off gene expression post-transcriptionally. This review focuses on splicing and its regulation in C. elegans. The fully-sequenced C. elegans genome combined with its elegant genetics offers unique advantages for exploring alternative splicing regulation in metazoans. The topics covered in this review include constitutive splicing factors, identification of alternatively spliced genes, examples of alternative splicing in C. elegans, and alternative splicing regulation. Key genes whose regulated alternative splicing are reviewed include let-2, unc-32, unc-52, egl-15 and xol-1. Factors involved in alternative splicing that are discussed include mec-8, smu-1, smu-2, fox-1, exc-7 and unc-75. PMID:18050427

  19. Chromatin and alternative splicing.

    PubMed

    All, M; Schor, I E; Muoz, M J; de la Mata, M; Agirre, E; Valcrcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing. PMID:21289049

  20. The evolutionary fate of alternatively spliced homologous exons after gene duplication.

    PubMed

    Abascal, Federico; Tress, Michael L; Valencia, Alfonso

    2015-06-01

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. PMID:25931610

  1. The Evolutionary Fate of Alternatively Spliced Homologous Exons after Gene Duplication

    PubMed Central

    Abascal, Federico; Tress, Michael L.; Valencia, Alfonso

    2015-01-01

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. PMID:25931610

  2. Of urchins and men: Evolution of an alternative splicing unit in fibroblast growth factor receptor genes

    PubMed Central

    MISTRY, NEVILLE; HARRINGTON, WHITNEY; LASDA, ERIKA; WAGNER, ERIC J.; GARCIA-BLANCO, MARIANO A.

    2003-01-01

    Alternative splicing of mammalian transcripts, which yields many diverse protein products from one gene, is the rule and not the exception. Although the mechanisms that govern alternative splicing are being unraveled, little is known about the evolution of this critical engine of proteome diversity. Here we present a phylogenetic analysis from a sea urchin to humans of the alternative splicing unit encoding the third Ig domain of fibroblast growth factor receptors. The remarkable conservation of intronic control elements, both in structure and function, indicates that the mechanisms that regulate this alternative splicing unit evolved over 600 million years ago. PMID:12554864

  3. Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

    Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.

  4. Increasing the Coding Potential of Genomes Through Alternative Splicing: The Case of PARK2 Gene.

    PubMed

    La Cognata, Valentina; Iemmolo, Rosario; D'Agata, Velia; Scuderi, Soraya; Drago, Filippo; Zappia, Mario; Cavallaro, Sebastiano

    2014-06-01

    The completion of the Human Genome Project aroused renewed interest in alternative splicing, an efficient and widespread mechanism that generates multiple protein isoforms from individual genes. Although our knowledge about alternative splicing is growing exponentially, its real impact on cellular life is still to be clarified. Connecting all splicing features (genes, splice transcripts, isoforms, and relative functions) may be useful to resolve this tangle. Herein, we will start from the case of a single gene, Parkinson protein 2, E3 ubiquitin protein ligase (PARK2), one of the largest in our genome. This gene is implicated in the pathogenesis of autosomal recessive juvenile Parkinsonism and it has been recently linked to cancer, leprosy, autism, type 2 diabetes mellitus and Alzheimer's disease. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification and protein diversity in tissues and cells. This review will provide an update of all human PARK2 alternative splice transcripts and isoforms presently known, and correlate them to those in rat and mouse, two common animal models for studying human disease genes. Alternative splicing relies upon a complex process that could be easily altered by both cis and trans-acting mutations. Although the contribution of PARK2 splicing in human disease remains to be fully explored, some evidences show disruption of this versatile form of genetic regulation may have pathological consequences. PMID:24955028

  5. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  6. Hypoxia Regulates Alternative Splicing of HIF and non-HIF Target Genes

    PubMed Central

    Sena, Johnny A.; Wang, Liyi; Heasley, Lynn E.; Hu, Cheng-Jun

    2014-01-01

    Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1A) and 2? (HIF2?/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma (HCC) cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. PMID:24850901

  7. Analysis of Genetic Interaction Networks Shows That Alternatively Spliced Genes Are Highly Versatile

    PubMed Central

    Talavera, David; Sheoran, Ritika; Lovell, Simon C.

    2013-01-01

    Alternative splicing has the potential to increase the diversity of the transcriptome and proteome. Where more than one transcript arises from a gene they are often so different that they are quite unlikely to have the same function. However, it remains unclear if alternative splicing generally leads to a gene being involved in multiple biological processes or whether it alters the function within a single process. Knowing that genetic interactions occur between functionally related genes, we have used them as a proxy for functional versatility, and have analysed the sets of genes of two well-characterised model organisms: Caenorhabditis elegans and Drosophila melanogaster. Using network analyses we find that few genes are functionally homogenous (only involved in a few functionally-related biological processes). Moreover, there are differences between alternatively spliced genes and genes with a single transcript; specifically, genes with alternatively splicing are, on average, involved in more biological processes. Finally, we suggest that factors other than specific functional classes determine whether a gene is alternatively spliced. PMID:23409018

  8. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

    PubMed Central

    2014-01-01

    Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected. PMID:24885185

  9. Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

    PubMed Central

    Shen, J; Beall, C J; Hirsh, J

    1993-01-01

    The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default. Images PMID:8336700

  10. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease.

    PubMed

    lvarez, Enrique; Castell, Alfredo; Carrasco, Luis; Izquierdo, Jos M

    2011-10-14

    Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A(pro) modulating the alternative splicing of pre-mRNAs. Expression of 2A(pro) potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A(pro) abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A(pro), leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A(pro) on splicing is to selectively block the second catalytic step. PMID:21945619

  11. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  12. Coordinately Regulated Alternative Splicing of Genes Involved in Cholesterol Biosynthesis and Uptake

    PubMed Central

    Naidoo, Devesh; Rudel, Lawrence L.; Temel, Ryan E.; McDaniel, Allison L.; Marshall, Stephanie M.; Krauss, Ronald M.

    2011-01-01

    Genes involved in cholesterol biosynthesis and uptake are transcriptionally regulated in response to cellular sterol content in a coordinated manner. A number of these genes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and LDL receptor (LDLR), undergo alternative splicing, resulting in reductions of enzyme or protein activity. Here we demonstrate that cellular sterol depletion suppresses, and sterol loading induces, alternative splicing of multiple genes involved in the maintenance of cholesterol homeostasis including HMGCR and LDLR, the key regulators of cellular cholesterol biosynthesis and uptake, respectively. These changes were observed in both in vitro studies of the HepG2 human hepatoma derived cell line, as well as in vivo studies of St. Kitts vervets, also known as African green monkeys, a commonly used primate model for investigating cholesterol metabolism. These effects are mediated in part by sterol regulation of polypyrimidine tract binding protein 1 (PTBP1), since knock-down of PTBP1 eliminates sterol induced changes in alternative splicing of several of these genes. Single nucleotide polymorphisms (SNPs) that influence HMGCR and LDLR alternative splicing (rs3846662 and rs688, respectively), have been associated with variation in plasma LDL-cholesterol levels. Sterol-induced changes in alternative splicing are blunted in carriers of the minor alleles for each of these SNPs, indicating an interaction between genetic and non-genetic regulation of this process. Our results implicate alternative splicing as a novel mechanism of enhancing the robust transcriptional response to conditions of cellular cholesterol depletion or accumulation. Thus coordinated regulation of alternative splicing may contribute to cellular cholesterol homeostasis as well as plasma LDL levels. PMID:21559365

  13. Alternative splicing and genomic structure of the Wilms tumor gene WT1.

    PubMed Central

    Haber, D A; Sohn, R L; Buckler, A J; Pelletier, J; Call, K M; Housman, D E

    1991-01-01

    The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5' splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggests that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions. Images PMID:1658787

  14. Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis

    SciTech Connect

    Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha; Conboy, John G.

    2009-02-03

    Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

  15. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

    PubMed Central

    Gardina, Paul J; Clark, Tyson A; Shimada, Brian; Staples, Michelle K; Yang, Qing; Veitch, James; Schweitzer, Anthony; Awad, Tarif; Sugnet, Charles; Dee, Suzanne; Davies, Christopher; Williams, Alan; Turpaz, Yaron

    2006-01-01

    Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. PMID:17192196

  16. The exosome controls alternative splicing by mediating the gene expression and assembly of the spliceosome complex

    PubMed Central

    Zhang, Lin; Wan, Yufeng; Huang, Guobin; Wang, Dongni; Yu, Xinyang; Huang, Guocun; Guo, Jinhu

    2015-01-01

    The exosome is a complex with exoribonuclease activity that regulates RNA surveillance and turnover. The exosome also plays a role in regulating the degradation of precursor mRNAs to maintain the expression of splicing variants. In Neurospora, the silencing of rrp44, which encodes the catalytic subunit of the exosome, changed the expression of a set of spliceosomal snRNA, snRNP genes and SR protein related genes. The knockdown of rrp44 also affected the assembly of the spliceosome. RNA-seq analysis revealed a global change in bulk splicing events. Exosome-mediated splicing may regulate alternative splicing of NCU05290, NCU07421 and the circadian clock gene frequency (frq). The knockdown of rrp44 led to an increased ratio of splicing variants without intron 6 (I-6) and shorter protein isoform small FRQ (s-FRQ) as a consequence. These findings suggest that the exosome controls splicing events by regulating the degradation of precursor mRNAs and the gene expression, assembly and function of the spliceosome. PMID:26306464

  17. The exosome controls alternative splicing by mediating the gene expression and assembly of the spliceosome complex.

    PubMed

    Zhang, Lin; Wan, Yufeng; Huang, Guobin; Wang, Dongni; Yu, Xinyang; Huang, Guocun; Guo, Jinhu

    2015-01-01

    The exosome is a complex with exoribonuclease activity that regulates RNA surveillance and turnover. The exosome also plays a role in regulating the degradation of precursor mRNAs to maintain the expression of splicing variants. In Neurospora, the silencing of rrp44, which encodes the catalytic subunit of the exosome, changed the expression of a set of spliceosomal snRNA, snRNP genes and SR protein related genes. The knockdown of rrp44 also affected the assembly of the spliceosome. RNA-seq analysis revealed a global change in bulk splicing events. Exosome-mediated splicing may regulate alternative splicing of NCU05290, NCU07421 and the circadian clock gene frequency (frq). The knockdown of rrp44 led to an increased ratio of splicing variants without intron 6 (I-6) and shorter protein isoform small FRQ (s-FRQ) as a consequence. These findings suggest that the exosome controls splicing events by regulating the degradation of precursor mRNAs and the gene expression, assembly and function of the spliceosome. PMID:26306464

  18. Human Aldehyde Dehydrogenase Genes: Alternatively-Spliced Transcriptional Variants and Their Suggested Nomenclature

    PubMed Central

    Black, William J.; Stagos, Dimitrios; Marchitti, Satori A.; Nebert, Daniel W.; Tipton, Keith F.; Bairoch, Amos; Vasiliou, Vasilis

    2011-01-01

    OBJECTIVE The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)+-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjgren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimers disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. METHODS Data-mining methods were used to retrieve all human ALDH sequences. Alternatively-spliced transcriptional variants were determined based upon: a) criteria for sequence integrity and genomic alignment; b) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and c) if available, empirical evidence of variants from the literature. RESULTS AND CONCLUSION Alternatively-spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. In order to avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily. PMID:19823103

  19. Global variability in gene expression and alternative splicing is modulated by mitochondrial content

    PubMed Central

    Guantes, Raul; Rastrojo, Alberto; Neves, Ricardo; Lima, Ana; Aguado, Begoña; Iborra, Francisco J.

    2015-01-01

    Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype. PMID:25800673

  20. The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.

    PubMed Central

    Hanke, P D; Storti, R V

    1988-01-01

    The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed. Images PMID:2851721

  1. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    PubMed

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development. PMID:25983000

  2. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5? splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5? exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  3. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  4. A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

    PubMed

    Zhang, Junyu; Liu, Hongbin; Liu, Zhiyuan; Liao, Yong; Guo, Luo; Wang, Honglian; He, Lin; Zhang, Xiaodong; Xing, Qinghe

    2013-01-01

    Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1. PMID:23342054

  5. Review: Alternative Splicing (AS) of Genes As An Approach for Generating Protein Complexity

    PubMed Central

    Roy, Bishakha; Haupt, Larisa M; Griffiths, Lyn R

    2013-01-01

    Prior to the completion of the human genome project, the human genome was thought to have a greater number of genes as it seemed structurally and functionally more complex than other simpler organisms. This along with the belief of “one gene, one protein”, were demonstrated to be incorrect. The inequality in the ratio of gene to protein formation gave rise to the theory of alternative splicing (AS). AS is a mechanism by which one gene gives rise to multiple protein products. Numerous databases and online bioinformatic tools are available for the detection and analysis of AS. Bioinformatics provides an important approach to study mRNA and protein diversity by various tools such as expressed sequence tag (EST) sequences obtained from completely processed mRNA. Microarrays and deep sequencing approaches also aid in the detection of splicing events. Initially it was postulated that AS occurred only in about 5% of all genes but was later found to be more abundant. Using bioinformatic approaches, the level of AS in human genes was found to be fairly high with 35-59% of genes having at least one AS form. Our ability to determine and predict AS is important as disorders in splicing patterns may lead to abnormal splice variants resulting in genetic diseases. In addition, the diversity of proteins produced by AS poses a challenge for successful drug discovery and therefore a greater understanding of AS would be beneficial. PMID:24179441

  6. Identification of new alternative splice events in the TCIRG1 gene in different human tissues

    SciTech Connect

    Smirnova, Anna S.; Morgun, Andrey . E-mail: anemorgun@hotmail.com; Shulzhenko, Natalia; Silva, Ismael D.C.G.; Gerbase-DeLima, Maria

    2005-05-13

    Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.

  7. Alternative splicing of testis-specific lactate dehydrogenase C gene in mammals and pigeon.

    PubMed

    Huang, Lin; Lin, Yaqiu; Jin, Suyu; Liu, Wei; Xu, Yaou; Zheng, Yucai

    2012-04-01

    The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals. PMID:22537060

  8. Alternative splicing in the human interleukin enhancer binding factor 3 (ILF3) gene.

    PubMed

    Duchange, N; Pidoux, J; Camus, E; Sauvaget, D

    2000-12-31

    The Interleukin Enhancer Binding Factor 3 (ILF3) gene has been mapped to chromosome 19 in humans and to chromosome 9 in mice. Several reported double-stranded RNA binding proteins including NF90, ILF3, MPP4 and DRBP76 have been suggested to be isoforms of the ILF3 gene but this has not been clearly established. We isolated several ilf3 transcripts from a melanoma cDNA library and two corresponding genomic fragments, and report alternative splicing and polyadenylation site selection in the human ILF3 gene. We show the existence of an alternative splice site responsible for the sequence divergence in the 3' part of the transcripts. Another alternative splicing event at a site between the two double-stranded RNA binding motifs leads to the additional presence in some cases of a four amino acids NVKQ peptide. We also describe the utilization of three distinct polyadenylation signals and the generation of an ilf3 transcript with a long extended 3' UTR. The expression of the different transcripts was evaluated. We used a GenBank sequence for the part of chromosome 19 corresponding to the ILF3 gene to determine the exon-intron organization of the entire gene which spans 38 kb and is divided into 21 exons. PMID:11167023

  9. Alternative Splicing in the Human PMP22 Gene: Implications in CMT1A Neuropathy.

    PubMed

    Visigalli, Davide; Castagnola, Patrizio; Capodivento, Giovanna; Geroldi, Alessandro; Bellone, Emilia; Mancardi, Gianluigi; Pareyson, Davide; Schenone, Angelo; Nobbio, Lucilla

    2016-01-01

    CMT1A patients commonly share PMP22 genetic overloading but they show phenotypic heterogeneity and variability in PMP22 mRNA and protein expression. Moreover, PMP22 mRNA levels do not correlate with clinical outcome measures in these patients, suggesting their uselessness as a disease biomarker. Thus, in-depth analysis of PMP22 transcription and translation might help to define its pathogenic role in CMT1A. We focused on the alternative splicing of PMP22 gene to verify whether mRNA processing is altered in CMT1A. We identified three new PMP22 transcripts enriched in human sural nerve biopsies. One of them was an untranslated variant, whereas the other two originated from a PMP22 undescribed exon and encoded for a new putative protein localized in the endoplasmic reticulum. As splicing events in the PMP22 gene are differently regulated in tissues and during development, we analyzed the levels of PMP22 transcripts and their splicing pattern in human and experimental CMT1A. We found an altered PMP22 splicing ratio in the CMT1A rat. In addition, we showed a remarkable derangement in rat QKI expression, which is a critical regulator of splicing during myelination. Overall, our data suggest that an alteration of mRNA processing could be a pathogenic mechanism in CMT1A. PMID:26486801

  10. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    PubMed Central

    Shang, Jin; Fan, Xin; Shangguan, Lei; Liu, Huan; Zhou, Yue

    2015-01-01

    Low back pain (LBP) is a very prevalent disease and degenerative disc diseases (DDDs) usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0) to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale. PMID:26649308

  11. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells.

    PubMed

    Shang, Jin; Fan, Xin; Shangguan, Lei; Liu, Huan; Zhou, Yue

    2015-01-01

    Low back pain (LBP) is a very prevalent disease and degenerative disc diseases (DDDs) usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0) to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale. PMID:26649308

  12. Prolyl 4-hydroxylase genes are subjected to alternative splicing in roots of maize seedlings under waterlogging

    PubMed Central

    Zou, Xiling; Jiang, Yuanyuan; Zheng, Yonglian; Zhang, Meidong; Zhang, Zuxin

    2011-01-01

    Background In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize. Methods A comprehensive genome-wide analysis of P4H genes of maize (zmP4H genes) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Key Results Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites was strongly correlated with selection of donor and receptor sites, and ambiguous junction sites due to small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harboured a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. Reverse transcriptionPCR (RTPCR) indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4H genes were under specific control by post-transcriptional regulation under waterlogging stress in the line HZ32. Conclusions Our results provide a framework for future dissection of the function of the emerging zmP4H family and suggest that AS might have an important role in the regulation of the expression profile of this gene family under waterlogging stress. PMID:21969257

  13. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing “minigene” in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  14. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  15. ASTALAVISTA: dynamic and flexible analysis of alternative splicing events in custom gene datasets

    PubMed Central

    Foissac, Sylvain; Sammeth, Michael

    2007-01-01

    In the process of establishing more and more complete annotations of eukaryotic genomes, a constantly growing number of alternative splicing (AS) events has been reported over the last decade. Consequently, the increasing transcript coverage also revealed the real complexity of some variations in the exonintron structure between transcript variants and the need for computational tools to address complex AS events. ASTALAVISTA (alternative splicing transcriptional landscape visualization tool) employs an intuitive and complete notation system to univocally identify such events. The method extracts AS events dynamically from custom gene annotations, classifies them into groups of common types and visualizes a comprehensive picture of the resulting AS landscape. Thus, ASTALAVISTA can characterize AS for whole transcriptome data from reference annotations (GENCODE, REFSEQ, ENSEMBL) as well as for genes selected by the user according to common functional/structural attributes of interest: http://genome.imim.es/astalavista PMID:17485470

  16. RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum

    PubMed Central

    2013-01-01

    Background The genome of Fusarium graminearum has been sequenced and annotated previously, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum. Results We identified and revised 655 incorrectly predicted gene models, including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that many of these could be missed genes. Finally, we identified the 5′ UTR and/or 3′ UTR sequences of 7666 genes. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons. Conclusions We have developed novel and efficient strategies to identify alternatively spliced genes and incorrect gene models based on RNA-Seq data. Our study identified hundreds of alternatively spliced genes in F. graminearum and for the first time indicated that alternative splicing is developmentally regulated in filamentous fungi. In addition, hundreds of incorrect predicted gene models were identified and revised and thousands of nTARs were discovered in our study, which will be helpful for the future genomic and transcriptomic studies in F. graminearum. PMID:23324402

  17. ASGS: an alternative splicing graph web service

    PubMed Central

    Bollina, Durgaprasad; Lee, Bernett T. K.; Tan, Tin Wee; Ranganathan, Shoba

    2006-01-01

    Alternative transcript diversity manifests itself a prime cause of complexity in higher eukaryotes. The Alternative Splicing Graph Server (ASGS) is a web service facilitating the systematic study of alternatively spliced genes of higher eukaryotes by generating splicing graphs for the compact visual representation of transcript diversity from a single gene. Taking a set of transcripts in General Feature Format as input, ASGS identifies distinct reference and variable exons, generates a transcript splicing graph, an exon summary, splicing events classification and a single line graph to facilitate experimental analysis. This freely available web service can be accessed at . PMID:16845045

  18. Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer

    PubMed Central

    Aversa, Rosanna; Sorrentino, Anna; Esposito, Roberta; Ambrosio, Maria Rosaria; Amato, Angela; Zambelli, Alberto; Ciccodicola, Alfredo; D’Apice, Luciana; Costa, Valerio

    2016-01-01

    Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression. PMID:26784191

  19. Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

    PubMed

    Aversa, Rosanna; Sorrentino, Anna; Esposito, Roberta; Ambrosio, Maria Rosaria; Amato, Angela; Zambelli, Alberto; Ciccodicola, Alfredo; D'Apice, Luciana; Costa, Valerio

    2016-01-01

    Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations-in human breast cell lines and breast tumor biopsies-we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression. PMID:26784191

  20. Detection of Alternative Splice and Gene Duplication by RNA Sequencing in Japanese Flounder, Paralichthys olivaceus

    PubMed Central

    Wang, Wenji; Wang, Jing; You, Feng; Ma, Liman; Yang, Xiao; Gao, Jinning; He, Yan; Qi, Jie; Yu, Haiyang; Wang, Zhigang; Wang, Xubo; Wu, Zhihao; Zhang, Quanqi

    2014-01-01

    Japanese flounder (Paralichthys olivaceus) is one of the economic important fish in China. Sexual dimorphism, especially the different growth rates and body sizes between two sexes, makes this fish a good model to investigate mechanisms responsible for such dimorphism for both fundamental questions in evolution and applied topics in aquaculture. However, the lack of omics data has hindered the process. The recent advent of RNA-sequencing technology provides a robust tool to further study characteristics of genomes of nonmodel species. Here, we performed de novo transcriptome sequencing for a double haploid Japanese flounder individual using Illumina sequencing. A single lane of paired-end sequencing produced more than 27 million reads. These reads were assembled into 107,318 nonredundant transcripts, half of which (51,563; 48.1%) were annotated by blastx to public protein database. A total of 1051 genes that had potential alternative splicings were detected by Chrysalis implemented in Trinity software. Four of 10 randomly picked genes were verified truly containing alternative splicing by cloning and Sanger sequencing. Notably, using a doubled haploid Japanese flounder individual allow us to analyze gene duplicates. In total, 3940 single-nucleotide polymorphisms were detected form 1859 genes, which may have happened gene duplicates. This study lays the foundation for structural and functional genomics studies in Japanese flounder. PMID:25512620

  1. Detection of alternative splice and gene duplication by RNA sequencing in Japanese flounder, Paralichthys olivaceus.

    PubMed

    Wang, Wenji; Wang, Jing; You, Feng; Ma, Liman; Yang, Xiao; Gao, Jinning; He, Yan; Qi, Jie; Yu, Haiyang; Wang, Zhigang; Wang, Xubo; Wu, Zhihao; Zhang, Quanqi

    2014-12-01

    Japanese flounder (Paralichthys olivaceus) is one of the economic important fish in China. Sexual dimorphism, especially the different growth rates and body sizes between two sexes, makes this fish a good model to investigate mechanisms responsible for such dimorphism for both fundamental questions in evolution and applied topics in aquaculture. However, the lack of "omics" data has hindered the process. The recent advent of RNA-sequencing technology provides a robust tool to further study characteristics of genomes of nonmodel species. Here, we performed de novo transcriptome sequencing for a double haploid Japanese flounder individual using Illumina sequencing. A single lane of paired-end sequencing produced more than 27 million reads. These reads were assembled into 107,318 nonredundant transcripts, half of which (51,563; 48.1%) were annotated by blastx to public protein database. A total of 1051 genes that had potential alternative splicings were detected by Chrysalis implemented in Trinity software. Four of 10 randomly picked genes were verified truly containing alternative splicing by cloning and Sanger sequencing. Notably, using a doubled haploid Japanese flounder individual allow us to analyze gene duplicates. In total, 3940 "single-nucleotide polymorphisms" were detected form 1859 genes, which may have happened gene duplicates. This study lays the foundation for structural and functional genomics studies in Japanese flounder. PMID:25512620

  2. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  3. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  4. Functional characterization of two alternatively spliced transcripts of tomato ABSCISIC ACID INSENSITIVE3 (ABI3) gene.

    PubMed

    Gao, Yongfeng; Liu, Jikai; Zhang, Zhiguo; Sun, Xiaochun; Zhang, Ni; Fan, Jing; Niu, Xiangli; Xiao, Fangming; Liu, Yongsheng

    2013-05-01

    Alternative splicing can produce transcripts that encode proteins with altered functions. The transcripts of the ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) gene, which is an important component in abscisic acid (ABA) signaling, are subjected to alternative splicing in both monocotyledons and dicotyledons. We identified two alternatively spliced tomato (Solanum lycopersicum) SlABI3 transcripts, SlABI3-F and SlABI3-T, which encode the nucleus-localized full-length and truncated proteins, respectively. The tissue-specific accumulation of SlABI3-F and SlABI3-T was determined, particularly in seeds at different developmental stages and in response to phytohormonal and abiotic stress. Ectopic over-expression of SlABI3-F and SlABI3-T resulted in the induction of seed-specific genes SlSOM, SlEM1 and SlEM6 in vegetative tissues. However, over-expression of SlABI3-F, but not SlABI3-T, activated expression of the downstream gene SlABI5 and conferred hypersensitivity to exogenous ABA during seed germination and primary root growth. In addition, the SlABI3-F protein interacted with SlABI5 much stronger than SlABI3-T did in the yeast two-hybrid assay. These results suggest that SlABI3-F and SlABI3-T have similar and distinct functionality in the ABA signaling, dependent on which tissue/organ they accumulate in. PMID:23504452

  5. Alternative splicing, gene localization, and binding of SH2-B to the insulin receptor kinase domain.

    PubMed

    Nelms, K; O'Neill, T J; Li, S; Hubbard, S R; Gustafson, T A; Paul, W E

    1999-12-01

    The SH2-B protein is an SH2-domain-containing molecule that interacts with a number of phosphorylated kinase and receptor molecules including the insulin receptor. Two isoforms of the SH2-B have been identified and have been proposed to arise through alternate splicing. Here we have identified a third isoform of the SH2-B protein, SH2-Bgamma, that interacts specifically with the insulin receptor. This interaction required phosphorylation of residue Y1146 in the triple tyrosine motif within the activation loop of the IR kinase and is one of only two signaling molecules shown to interact directly with this residue of the insulin receptor kinase domain. The intron/exon structure of the SH2-B gene was determined. Alternate splice sites utilized to generate the different isoforms of the SH2-B protein were identified in the 3' end of the SH2-B gene immediately downstream of the exon encoding the core of the SH2 domain. Additionally, the chromosomal location of the SH2-B gene was determined to be the distal arm of mouse Chromosome (Chr) 7 in a region linked to obesity in mice. PMID:10594240

  6. Splicing predictions reliably classify different types of alternative splicing.

    PubMed

    Busch, Anke; Hertel, Klemens J

    2015-05-01

    Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5' or 3' splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

  7. Complete nucleotide sequence of the fast skeletal troponin T gene. Alternatively spliced exons exhibit unusual interspecies divergence.

    PubMed

    Breitbart, R E; Nadal-Ginard, B

    1986-04-01

    The continuous nucleotide sequence of the rat fast skeletal muscle troponin T gene is reported, complementing the previous determinations of its structural organization and its capacity to encode multiple isoforms via alternative RNA splicing. Canonical promoter elements, as well as consensus sequences that may be involved in the 3' processing of the primary transcript, are present. All exons are flanked by conventional donor and acceptor splice sites, which can hybridize to U1 RNA. Extensive computer-assisted analyses of the genomic sequence do not reveal cis elements that unambiguously distinguish alternative from constitutive exons. Local RNA secondary structures can be predicted, however, that sequester exons or their splice sites in stem-and-loop formations, and which may also pair with small nuclear RNAs. These interactions might, in theory, contribute to differential exon usage. The structural features of exon organization that characterize this rat skeletal gene are closely conserved in the chicken cardiac troponin T gene, but the former exhibits a more diversified capacity for differential splicing. Implications for the mechanisms of alternative RNA splicing are considered. Comparisons of troponin T amino acid sequences among several species reveal striking dissimilarities, in contrast to the otherwise highly conserved contractile proteins. These divergences involve entire peptide subsegments and are concentrated in the same domains as are encoded by alternatively spliced exons, suggesting that exon shuffling may have contributed to the evolution of troponin T. PMID:3735424

  8. Alternatively spliced products lacking exon 12 dominate the expression of fragile X mental retardation 1 gene in human tissues.

    PubMed

    Fu, Xianguo; Zheng, Dezhu; Liao, Juan; Li, Qingqin; Lin, Yuxiang; Zhang, Duo; Yan, Aizhen; Lan, Fenghua

    2015-08-01

    Fragile X mental retardation 1 gene (FMR1) expression is associated with fragile X syndrome (FXS) and exhibits several splicing products. However, the proportion of spliced isoforms that are expressed in different tissues remains unclear. In the present study, long-chain reverse transcription-polymerase chain reaction with a T cloning-sequencing method was conducted in order to analyze the entire coding region of the FMR1 gene in human tissues. In particular, FXS-associated tissues were analyzed, including the brain and testis. Twenty alternatively spliced isoforms were observed among 271 recombinants, including six novel ones. The isoform that consisted of the entire FMR1 coding region (ISO1) accounted for a small proportion of all isoforms. Isoforms lacking exon 12 were the most abundant. In particular, spliced isoforms ISO7 and ISO17 were the most abundant. However, their relative abundance varied between the peripheral blood cells, and the testis and brain tissues. Bioinformatic analyses suggested that exon 12 may be the sole exon undergoing positive selection. The results of the present study suggested that the mechanisms underlying alternative splicing (AS) of the FMR1 gene may be more complex. Furthermore, the functions of alternatively spliced products lacking exon 12 require further investigation. The results of the present study provide novel insights into the association between AS and the structure and function of the FMR1 gene. PMID:25847585

  9. Selecting for Functional Alternative Splices in ESTs

    PubMed Central

    Kan, Zhengyan; States, David; Gish, Warren

    2002-01-01

    The expressed sequence tag (EST) collection in dbEST provides an extensive resource for detecting alternative splicing on a genomic scale. Using genomically aligned ESTs, a computational tool (TAP) was used to identify alternative splice patterns for 6400 known human genes from the RefSeq database. With sufficient EST coverage, one or more alternatively spliced forms could be detected for nearly all genes examined. To identify high (>95%) confidence observations of alternative splicing, splice variants were clustered on the basis of having mutually exclusive structures, and sample statistics were then applied. Through this selection, alternative splices expected at a frequency of >5% within their respective clusters were seen for only 17%28% of genes. Although intron retention events (potentially unspliced messages) had been seen for 36% of the genes overall, the same statistical selection yielded reliable cases of intron retention for <5% of genes. For high-confidence alternative splices in the human ESTs, we also noted significantly higher rates both of cross-species conservation in mouse ESTs and of validation in the GenBank mRNA collection. We suggest quantitative analytical approaches such as these can aid in selecting useful targets for further experimental characterization and in so doing may help elucidate the mechanisms and biological implications of alternative splicing. PMID:12466287

  10. Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

    PubMed

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A; Vajja, Venkata G; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K; Levi, Amnon; Wehner, Todd; Reddy, Umesh K

    2015-03-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  11. Differential gene expression and alternative splicing between diploid and tetraploid watermelon

    PubMed Central

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A.; Vajja, Venkata G.; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K.; Levi, Amnon; Wehner, Todd; Reddy, Umesh K.

    2015-01-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  12. Chromatin's thread to alternative splicing regulation.

    PubMed

    Iannone, Camilla; Valcrcel, Juan

    2013-12-01

    Intron removal (pre-mRNA splicing) is a necessary step for expression of most genes in higher eukaryotes. Alternative splice site selection is a prevalent mechanism that diversifies genome outputs and offers ample opportunities for gene regulation in these organisms. Pre-mRNA splicing occurs co-transcriptionally and is influenced by features in chromatin structure, including nucleosome density and epigenetic modifications. We review here the molecular mechanisms by which the reciprocal interplay between chromatin and RNA processing can contribute to alternative splicing regulation. PMID:23912688

  13. Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.

    PubMed

    Lefebvre, Jonathan; Clarkson, Michael; Massa, Filippo; Bradford, Stephen T; Charlet, Aurelie; Buske, Fabian; Lacas-Gervais, Sandra; Schulz, Herbert; Gimpel, Charlotte; Hata, Yutaka; Schaefer, Franz; Schedl, Andreas

    2015-08-01

    The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2? is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2? as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders. PMID:25993318

  14. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  15. Identification of alternative 5?/3? splice sites based on the mechanism of splice site competition

    PubMed Central

    Xia, Huiyu; Bi, Jianning; Li, Yanda

    2006-01-01

    Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5?/3? splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified ?70% of the splice sites into alternative and constitutive, as well as ?80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology. PMID:17098928

  16. Structure of the human laminin {gamma}2 chain gene (LAMC2): Alternative splicing with different tissue distribution of two transcripts

    SciTech Connect

    Airenne, T.; Haakana, H.; Kallunki, T.

    1996-02-15

    This article discusses the exon-intron structure and tissue distribution of the laminin {gamma}2 chain (LAMC2) gene, which is mutated in some cases of junctional epidermolysis bullosa. The article also discusses the transcription and splicing of this gene, which result in alternative uses of the last two exons of the gene. The different tissue distributions of the transcripts indicate different functions for the gene in vivo. 36 refs., 8 figs., 3 tabs.

  17. Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene

    SciTech Connect

    Cybulsky, M.I.; Fries, J.W.U.; Williams, A.J.; Sultan, P.; Gimbrone, M.A. Jr.; Collins, T. ); Eddy, R.; Byers, M.; Shows, T. )

    1991-09-01

    Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning {approx}25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-{kappa}B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression.

  18. Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene.

    PubMed

    Cybulsky, M I; Fries, J W; Williams, A J; Sultan, P; Eddy, R; Byers, M; Shows, T; Gimbrone, M A; Collins, T

    1991-09-01

    Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning approximately 25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-kappa B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression. PMID:1715583

  19. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis

    PubMed Central

    Kalyna, Maria; Simpson, Craig G.; Syed, Naeem H.; Lewandowska, Dominika; Marquez, Yamile; Kusenda, Branislav; Marshall, Jacqueline; Fuller, John; Cardle, Linda; McNicol, Jim; Dinh, Huy Q.; Barta, Andrea; Brown, John W. S.

    2012-01-01

    Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT–PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5′ or 3′UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3′-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes. PMID:22127866

  20. Alternative Splicing in Alzheimer’s Disease

    PubMed Central

    Love, Julia E.; Hayden, Eric J.; Rohn, Troy T.

    2015-01-01

    Neurodegenerative diseases have a variety of different genes contributing to their underlying pathology. Unfortunately, for many of these diseases it is not clear how changes in gene expression affect pathology. Transcriptome analysis of neurodegenerative diseases using ribonucleic acid sequencing (RNA Seq) and real time quantitative polymerase chain reaction (RT-qPCR) provides for a platform to allow investigators to determine the contribution of various genes to the disease phenotype. In Alzheimer’s disease (AD) there are several candidate genes reported that may be associated with the underlying pathology and are, in addition, alternatively spliced. Thus, AD is an ideal disease to examine how alternative splicing may affect pathology. In this context, genes of particular interest to AD pathology include the amyloid precursor protein (APP), TAU, and apolipoprotein E (APOE). Here, we review the evidence of alternative splicing of these genes in normal and AD patients, and recent therapeutic approaches to control splicing. PMID:26942228

  1. Cloning, genomic organization, alternative splicing and expression analysis of the human gene WNK3 (PRKWNK3).

    PubMed

    Holden, Simon; Cox, James; Raymond, F Lucy

    2004-06-23

    We report the isolation of a full length coding WNK3 cDNA from human fetal brain. The WNK3 transcript has an open reading frame of 5403 nucleotides and encodes a putative protein of 1800 amino acids. The human WNK3 gene comprises 24 exons and lies within a 559 kb genomic segment on chromosome Xp11.22 which has conserved synteny with a 705 kb genomic segment of human chromosome 9q22.31 which contains WNK2. The WNK3 transcript is expressed in several human fetal and adult tissues and has at least two splice isoforms generated by the alternative splicing of exon 18 and exon 22 which maintain the open reading frame. Usage of exon 18b is restricted to brain and introduces an additional 47 amino acids into the predicted protein. The predicted WNK3 protein has a similar structural organization to the other human WNK kinases. Significant homology between these proteins is confined to three conserved regions of their amino acid sequences which we have designated CR1, CR2 and CR3. CR1 and CR3 contain highly conserved residues which have been shown to be important for the normal function of WNK1 and WNK4, and CR2 contains a highly conserved 22 amino acid motif specific to chordate species. WNK3 lies within the critical linkage interval for several human monogenic disorders, including X-linked mental retardation. The function of mammalian WNK3 kinase remains to be investigated. PMID:15194194

  2. Splicing and alternative splicing in rice and humans.

    PubMed

    E, Zhiguo; Wang, Lei; Zhou, Jianhua

    2013-09-01

    Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. PMID:24064058

  3. Computational identification of tissue-specific alternative splicing elements in mouse genes from RNA-Seq

    PubMed Central

    Wen, Ji; Chiba, Akira; Cai, Xiaodong

    2010-01-01

    Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations. PMID:20685814

  4. Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae).

    PubMed

    Zhang, Bo; Liu, Zhi-Xiong; Ma, Jiang; Song, Yi; Chen, Fa-Ju

    2015-12-01

    Magnolia stellata is a woody ornamental shrub with more petaloid tepals than related plants from family Magnoliaceae. Recent studies revealed that expression changes in an AGAMOUS (AG) orthologous gene could resulted in double flowers with increased numbers of petals. We isolated three transcripts encoding different isoforms of a single AG orthologous gene, MastAG, mastag_2 and mastag_3, from M. stellata. Sequence alignments and Southern blot analyses suggested that MastAG was a single-copy gene in M. stellata genomes, and that mastag_2 and mastag_3 were abnormally spliced isoforms of MastAG. An 144bp exon skipping in MastAG results in the truncated mastag_2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas an 165bp exon skipping of MastAG produces a truncated mastag_3 protein lacking 6 aa of the K3 subdomain and the completely C terminal region. Expression analyses showed that three alternative splicing (AS) isoforms expressed only in developing stamens and carpels. Functional analyses revealed that MastAG could mimic the endogenous AG to specify carpel identity, but failed to regulate stamen development in an Arabidopsis ag-1 mutant. Moreover, the key domain or subdomain deletions represented by mastag_2 and mastag_3 resulted in loss of C-function. However, ectopic expression of mastag_2 in Arabidopsis produced flowers with sepals converted into carpeloid organs, but without petals and stamens, whereas ectopic expression of mastag_3 in Arabidopsis could mimic the flower phenotype of the ag mutant and produced double flowers with homeotic transformation of stamens into petals and carpels into another ag flower. Our results also suggest that mastag_3 holds some potential for biotechnical engineering to create multi-petal phenotypes in commercial ornamental cultivars. PMID:26706078

  5. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    EPA Science Inventory

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

    Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

    Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  6. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  7. The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function

    PubMed Central

    Khan, Sikandar G.; Muniz-Medina, Vanessa; Shahlavi, Tala; Baker, Carl C.; Inui, Hiroki; Ueda, Takahiro; Emmert, Steffen; Schneider, Thomas D.; Kraemer, Kenneth H.

    2002-01-01

    XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82882 bp) and 15 introns (0.085.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcriptionpolymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in ?0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in ?0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (?2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility. PMID:12177305

  8. A Mutation in the Srrm4 Gene Causes Alternative Splicing Defects and Deafness in the Bronx Waltzer Mouse

    PubMed Central

    Nakano, Yoko; Jahan, Israt; Bonde, Gregory; Sun, Xingshen; Hildebrand, Michael S.; Engelhardt, John F.; Smith, Richard J. H.; Cornell, Robert A.; Fritzsch, Bernd; Bnfi, Botond

    2012-01-01

    Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of premRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and proteinprotein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development. PMID:23055939

  9. A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse.

    PubMed

    Nakano, Yoko; Jahan, Israt; Bonde, Gregory; Sun, Xingshen; Hildebrand, Michael S; Engelhardt, John F; Smith, Richard J H; Cornell, Robert A; Fritzsch, Bernd; Bnfi, Botond

    2012-01-01

    Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre-mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein-protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development. PMID:23055939

  10. Global Disruption of Alternative Splicing and Neurodegeneration Is Caused by Mutation of a U2 snRNA Gene

    PubMed Central

    Jia, Yichang; Mu, John C.; Ackerman, Susan L.

    2012-01-01

    SUMMARY Although uridine-rich small nuclear RNAs (U-snRNAs) are essential for pre-mRNA splicing, little is known regarding their function in the regulation of alternative splicing or of the biological consequences of their dysfunction in mammals. Here, we demonstrate that mutation of Rnu28, one of the mouse multicopy U2 snRNA genes, causes ataxia and neurodegeneration. Coincident with the observed pathology, the level of mutant U2 RNAs was highest in the cerebellum and increased after granule neuron maturation. Furthermore, neuron loss was strongly dependent on the dosage of mutant and wild type snRNA genes. Comprehensive transcriptome analysis identified a group of alternative splicing events, including the splicing of small introns, which were disrupted in the mutant cerebellum. Our results suggest that the expression of mammalian U2 snRNA genes, previously presumed to be ubiquitious, is spatially and temporally regulated, and dysfunction of a single U2 snRNA causes neuron degeneration through distortion of pre-mRNA splicing. PMID:22265417

  11. Alternative Splicing of the Human Rab6A Gene Generates Two Close but Functionally Different Isoforms

    PubMed Central

    Echard, Arnaud; Opdam, Frank J.M.; de Leeuw, Hubert J.P.C.; Jollivet, Florence; Savelkoul, Paul; Hendriks, Wiljan; Voorberg, Jan; Goud, Bruno; Fransen, Jack A.M.

    2000-01-01

    Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A?, which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A? (Rab6A? Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A? Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A? is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A? interacts with two Rab6A partners, GAPCenA and clone 1, but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A? are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors. PMID:11071909

  12. Alternative splicing of exon 10 in the tau gene as a target for treatment of tauopathies

    PubMed Central

    Zhou, Jianhua; Yu, Qingming; Zou, Tie

    2008-01-01

    Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in exon 10 inclusion into tau mRNA. Abnormal splicing with inclusion of exon 10 into tau mRNA has also been observed in PSP and AD patients. These results indicate that abnormal splicing of exon 10, leading to the production of tau with exon 10, is probably one of the mechanisms by which tau accumulates and aggregates in tauopathic brains. Therefore, modulation of exon 10 splicing in the tau gene could potentially be targeted to prevent tauopathies. To identify small molecules or compounds that could potentially be developed into drugs to treat tauopathies, we established a cell-based high-throughput screening assay. In this review, we will discuss how realistic, specific biological molecules can be found to regulate exon 10 splicing in the tau gene for potential treatment of tauopathies. PMID:19090983

  13. A Japanese plum ?-l-arabinofuranosidase/?-D-xylosidase gene is developmentally regulated by alternative splicing.

    PubMed

    Di Santo, M Carolina; Ilina, Natalia; Pagano, Eduardo A; Sozzi, Gabriel O

    2015-02-01

    A full-length cDNA clone named PsARF/XYL was obtained from Prunus salicina Lindl., and determined to encode a putative ?-l-arabinofuranosidase/?-d-xylosidase belonging to glycoside hydrolase (GH, EC 3.2.1.-) family 3. Two related PsARF/XYL cDNAs were amplified, one from a fully-spliced transcript (PsARF/XYLa) and another one from an intron-retained transcript (PsARF/XYLb). The protein deduced from PsARF/XYLb is a truncated peptide at C-terminus that conserves the active-site amino acid sequence. High levels of PsARF/XYLa and PsARF/XYLb transcripts are detectable in several plant tissues. PsARF/XYLb transcripts accumulate progressively during the phase of exponential fruit growth but they become barely noticeable during on-tree ripening, or after a 6-h exposure of preclimacteric full-size plums to ethylene. In contrast, PsARF/XYLa is expressed throughout fruit development, and transcript accumulation parallels the climacteric rise in ethylene production during ripening. PsARF/XYLa expression is strongly induced in preclimacteric full-size plums after a 6-h treatment with physiologically active concentrations of ethylene. These findings suggest that PsARF/XYL gene is post-transcriptionally regulated by alternative splicing during development and that ethylene may be involved in this regulation. The isolation of a partial cDNA clone, PsARF1, is also reported. It encodes a putative cell-wall ?-l-arabinofuranosidase, and its transcription is rapidly inhibited by ethylene in mature green plums. PMID:25576002

  14. TWO ISOFORMS OF RUBISCO ACTIVASE IN COTTON, THE PRODUCTS OF SEPARATE GENES NOT ALTERNATIVE SPLICING.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In several plants, Rubisco activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA. Two forms of activase corresponding to the longer, alpha and the shorter, beta forms were detected in cotton (Gossypium hirsutum L.) leaves, but their N-termini differed. The cDNAs...

  15. Identification of novel alternative splicing transcript and expression analysis of bovine TMEM95 gene.

    PubMed

    Zhang, Sihuan; Cai, Hanfang; Yang, Qing; Shi, Tao; Pan, Chuanying; Lei, Chuzhao; Dang, Ruihua; Chen, Hong; Lan, Xianyong

    2016-01-10

    Transmembrane protein 95 (TMEM95) is closely related to male reproductive performance in cattle, but does not affect semen quality. Alternative splicing plays an important role in regulating biological function as well as in generating proteomic and functional diversity in metazoan organisms. Thus, the aim of this study was to clone and identify transcripts of the TMEM95 gene in cattle using RT-PCR, characterize them via bioinformatics analysis, and detect their expression patterns using qRT-PCR. Two transcripts of TMEM95 were identified in cattle, including TMEM95-SV1 and TMEM95-SV2. Bioinformatics predicted that TMEM95-SV1 has a leucine-rich repeat C-terminal domain and a Pfam: IZUMO. These regions are closely related to protein interactions and the acrosome reaction, respectively. Interestingly, the two transcripts were exclusively expressed in the testes and brain in male fetus cattle, and TMEM95-SV1 was expressed in the brain at significantly higher levels than in the testis (P<0.05, 4.06-fold) and TMEM95-SV2 in the brain (P<0.05, 4.95-fold). These findings enrich the understanding of the TMEM95 gene function and benefit for enhancing male reproduction in cattle industry. PMID:26385321

  16. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  17. Alternative non-coding splice variants of Nespas, an imprinted gene antisense to Nesp in the Gnas imprinting cluster.

    PubMed

    Williamson, Christine M; Skinner, Judith A; Kelsey, Gavin; Peters, Josephine

    2002-02-01

    The Gnas locus on mouse Chr 2 represents a unique cluster of overlapping imprinted genes. Three of these in the order Nesp--Gnasxl--Gnas are transcribed in the sense direction with Nesp having maternal-specific expression, Gnasxl having paternal expression, and Gnas as being biallelically expressed in most tissues. A fourth imprinted gene, Nespas, is paternally expressed, lies antisense to Nesp, and expresses an unspliced transcript. Large unspliced antisense transcripts are emerging as a feature of imprinted gene clusters, and such non-coding RNAs may have a cis-regulatory function. Here we show that, in addition to an unspliced form of Nepas, we can detect five alternatively spliced forms of Nespas up to 1.4 kb in length that are non-coding. The splice variants are paternally expressed; they start approximately 2 kb upstream of Gnasxl in a region of maternal methylation and end 2.5 kb beyond the ATG of Nesp. These variants do not correspond to exons of the human antisense transcript although they start in the same region; the Nespas transcript, like its human counterpart, is spliced in various alternative patterns. The identification of a set of small spliced imprinted transcripts in the human and now in the mouse suggests that these antisense transcripts are functionally important. PMID:11889554

  18. Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

    PubMed Central

    Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

    2015-01-01

    Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975

  19. Alternatively Spliced Genes as Biomarkers for Schizophrenia, Bipolar Disorder and Psychosis: A Blood-Based Spliceome-Profiling Exploratory Study

    PubMed Central

    Glatt, S.J.; Chandler, S.D.; Bousman, C.A.; Chana, G.; Lucero, G.R.; Tatro, E.; May, T.; Lohr, J.B.; Kremen, W.S.; Everall, I.P.; Tsuang, M.T.

    2010-01-01

    Objective Transcriptomic biomarkers of psychiatric diseases obtained from a query of peripheral tissues that are clinically accessible (e.g., blood cells instead of post-mortem brain tissue) have substantial practical appeal to discern the molecular subtypes of common complex diseases such as major psychosis. To this end, spliceome-profiling is a new methodological approach that has considerable conceptual relevance for discovery and clinical translation of novel biomarkers for psychiatric illnesses. Advances in microarray technology now allow for improved sensitivity in measuring the transcriptome while simultaneously querying the exome (all exons) and spliceome (all alternatively spliced variants). The present study aimed to evaluate the feasibility of spliceome-profiling to discern transcriptomic biomarkers of psychosis. Methods We measured exome and spliceome expression in peripheral blood mononuclear cells from 13 schizophrenia patients, nine bipolar disorder patients, and eight healthy control subjects. Each diagnostic group was compared to each other, and the combined group of bipolar disorder and schizophrenia patients was also compared to the control group. Furthermore, we compared subjects with a history of psychosis to subjects without such history. Results After applying Bonferroni corrections for the 21,866 full-length gene transcripts analyzed, we found significant interactions between diagnostic group and exon identity, consistent with group differences in rates or types of alternative splicing. Relative to the control group, 18 genes in the bipolar disorder group, eight genes in the schizophrenia group, and 15 genes in the combined bipolar disorder and schizophrenia group appeared differentially spliced. Importantly, thirty-three genes showed differential splicing patterns between the bipolar disorder and schizophrenia groups. More frequent exon inclusion and/or over-expression was observed in psychosis. Finally, these observations are reconciled with an analysis of the ontologies, the pathways and the protein domains significantly over-represented among the alternatively spliced genes, several of which support prior discoveries. Conclusions To our knowledge, this is the first blood-based spliceome-profiling study of schizophrenia and bipolar disorder to be reported. The battery of alternatively spliced genes and exons identified in this discovery-oriented exploratory study, if replicated, may have potential utility to discern the molecular subtypes of psychosis. Spliceome-profiling, as a new methodological approach in transcriptomics, warrants further work to evaluate its utility in personalized medicine. Potentially, this approach could also permit the future development of tissue-sampling methodologies in a form that is more acceptable to patients and thereby allow monitoring of dynamic and time-dependent plasticity in disease severity and response to therapeutic interventions in clinical psychiatry. PMID:21532980

  20. Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

    PubMed Central

    Jiménez-López, Claudia; Lorenz, Michael C.; van Hoof, Ambro

    2013-01-01

    Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes. PMID:23516382

  1. Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible

    PubMed Central

    Lareau, Liana F.; Brenner, Steven E.

    2015-01-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

  2. Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible.

    PubMed

    Lareau, Liana F; Brenner, Steven E

    2015-04-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

  3. Genomics of alternative splicing: evolution, development and pathophysiology.

    PubMed

    Gamazon, Eric R; Stranger, Barbara E

    2014-06-01

    Alternative splicing is a major cellular mechanism in metazoans for generating proteomic diversity. A large proportion of protein-coding genes in multicellular organisms undergo alternative splicing, and in humans, it has been estimated that nearly 90 % of protein-coding genes-much larger than expected-are subject to alternative splicing. Genomic analyses of alternative splicing have illuminated its universal role in shaping the evolution of genomes, in the control of developmental processes, and in the dynamic regulation of the transcriptome to influence phenotype. Disruption of the splicing machinery has been found to drive pathophysiology, and indeed reprogramming of aberrant splicing can provide novel approaches to the development of molecular therapy. This review focuses on the recent progress in our understanding of alternative splicing brought about by the unprecedented explosive growth of genomic data and highlights the relevance of human splicing variation on disease and therapy. PMID:24378600

  4. TUMOR-SPECIFIC EXPRESSION AND ALTERNATIVE SPLICING OF THE COL6A3 GENE IN PANCREATIC CANCER

    PubMed Central

    Arafat, Hwyda; Lazar, Melissa; Salem, Khalifa; Chipitsyna, Galina; Gong, Qiaoke; Pan, Te-Cheng; Zhang, Rui-Zhu; Yeo, Charles J.; Chu, Mon-Li

    2011-01-01

    Introduction Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. Conclusions We describe, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events. PMID:21719059

  5. Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans

    PubMed Central

    Mondal, Ashis K.; Das, Swapan K.; Baldini, Giulia; Chu, Winston S.; Sharma, Neeraj K.; Hackney, Oksana G.; Zhao, Jianhua; Grant, Struan F. A.; Elbein, Steven C.

    2010-01-01

    Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5′ and 3′ untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3′ untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P < 0.01) and rs7903146 genotype (P < 0.026) in sc adipose. Insulin (5–10 nm) suppressed all TCF7L2 isoforms in SGBS cells but suppressed exon 13a-containing isoforms most significantly (P < 0.001). The isoform distribution differed throughout SGBS cell differentiation. Isoforms with predicted early stop codons yielded stable proteins of the predicted size, bound β-catenin, and targeted correctly to the nucleus. Conclusions: Intronic TCF7L2 variants may regulate alternative transcript isoforms, which in turn may have distinct physiologic roles. PMID:20097709

  6. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine

    PubMed Central

    Sheng, Zhen; Sun, Yi; Zhu, Ruixin; Jiao, Na; Tang, Kailin; Cao, Zhiwei; Ma, Chao

    2015-01-01

    Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs. PMID:26606055

  7. Alternate promoters and alternate splicing of human tenascin-X, a gene with 5' and 3' ends buried in other genes.

    PubMed

    Speek, M; Barry, F; Miller, W L

    1996-11-01

    Tenascin-X (TN-X) is an extracellular matrix protein encoded by a large gene that overlaps the steroid 21-hydroxylase (P450c21) gene in the HLA locus on chromosome 6p21.3. This may be the most complex locus in the human genome identified to date, containing 13 overlapping transcription units in 160 kb of DNA. Previous studies determined the sequence of 39 TN-X exons, encoding a 12 kb open reading frame, but the promoter(s) of the gene had not been located. We identify the principal TN-X promoter and a previously unknown 5' untranslated exon that lies more than 10 kb upstream from the previously known exons. This promoter, which is substantially different from the promoter for TN-C, initiates transcription in human fetal adrenal and muscle, but expression in human NCI-H295 adrenocortical carcinoma cells is initiated by two other promoters lying further upstream. One of these is the same as the promoter for a recently identified Creb-related protein (Creb-rp), but transcripts initiated form this promoter in human adrenal NCI-H295 tumor cells are spliced differently from Creb-rp, and are largely retained in the nuclei of these cells. By analogy with the other two members of the tenascin family, TN-C and TN-R, it has been predicted that TN-X should undergo alternate splicing in its fibronectin-like domains. RACE cloning and RNase protection experiments reveal no such alternate splicing. The TN-X gene appears to be unique in having both its 5' and 3' ends buried in other genes. PMID:8923003

  8. Alternative splicing and mRNA expression analysis of bovine SLAMF7 gene in healthy and mastitis mammary tissues.

    PubMed

    Ju, Zhihua; Wang, Changfa; Li, Qiuling; Hou, Minghai; Gao, Shuai; Hou, Qinlei; Li, Jianbin; Huang, Jinming; Zhong, Jifeng

    2012-04-01

    The signaling lymphocyte-activating molecule family 7 (SLAMF7) proteins serve as adhesion molecules on the surface of a variety of mature hematopoietic cells, and also partially control certain innate and adaptive immune responses. We characterized three novel bovine SLAMF7 splice variants, designated as SLAMF7-AS1, AS2, and AS3. All three novel SLAMF7 isoforms are derived from the complete transcripts (SLAMF7-complete) via alternative splicing (AS). The patterns of the three splice variants are exon skipping and alternative 5' splice sites. Bovine SLAMF7 transcripts are expressed in mammary tissue, as demonstrated by real-time PCR. The levels of the complete transcript expression in the normal mammary tissues were higher than that in Staphylococcus aureus (Staph. aureus)-induced mastitis mammary tissues. However, it was not significant for the mRNA expression level comparison between these two kinds of mammary. The SLAMF-AS2 isoforms are expressed the lowest levels among the three transcripts in both normal and infected mammary tissues. This study provides clues for a better understanding of bovine SLAMF7 gene function. PMID:21769477

  9. Alternative splicing at GYNNGY 5? splice sites: more noise, less regulation

    PubMed Central

    Wang, Meng; Zhang, Peiwei; Shu, Yang; Yuan, Fei; Zhang, Yuchao; Zhou, You; Jiang, Min; Zhu, Yufei; Hu, Landian; Kong, Xiangyin; Zhang, Zhenguo

    2014-01-01

    Numerous eukaryotic genes are alternatively spliced. Recently, deep transcriptome sequencing has skyrocketed proportion of alternatively spliced genes; over 95% human multi-exon genes are alternatively spliced. One fundamental question is: are all these alternative splicing (AS) events functional? To look into this issue, we studied the most common form of alternative 5? splice sitesGYNNGYs (Y = C/T), where both GYs can function as splice sites. Global analyses suggest that splicing noise (due to stochasticity of splicing process) can cause AS at GYNNGYs, evidenced by higher AS frequency in non-coding than in coding regions, in non-conserved than in conserved genes and in lowly expressed than in highly expressed genes. However, ?20% AS GYNNGYs in humans and ?3% in mice exhibit tissue-dependent regulation. Consistent with being functional, regulated GYNNGYs are more conserved than unregulated ones. And regulated GYNNGYs have distinctive sequence features which may confer regulation. Particularly, each regulated GYNNGY comprises two splice sites more resembling each other than unregulated GYNNGYs, and has more conserved downstream flanking intron. Intriguingly, most regulated GYNNGYs may tune gene expression through coupling with nonsense-mediated mRNA decay, rather than encode different proteins. In summary, AS at GYNNGY 5? splice sites is primarily splicing noise, and secondarily a way of regulation. PMID:25428370

  10. Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

    PubMed Central

    Saravanaperumal, Siva Arumugam; Pediconi, Dario; Renieri, Carlo; La Terza, Antonietta

    2012-01-01

    Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals. PMID:22719917

  11. Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

    PubMed

    Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

    2016-02-01

    Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms. PMID:26194054

  12. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. PMID:25720307

  13. RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts

    PubMed Central

    Sorber, Katherine; Dimon, Michelle T.; DeRisi, Joseph L.

    2011-01-01

    Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5? GU-AG 3? and 5 new 5? GC-AG 3? junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasites transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasites transcriptome. PMID:21245033

  14. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing.

    PubMed Central

    Martin, J F; Miano, J M; Hustad, C M; Copeland, N G; Jenkins, N A; Olson, E N

    1994-01-01

    Members of the myocyte-specific enhancer-binding factor 2 (MEF2) family of transcription factors bind a conserved A/T-rich sequence in the control regions of numerous muscle-specific genes. Mammalian MEF2 proteins have been shown previously to be encoded by three genes, Mef2, xMef2, and Mef2c, each of which gives rise to multiple alternatively spliced transcripts. We describe the cloning of a new member of the MEF2 family from mice, termed MEF2D, which shares extensive homology with other MEF2 proteins but is the product of a separate gene. MEF2D binds to and activates transcription through the MEF2 site and forms heterodimers with other members of the MEF2 family. Deletion mutations show that the carboxyl terminus of MEF2D is required for efficient transactivation. MEF2D transcripts are widely expressed, but alternative splicing of MEF2D transcripts gives rise to a muscle-specific isoform which is induced during myoblast differentiation. The mouse Mef2, Mef2c, and Mef2d genes map to chromosomes 7, 13, and 3, respectively. The complexity of the MEF2 family of regulatory proteins provides the potential for fine-tuning of transcriptional responses as a consequence of combinatorial interactions among multiple MEF2 isoforms encoded by the four Mef2 genes. Images PMID:8114702

  15. RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells

    PubMed Central

    Akin, Debra; Newman, Jeremy R.B.; McIntyre, Lauren M.

    2016-01-01

    Purpose The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. Methods Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. Results Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. Conclusions These data suggest that lowering of PNN levels in epithelial cells results in dramatic transformation in the number and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce. Many of the genes affected by PNN knockdown are known to affect the epithelial phenotype. This window into the complexity of RNA splicing in the corneal epithelium implies that PNN exerts broad influence over the regulation and maintenance of the epithelial cell phenotype. PMID:26900324

  16. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  17. RNA splicing and genes

    SciTech Connect

    Sharp, P.A.

    1988-11-25

    The splicing of long transcripts RNA (copied from DNA in the cell nucleus) into smaller specific mRNA is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing 5' and 3' splice sites. This complex contains the evolutionary highly conserved small nuclear RNAs (snRNAs) Us, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRANs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

  18. Differential Expressions of the Alternatively Spliced Variant mRNAs of the Opioid Receptor Gene, OPRM1, in Brain Regions of Four Inbred Mouse Strains

    PubMed Central

    Xu, Jin; Lu, Zhigang; Xu, Mingming; Rossi, Grace C.; Kest, Benjamin; Waxman, Amanda R.; Pasternak, Gavril W.; Pan, Ying-Xian

    2014-01-01

    The opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains. PMID:25343478

  19. Human Pot1 (Protection of Telomeres) Protein: Cytolocalization, Gene Structure, and Alternative Splicing

    PubMed Central

    Baumann, Peter; Podell, Elaine; Cech, Thomas R.

    2002-01-01

    Fission yeast Pot1 (protection of telomeres) is a single-stranded telomeric DNA binding protein with a critical role in ensuring chromosome stability. A putative human homolog (hPot1) was previously identified, based on moderate sequence similarity with fission yeast Pot1 and telomere end-binding proteins from ciliated protozoa. Using indirect immunofluorescence, we show here that epitope-tagged hPot1 localizes to telomeres in interphase nuclei of human cells, consistent with a direct role in telomere end protection. The hPOT1 gene contains 22 exons, most of which are present in all cDNAs examined. However, four exons are subject to exon skipping in some transcripts, giving rise to five splice variants. Four of these are ubiquitously expressed, whereas the fifth appears to be specific to leukocytes. The resultant proteins vary significantly in their ability to form complexes with single-stranded telomeric DNA as judged by electrophoretic mobility shift assays. In addition to these splice variants, the Pot1 family is expanded by the identification of six more genes from diverse species. Pot1-like proteins have now been found in plants, animals, yeasts, and microsporidia. PMID:12391173

  20. The evolution of novelty in conserved genes; evidence of positive selection in the Drosophila fruitless gene is localised to alternatively spliced exons

    PubMed Central

    Parker, D J; Gardiner, A; Neville, M C; Ritchie, M G; Goodwin, S F

    2014-01-01

    There has been much debate concerning whether cis-regulatory or coding changes are more likely to produce evolutionary innovation or adaptation in gene function, but an additional complication is that some genes can dramatically diverge through alternative splicing, increasing the diversity of gene function within a locus. The fruitless gene is a major transcription factor with a wide range of pleiotropic functions, including a fundamental conserved role in sexual differentiation, species-specific morphology and an important influence on male sexual behaviour. Here, we examine the structure of fruitless in multiple species of Drosophila, and determine the patterns of selective constraint acting across the coding region. We found that the pattern of selection, estimated from the ratio of non-synonymous to synonymous substitutions, varied considerably across the gene, with most regions of the gene evolutionarily conserved but with several regions showing evidence of divergence as a result of positive selection. The regions that showed evidence of positive selection were found to be localised to relatively consistent regions across multiple speciation events, and are associated with alternative splicing. Alternative splicing may thus provide a route to gene diversification in key regulatory loci. PMID:24149653

  1. The human LEF-1 gene contains a promoter preferentially active in lymphocytes and encodes multiple isoforms derived from alternative splicing

    PubMed Central

    Hovanes, K.; Li, T. W. H.; Waterman, M. L.

    2000-01-01

    Lymphoid Enhancer Factor-1 (LEF-1) is a member of a family of transcription factors that function as downstream mediators of the Wnt signal transduction pathway. In the absence of Wnt signals, specific LEF/TCF isoforms repress rather than activate gene targets through recruitment of the co-repressor CtBP. Characterization of the full-length human LEF-1 gene locus and its complete set of mRNA products shows that this family member exists as a unique set of alternatively spliced isoforms; none are homologous to TCF-1E/TCF-4E. Therefore LEF-1 is distinct from its TCF family members in that it cannot engage in activities specific to this isoform such as recruitment of the co-repressor CtBP. Expression of alternatively spliced LEF-1 isoforms are driven by a promoter that is highly active in lymphocyte cell lines. Transcription initiates within a TATA-less core promoter region that contains consensus binding sites for Sp1, an E box, an Initiator element and a LEF/TCF binding site, all juxtaposed to the start sites of transcription. The promoter is most active in a B lymphocyte cell line (Raji) in which the endogenous LEF-1 gene is silent, suggesting that the promoter region is actively repressed by a silencing mechanism. PMID:10756202

  2. New Modularity of DAP-Kinases: Alternative Splicing of the DRP-1 Gene Produces a ZIPk-Like Isoform

    PubMed Central

    Shoval, Yishay; Berissi, Hanna; Kimchi, Adi; Pietrokovski, Shmuel

    2011-01-01

    DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase (DAP-kinase) family, which function in different settings of cell death including autophagy. DAP kinases are very similar in their catalytic domains but differ substantially in their extra-catalytic domains. This difference is crucial for the significantly different modes of regulation and function among DAP kinases. Here we report the identification of a novel alternatively spliced kinase isoform of the DRP-1 gene, termed DRP-1?. The alternative splicing event replaces the whole extra catalytic domain of DRP-1 with a single coding exon that is closely related to the sequence of the extra catalytic domain of ZIPk. As a consequence, DRP-1? lacks the calmodulin regulatory domain of DRP-1, and instead contains a leucine zipper-like motif similar to the protein binding region of ZIPk. Several functional assays proved that this new isoform retained the biochemical and cellular properties that are common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and activation of membrane blebbing and autophagy. In addition, DRP-1? also acquired binding to the ATF4 transcription factor, a feature characteristic of ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like isoform. DRP-1? is highly conserved in evolution, present in all known vertebrate DRP-1 loci. We detected the corresponding mRNA and protein in embryonic mouse brains and in human embryonic stem cells thus confirming the in vivo utilization of this isoform. The discovery of module conservation within the DAPk family members illustrates a parsimonious way to increase the functional complexity within protein families. It also provides crucial data for modeling the expansion and evolution of DAP kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely evolved from their ancient ancestor gene DAPk by two gene duplication events that occurred close to the emergence of vertebrates. PMID:21408167

  3. Misregulation of pre-mRNA alternative splicing in cancer

    PubMed Central

    Zhang, Jian; Manley, James L.

    2013-01-01

    Alternative splicing of mRNA precursors enables one gene to produce multiple protein isoforms with differing functions. Under normal conditions, this mechanism is tightly regulated in order for the human genome to generate proteomic diversity sufficient for the functional requirements of complex tissues. When deregulated, however, cancer cells take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Here we discuss aspects of alternative splicing misregulation in cancer, focusing on splicing events affected by deregulation of regulatory splicing factors and also recent studies identifying mutated components of the splicing machinery. PMID:24145039

  4. Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells.

    PubMed

    Ergin, Volkan; Erdogan, Mutlu; Menevse, Adnan

    2015-01-01

    Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis. PMID:26648138

  5. Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells

    PubMed Central

    Ergin, Volkan; Erdogan, Mutlu; Menevse, Adnan

    2015-01-01

    Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis. PMID:26648138

  6. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  7. The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer

    PubMed Central

    2010-01-01

    Background Dietary or therapeutic interventions to counteract the loss of PTEN expression could contribute to the prevention of prostate carcinogenesis or reduce the rate of cancer progression. In this study, we investigate the interaction between sulforaphane, a dietary isothiocyanate derived from broccoli, PTEN expression and gene expression in pre malignant prostate tissue. Results We initially describe heterogeneity in expression of PTEN in non-malignant prostate tissue of men deemed to be at risk of prostate cancer. We subsequently use the mouse prostate-specific PTEN deletion model, to show that sulforaphane suppresses transcriptional changes induced by PTEN deletion and induces additional changes in gene expression associated with cell cycle arrest and apoptosis in PTEN null tissue, but has no effect on transcription in wild type tissue. Comparative analyses of changes in gene expression in mouse and human prostate tissue indicate that similar changes can be induced in humans with a broccoli-rich diet. Global analyses of exon expression demonstrated that sulforaphane interacts with PTEN deletion to modulate alternative gene splicing, illustrated through a more detailed analysis of DMBT1 splicing. Conclusion To our knowledge, this is the first report of how diet may perturb changes in transcription induced by PTEN deletion, and the effects of diet on global patterns of alternative gene splicing. The study exemplifies the complex interaction between diet, genotype and gene expression, and the multiple modes of action of small bioactive dietary components. PMID:20626841

  8. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  9. An alternatively spliced domain of the NDC1 NAD(P)H dehydrogenase gene strongly influences the expression of the ACTIN2 reference gene in Arabidopsis thaliana.

    PubMed

    Wallstrm, Sab V; Aidemark, Mari; Escobar, Matthew A; Rasmusson, Allan G

    2012-02-01

    In plant respiratory chains, alternative pathways for NAD(P)H oxidation are mediated by type II NAD(P)H dehydrogenases belonging to the NDA, NDB, and NDC families. For the latter type, Arabidopsis thaliana contains a single gene, NDC1, whose functional role has not previously been analyzed in the plant. We found that A. thaliana NDC1 is alternatively spliced. Four base pairs at the 3' end of intron 5 are spliced out in NDC1-1, but retained in the NDC1-2 mRNA, which therefore contains a truncated reading frame. Both variants are conserved in dicotyledonous and monocotyledonous plants and their relative abundance varies between organs and in response to light. Three analyzed NDC1 T-DNA insertion lines all displayed an early bolting phenotype. A dramatic upregulation of ACTIN2 was characteristic of two lines containing T-DNA inserts upstream of intron 5, whereas a line with an insertion downstream of the NDC1-2 reading frame had an ACTIN2 expression level identical to the wildtype. Thus, the alternatively spliced 5' domain of NDC1 strongly influences the expression of the functionally unrelated ACTIN2, which is a common reference gene for quantitative RT-PCR. Also for other reference genes, strong expressional effects were observed when comparing various mutants and wildtypes in microarray databases. PMID:22195593

  10. Efficient prediction of alternative splice forms using protein domain homology.

    PubMed

    Hiller, Michael; Backofen, Rolf; Heymann, Stephan; Busch, Anke; Glaesser, Timo Mika; Freytag, Johann-Christoph

    2004-01-01

    Alternative splicing can yield manifold different mature mRNAs from one precursor. New findings indicate that alternative splicing occurs much more often than previously assumed. A major goal of functional genomics lies in elucidating and characterizing the entire spectrum of alternative splice forms. Existing approaches such as EST-alignments focus only on the mRNA sequence to detect alternative splice forms. They do not consider function and characteristics of the resulting proteins. One important example of such functional characterization is homology to a known protein domain family. A powerful description of protein domains are profile Hidden Markov models (HMM) as stored in the Pfam database. In this paper we address the problem of identifying the splice form with the highest similarity to a protein domain family. Therefore, we take into consideration all possible splice forms. As demonstrated here for a number of genes, this homology based approach can be used successfully for predicting partial gene structures. Furthermore, we present some novel splice form predictions with high-scoring protein domain homology and point out that the detection of splice form specific protein domains helps to answer questions concerning hereditary diseases. Simple approaches based on a BLASTP search cannot be applied here, since the number of possible splice forms increases exponentially with the number of exons. To this end, we have developed an efficient polynomial-time algorithm, called ASFPred (Alternative Splice Form Prediction). This algorithm needs only a set of exons as input. PMID:15107023

  11. Global analysis of alternative splicing differences between humans and chimpanzees

    PubMed Central

    Calarco, John A.; Xing, Yi; Cceres, Mario; Calarco, Joseph P.; Xiao, Xinshu; Pan, Qun; Lee, Christopher; Preuss, Todd M.; Blencowe, Benjamin J.

    2007-01-01

    Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees. Surprisingly, 6%8% of profiled orthologous exons display pronounced splicing level differences in the corresponding tissues from the two species. Little overlap is observed between the genes associated with alternative splicing differences and the genes that display steady-state transcript level differences, indicating that these layers of regulation have evolved rapidly to affect distinct subsets of genes in humans and chimpanzees. The alternative splicing differences we detected are predicted to affect diverse functions including gene expression, signal transduction, cell death, immune defense, and susceptibility to diseases. Differences in expression at the protein level of the major splice variant of Glutathione S-transferase omega-2 (GSTO2), which functions in the protection against oxidative stress and is associated with human aging-related diseases, suggests that this enzyme is less active in human cells compared with chimpanzee cells. The results of this study thus support an important role for alternative splicing in establishing differences between humans and chimpanzees. PMID:17978102

  12. Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

    PubMed Central

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R.; Pérez, Belén; Alves, Sandra

    2015-01-01

    This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in “Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II” Matos et al. (2015) [1]. PMID:26693516

  13. Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonalves, Vnia; Pinto, Eugnia; Laranjeira, Francisco; Prata, Maria Joo; Jordan, Peter; Desviat, Lourdes R; Prez, Beln; Alves, Sandra

    2015-12-01

    This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1]. PMID:26693516

  14. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae

    PubMed Central

    Schreiber, Konrad; Csaba, Gergely; Haslbeck, Martin; Zimmer, Ralf

    2015-01-01

    mRNA splicing is required in about 4% of protein coding genes in Saccharomyces cerevisiae. The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3 side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. PMID:26469855

  15. Platelet-derived growth factor A chain: gene structure, chromosomal location, and basis for alternative mRNA splicing.

    PubMed

    Bonthron, D T; Morton, C C; Orkin, S H; Collins, T

    1988-03-01

    Genomic clones encoding the A chain of platelet-derived growth factor (PDGF) have been isolated. The gene contains seven exons spanning about 24 kilobases of DNA. The positions of intervening sequences closely match those of the related B-chain (c-sis) gene on chromosome 22. In situ hybridization was used to localize the PDGF A-chain gene to the distal portion of the short arm of chromosome 7 (7p21-p22). Within the (G + C)-rich 5' region, a single transcriptional start site was identified approximately equal to 36 base pairs downstream of a TATAA consensus promoter element. The three size classes of A-chain mRNA probably arise by selection of alternative poly(A) sites in exon 7, but only a single consensus AATAAA signal was identified in this region. Two functionally different A-chain precursors, which differ by the presence or absence of a basic C terminus, are generated as a result of alternative mRNA splicing events, which include or exclude exon 6. This and other structural features of the A-chain gene suggest that PDGF expression may be modulated at transcriptional and post-transcriptional levels. PMID:3422746

  16. Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

    PubMed Central

    Read, D; Manley, J L

    1992-01-01

    A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms. Images PMID:1372245

  17. Transcriptome profiling and sequencing of differentiated human hematopoietic stem cells reveal lineage-specific expression and alternative splicing of genes

    PubMed Central

    Liu, Poching; Barb, Jennifer; Woodhouse, Kimberly; Taylor, James G.; Munson, Peter J.

    2011-01-01

    Hematopoietic differentiation is strictly regulated by complex network of transcription factors that are controlled by ligands binding to cell surface receptors. Disruptions of the intricate sequences of transcriptional activation and suppression of multiple genes cause hematological diseases, such as leukemias, myelodysplastic syndromes, or myeloproliferative syndromes. From a clinical standpoint, deciphering the pattern of gene expression during hematopoiesis may help unravel disease-specific mechanisms in hematopoietic malignancies. Herein, we describe a human in vitro hematopoietic model system where lineage-specific differentiation of CD34+ cells was accomplished using specific cytokines. Microarray and RNAseq-based whole transcriptome and exome analysis was performed on the differentiated erythropoietic, granulopoietic, and megakaryopoietic cells to delineate changes in expression of whole transcripts and exons. Analysis on the Human 1.0 ST exon arrays indicated differential expression of 172 genes (P < 0.0000001) and significant alternate splicing of 86 genes during differentiation. Pathway analysis identified these genes to be involved in Rac/RhoA signaling, Wnt/B-catenin signaling and alanine/aspartate metabolism. Comparison of the microarray data to next generation RNAseq analysis during erythroid differentiation demonstrated a high degree of correlation in gene (R = 0.72) and exon (R = 0.62) expression. Our data provide a molecular portrait of events that regulate differentiation of hematopoietic cells. Knowledge of molecular processes by which the cells acquire their cell-specific fate would be beneficial in developing cell-based therapies for human diseases. PMID:21828245

  18. RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays

    PubMed Central

    Seok, Junhee; Xu, Weihong; Davis, Ronald W.; Xiao, Wenzhong

    2015-01-01

    Human transcriptome arrays (HTA) have recently been developed for high-throughput alternative splicing analysis by measuring signals not only from exons but also from exon-exon junctions. Effective use of these rich signals requires the development of computational methods for better gene and alternative splicing analyses. In this work, we introduce a computational method, Robust Alternative Splicing Analysis (RASA), for the analysis of the new transcriptome arrays by effective integration of the exon and junction signals. To increase robustness, RASA calculates the expression of each gene by selecting exons classified as not alternatively spliced. It then identifies alternatively spliced exons that are supported by both exon and junction signals to reduce the false positives. Finally, it detects additional alternative splicing candidates that are supported by only exon signals because the signals from the corresponding junctions are not well detected. RASA was demonstrated with Affymetrix HTAs and its performance was evaluated with mRNA-Seq and RT-PCR. The validation rate is 52.4%, which is a 60% increase when compared with previous methods that do not use selected exons for gene expression calculation and junction signals for splicing detection. These results suggest that RASA significantly improves alternative splicing analyses on HTA platforms. PMID:26145443

  19. RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays.

    PubMed

    Seok, Junhee; Xu, Weihong; Davis, Ronald W; Xiao, Wenzhong

    2015-01-01

    Human transcriptome arrays (HTA) have recently been developed for high-throughput alternative splicing analysis by measuring signals not only from exons but also from exon-exon junctions. Effective use of these rich signals requires the development of computational methods for better gene and alternative splicing analyses. In this work, we introduce a computational method, Robust Alternative Splicing Analysis (RASA), for the analysis of the new transcriptome arrays by effective integration of the exon and junction signals. To increase robustness, RASA calculates the expression of each gene by selecting exons classified as not alternatively spliced. It then identifies alternatively spliced exons that are supported by both exon and junction signals to reduce the false positives. Finally, it detects additional alternative splicing candidates that are supported by only exon signals because the signals from the corresponding junctions are not well detected. RASA was demonstrated with Affymetrix HTAs and its performance was evaluated with mRNA-Seq and RT-PCR. The validation rate is 52.4%, which is a 60% increase when compared with previous methods that do not use selected exons for gene expression calculation and junction signals for splicing detection. These results suggest that RASA significantly improves alternative splicing analyses on HTA platforms. PMID:26145443

  20. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicols; Giono, Luciana E; Cambindo Botto, Adrin E; Muoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing. PMID:26296319

  1. Should pharmacologists care about alternative splicing? IUPHAR Review 4

    PubMed Central

    Bonner, T I

    2014-01-01

    Alternative splicing of mRNAs occurs in the majority of human genes, and most differential splicing results in different protein isoforms with possibly different functional properties. However, there are many reported splicing variations that may be quite rare, and not all combinatorially possible variants of a given gene are expressed at significant levels. Genes of interest to pharmacologists are frequently expressed at such low levels that they are not adequately represented in genome-wide studies of transcription. In single-gene studies, data are commonly available on the relative abundance and functional significance of individual alternatively spliced exons, but there are rarely data that quantitate the relative abundance of full-length transcripts and define which combinations of exons are significant. A number of criteria for judging the significance of splice variants and suggestions for their nomenclature are discussed. PMID:24670145

  2. Genome-wide profiling of alternative splicing in Alzheimer's disease.

    PubMed

    Lai, Mitchell K P; Esiri, Margaret M; Tan, Michelle G K

    2014-12-01

    Alternative splicing is a highly regulated process which generates transcriptome and proteome diversity through the skipping or inclusion of exons within gene loci. Identification of aberrant alternative splicing associated with human diseases has become feasible with the development of new genomic technologies and powerful bioinformatics. We have previously reported genome-wide gene alterations in the neocortex of a well-characterized cohort of Alzheimer's disease (AD) patients and matched elderly controls using a commercial exon microarray platform [1]. Here, we provide detailed description of analyses aimed at identifying differential alternative splicing events associated with AD. PMID:26484111

  3. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  4. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications

    PubMed Central

    Niklas, Karl J.; Bondos, Sarah E.; Dunker, A. Keith; Newman, Stuart A.

    2015-01-01

    Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences. PMID:25767796

  5. Alternative splicing generates secretory isoforms of human CD1.

    PubMed Central

    Woolfson, A; Milstein, C

    1994-01-01

    Human CD1 genes are a family of five non-polymorphic genes that, although homologous to both class I and II major histocompatibility complex genes, map to chromosome 1. Only three of the antigens, CD1a, -b, and -c, have been clustered with monoclonal antibodies. They are noncovalently associated with beta 2-microglobulin and may function as nonclassical antigen-presenting molecules. Here we analyze their expression in mouse myeloma transfectants and human thymocytes and find mRNA splicing complexity. This manifests itself as incomplete splicing, alternative splicing, utilization of cryptic splice sites, and the generation of alternative reading frames. In the case of CD1A transfectants, we demonstrate that the major protein product is secreted and show by amino acid sequence analysis that this is derived from an unspliced transcript. A second major CD1a component appears to be retained intracellularly. The production of alternatively spliced transcripts in the thymus is not a feature of all CD1 genes. Although in the case of CD1A only the transcript encoding the cell surface CD1a isoform is found, CD1C and -E produce complex intrathymic splicing patterns. The CD1C transcripts predict the expression of a secreted CD1c isoform in the human thymus, which we detect in CD1C transfectant culture supernatants. CD1 gene expression is thus characterized by considerable splicing complexity, and the difference between the splicing patterns found in different environments suggests that this is tissue specific. Images PMID:7517559

  6. Genome-wide survey of Alternative Splicing in Sorghum Bicolor.

    PubMed

    Panahi, Bahman; Abbaszadeh, Bahram; Taghizadeghan, Mehdi; Ebrahimie, Esmaeil

    2014-07-01

    Sorghum bicolor is a member of grass family which is an attractive model plant for genome study due to interesting genome features like low genome size. In this research, we performed comprehensive investigation of Alternative Splicing and ontology aspects of genes those have undergone these events in sorghum bicolor. We used homology based alignments between gene rich transcripts, represented by tentative consensus (TC) transcript sequences, and genomic scaffolds to deduce the structure of genes and identify alternatively spliced transcripts in sorghum. Using homology mapping of assembled expressed sequence tags with genomics data, we identified 2,137 Alternative Splicing events in S. bicolor. Our study showed that complex events and intron retention are the main types of Alternative Splicing events in S. bicolor and highlights the prevalence of splicing site recognition for definition of introns in this plant. Annotations of the alternatively spliced genes revealed that they represent diverse biological process and molecular functions, suggesting a fundamental role for Alternative Splicing in affecting the development and physiology of S. bicolor. PMID:25049459

  7. The role of gene polymorphism in HLA class I splicing.

    PubMed

    Voorter, C E M; Gerritsen, K E H; Groeneweg, M; Wieten, L; Tilanus, M G J

    2016-04-01

    Among the large number of human leucocyte antigen (HLA) alleles, only a few have been identified with a nucleotide polymorphism impairing correct splicing. Those alleles show aberrant expression levels, due to either a direct effect of the polymorphism on the normal splice site or to the creation of an alternative splice site. Furthermore, in several studies, the presence of alternatively spliced HLA transcripts co-expressed with the mature spliced transcripts was reported. We evaluated the splice site sequences of all known HLA class I alleles and found that, beside the consensus GT and AG sequences at the intron borders, there were some other highly conserved nucleotides for the different class I genes. In this review, we summarize the splicing mechanism and evaluate what is known today about alternative splicing of HLA class I genes. PMID:26920492

  8. Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels

    PubMed Central

    Young, Janet M; Shykind, Benjamin M; Lane, Robert P; Tonnes-Priddy, Lori; Ross, Joseph A; Walker, Megan; Williams, Eleanor M; Trask, Barbara J

    2003-01-01

    Background The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions. Results We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity. Conclusions Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes. PMID:14611657

  9. Mechanisms and Regulation of Alternative Pre-mRNA Splicing

    PubMed Central

    Lee, Yeon

    2015-01-01

    Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

  10. Mechanisms and Regulation of Alternative Pre-mRNA Splicing.

    PubMed

    Lee, Yeon; Rio, Donald C

    2015-01-01

    Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

  11. A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing.

    PubMed

    Nishimura, Hitoshi; Fujimoto, Atsushi; Tamura, Naoyuki; Yajima, Toshiki; Wajjwalku, Worawidh; Yoshikai, Yasunobu

    2005-01-01

    There are several isoforms of interleukin (IL) -15 generated by alternating splicing. We reported previously that alternative IL-15 transgenic (Tg) mice expressing an IL-15 cDNA isoform encoding nonsecretable IL-15 protein had an impaired ability to produce IL-15. In this study, we found that expression of endogenous IL-15 mRNA but not tumor necrosis factor alpha mRNA was severely impaired in response to lipopolysaccharide, not only in macrophages from alternative IL-15 Tg mice but also in RAW264.7 cells that had been transfected with alternative IL-15 together with IL-15 receptor alpha (IL-15Ralpha). IL-15 promoter activity was suppressed in the transfected cells. Although nuclear factor-kappaB activation was not impaired, the binding activity of nuclear extracts to the interferon-stimulated response element of the IL-15 promoter region was reduced in RAW264.7 cells, which had been cotransfected with alternative IL-15 and IL-15Ralpha. IL-15 was mainly colocalized with IL-15Ralpha at the cytoplasmic membrane of RAW264.7 cells, which had been cotransfected with normal IL-15, whereas nonsecretable IL-15 was colocalized with IL-15Ralpha in nucleus after cotransfection with alternative IL-15 and IL-15Ralpha. These results suggest that nonsecretable IL-15 generated by alternative splicing suppresses further IL-15 gene transcription, implying a novel autocrine regulatory mechanism for cytokine gene expression by alternative splicing. PMID:15629891

  12. Evidence for the Possible Biological Significance of the igf-1 Gene Alternative Splicing in Prostate Cancer

    PubMed Central

    Philippou, Anastassios; Armakolas, Athanasios; Koutsilieris, Michael

    2013-01-01

    Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of prostate cancer (PCa), since it plays a key role in cell proliferation, differentiation, and apoptosis. The IGF-I actions are mediated mainly via its binding to the type I IGF receptor (IGF-IR), however IGF-I signaling via insulin receptor (IR) and hybrid IGF-I/IR is also evident. Different IGF-I mRNA splice variants, namely IGF-IEa, IGF-IEb, and IGF-IEc, are expressed in human cells and tissues. These transcripts encode several IGF-I precursor proteins which contain the same bioactive product (mature IGF-I), however, they differ by the length of their signal peptides on the amino-terminal end and the structure of the extension peptides (E-peptides) on the carboxy-terminal end. There is an increasing interest in the possible different role of the IGF-I transcripts and their respective non-(mature)IGF-I products in the regulation of distinct biological activities. Moreover, there is strong evidence of a differential expression profile of the IGF-I splice variants in normal versus PCa tissues and PCa cells, implying that the expression pattern of the various IGF-I transcripts and their respective protein products may possess different functions in cancer biology. Herein, the evidence that the IGF-IEc transcript regulates PCa growth via Ec peptide specific and IGF-IR/IR-independent signaling is discussed. PMID:23519101

  13. Multiple effects of digoxin on subsets of cancer-associated genes through the alternative splicing pathway.

    PubMed

    Lu, Guan-Yu; Liu, Shu-Ting; Huang, Shih-Ming; Chang, Yung-Lung; Lin, Wei-Shiang

    2014-11-01

    The signaling characteristics of Na(+)/K(+)-ATPase are distinct from its ion pumping activity. Cardiac glycosides modulate the Na(+)/K(+)-ATPase protein complex upon binding, activate downstream signaling pathways and increase [Ca(2+)]i. Recent studies demonstrate that the depletion of p53 and hypoxia-induced factor 1? proteins is caused by cardiac glycosides. However, the detailed mechanisms governing this process are not well known. In this study, we showed that the depletion of p53 proteins by digoxin involved not only inhibition of protein synthesis but also inhibition at the post-transcriptional level. Post-transcriptional regulation occurs via down-regulation of SRSF3, the primary splicing factor responsible for the switch from p53? to the p53? isoform. Digoxin also modulated G2/M arrest, DNA damage and apoptosis through the p53-dependent pathway in HeLa cells. In addition, digoxin was involved in epithelial-mesenchymal-transition progression via E-cadherin reduction and snail induction. Digoxin had similar effects to caffeine, another SRSF3-reduced agent, on the cell cycle profile and DNA damage of cells. Interestingly, combined digoxin and caffeine treatment blocked cell cycle progression and conferred resistance to cell death via snail induction. These findings demonstrate that down-regulation of splicing factor, such as SRSF3, to alter cell cycle progression, cell death and invasion is a potential target for the drug repositioning of cardiac glycosides. PMID:25193633

  14. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza.

    PubMed

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  15. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

    PubMed Central

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  16. Genome-wide mapping of alternative splicing in Arabidopsis thaliana

    PubMed Central

    Filichkin, Sergei A.; Priest, Henry D.; Givan, Scott A.; Shen, Rongkun; Bryant, Douglas W.; Fox, Samuel E.; Wong, Weng-Keen; Mockler, Todd C.

    2010-01-01

    Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ?42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC+ isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC+ isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC+ and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression. PMID:19858364

  17. Alternatively spliced transcripts of Pi-ta blast resistance gene in Oryza sativa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Pi-ta gene in rice (Oryza sativa L.) confers resistance to races of Magnaporthe oryzae containing its cognate avirulence gene AVR-Pita. Pi-ta is a single-copy gene belonging to the nucleotide-binding site leucine-rich repeat (NBS-LRR) class of plant resistance (R) genes. In the present study, w...

  18. Identification of novel alternative splice variants of the BCL2L12 gene in human cancer cells using next-generation sequencing methodology.

    PubMed

    Adamopoulos, Panagiotis G; Kontos, Christos K; Tsiakanikas, Panagiotis; Scorilas, Andreas

    2016-04-01

    The next-generation sequencing (NGS) technology has enabled genome-wide studies, providing massively parallel DNA sequencing. NGS applications constitute a revolution in molecular biology and genetics and have already paved new ways in cancer research. BCL2L12 is an apoptosis-related gene, previously cloned from members of our research group. Like most members of the BCL2 gene family, it is highly implicated in various types of cancer and hematological malignancies. In the present study, we used NGS to discover novel alternatively spliced variants of the apoptosis-related BCL2L12 gene in many human cancer cell lines, after 3'-RACE nested PCR. Extensive computational analysis uncovered new alternative splicing events and patterns, resulting in novel alternative transcripts of the BCL2L12 gene. PCR was then performed to validate NGS data and identify the derived novel transcripts of the BCL2L12 gene. Therefore, 50 novel BCL2L12 splice variants were discovered. Since BCL2L12 is involved in the apoptotic machinery, the quantification of distinct BCL2L12 transcripts in human samples may have clinical applications in different types of cancer. PMID:26797417

  19. Regulation of alternative splicing in obesity and weight loss

    PubMed Central

    Kaminska, Dorota; Pihlajamäki, Jussi

    2013-01-01

    Alternative splicing (AS) is a mechanism by which multiple mRNA transcripts are generated from a single gene. According to recent reports approximately 95–100% of human multi-exon genes undergo AS. This increases the amount of functionally different protein isoforms, and in some cases leads to metabolic diseases. Herein we provide a brief overview of the basic aspects of splicing regulation in obesity and insulin resistance with specific examples. In addition, we review our recent findings demonstrating that weight loss regulates AS of TCF7L2 gene in both liver and adipose tissue, and that this splicing associates with changes in fatty acid and glucose metabolism. Future studies using global analysis of transcript variants and splicing regulators are needed for exploring the association of AS with metabolic alterations in obesity and type 2 diabetes (T2D). Understanding of the molecular mechanisms behind the aberrantly spliced transcripts may also provide opportunities for new diagnostic approaches. PMID:23991360

  20. The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

    PubMed Central

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Aceto, Serena; Nimmo, Derric; Neira, Marco; Alphey, Luke; Polito, Lino C.; Saccone, Giuseppe

    2013-01-01

    In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control. PMID:23418412

  1. Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease.

    PubMed

    Cogn, M; Bakhshi, A; Korsmeyer, S J; Guglielmi, P

    1988-09-01

    The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells. PMID:3137264

  2. Aberrant Splicing of an Alternative Exon in the Drosophila Troponin-T Gene Affects Flight Muscle Development

    PubMed Central

    Nongthomba, Upendra; Ansari, Maqsood; Thimmaiya, Divesh; Stark, Meg; Sparrow, John

    2007-01-01

    During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up1 muscles lack normal sarcomeres and contain zebra bodies, a phenotypic feature of human nemaline myopathies. We show that the up1 mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins. PMID:17603127

  3. Human [delta]-aminolevulinate dehydratase (ALAD) gene: Structure and alternative splicing of the erythroid and housekeeping mRNAs

    SciTech Connect

    Kaya, A.H.; Plewinska, M.; Wong, D.M.; Desnick, R.J.; Wetmur, J.G. )

    1994-01-15

    Genomic clones containing ALAD, the second enzyme in the heme pathway, were isolated, and the entire sequence was determined in both orientations. The gene contained two alternative noncoding exons, 1A and 1B, and 1q coding exons, 2-12. Ten Alu-repetitive elements were within the gene, including an inverted repeat that may have resulted from gene conversion. The housekeeping transcript, which included exon 1A and not 1B, was identified in a human adult liver cDNA library, while an erythroid-specific transcript, which contained exon 1B and not 1A, was detected in a human K562 erythroleukemia cDNA library. The promoter region upstream of housekeeping exon 1A was GC-rich and contained three potential Sp1 elements and a CCAAT box. Further upstream, there were three potential GATA-1 binding sites and an AP1 site. The promoter region upstream of erythroid-specific exon 1B had several CACCC boxes and two potential GATA-1 binding sites. To assess the tissue-specific expression of exons 1A and 1B, HeLa and K562 cells were transduced with CAT constructs containing either exon 1A or 1B and their respective upstream promoter region. Two housekeeping CAT constructs, with 450 and 1400 bp upstream of exon 1A, were expressed at similar levels in HeLa cells, whereas the erythroid-specific construct, containing the entire 450-bp promoter region upstream of exon 1B, was not. In contrast, the housekeeping and erythroid constructs were both expressed in K562 cells. These findings demonstrate that the human ALAD gene contains two promoter regions that generate housekeeping and erythroid-specific transcripts by alternative splicing, analogous to the expression of the human hydroxymethylbilane synthase gene, which encodes the third enzyme of the heme biosynthetic pathway. The expression of housekeeping and erythroid-specific transcripts apparently evolved to ensure sufficient heme biosynthesis for the high-level tissue-specific production of hemoglobin required. 39 refs., 5 figs.

  4. Dual REST-dependence of L1CAM: from gene expression to alternative splicing governed by Nova2 in neural cells.

    PubMed

    Mikulak, Joanna; Negrini, Sara; Klajn, Andrijana; D'Alessandro, Rosalba; Mavilio, Domenico; Meldolesi, Jacopo

    2012-03-01

    L1 cell adhesion molecule (L1CAM), an adhesion/signaling protein encoded by a gene target of the transcription repressor RE-1-Silencing Transcription factor (REST), is expressed in two alternatively spliced isoforms. The full-length isoform, typical of low-REST neural cells, plays key roles in survival/migration, outgrowth/fasciculation/regeneration of axons, synaptic plasticity; the isoform missing two mini-exons, abundant in a few high-REST non-neural cells, maintains some effect on migration and proliferation. To investigate whether and how L1CAM alternative splicing depends on REST we used neural cell models expressing low or high levels of REST (PC12, SH-SY5Y, differentiated NT2/D1 and primary neurons transduced or not with REST). The short isoform was found to rise when the low-REST levels of neural cells were experimentally increased, while the full-length isoform increased in high-REST cells when the repressor tone was attenuated. These results were due to Nova2, a neural cell-specific splicing factor shown here to be repressed by REST. REST control of L1CAM occurs therefore by two mechanisms, transcription and alternative splicing. The splicing mechanism, affecting not only L1CAM but all Nova2 targets (∼7% of brain-specific splicing, including the mRNAs of other adhesion and synaptic proteins) is expected to be critical during development and important also for the structure and function of mature neural cells. PMID:22176577

  5. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remainsunknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms"). PMID:26871637

  6. Cross-kingdom patterns of alternative splicing and splice recognition

    PubMed Central

    McGuire, Abigail M; Pearson, Matthew D; Neafsey, Daniel E; Galagan, James E

    2008-01-01

    Background Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently identifies intron-exon boundaries, and cassette exons (CEs) are often caused by variable recognition of splice junctions by the exon definition (ED) mechanism. We have performed a comprehensive survey of alternative splicing across 42 eukaryotes to gain insight into how spliceosomal introns are recognized. Results All eukaryotes we studied exhibit RIs, which appear more frequently than previously thought. CEs are also present in all kingdoms and most of the organisms in our analysis. We observe that the ratio of CEs to RIs varies substantially among kingdoms, while the ratio of competing 3' acceptor and competing 5' donor sites remains nearly constant. In addition, we find the ratio of CEs to RIs in each organism correlates with the length of its introns. In all 14 fungi we examined, as well as in most of the 9 protists, RIs far outnumber CEs. This differs from the trend seen in 13 multicellular animals, where CEs occur much more frequently than RIs. The six plants we analyzed exhibit intermediate proportions of CEs and RIs. Conclusion Our results suggest that most extant eukaryotes are capable of recognizing splice sites via both ID and ED, although ED is most common in multicellular animals and ID predominates in fungi and most protists. PMID:18321378

  7. Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals.

    PubMed

    Zhang, Yong; Liu, Yun; Huang, Xigui; Liu, Xiaochun; Jiao, Baowei; Meng, Zining; Zhu, Pei; Li, Shuisheng; Lin, Haoran; Cheng, Christopher H K

    2008-12-01

    Two GPR39 transcripts, designated as sbGPR39-1a and sbGPR39-1b, were identified in black seabream (Acanthopagrus schlegeli). The deduced amino acid (aa) sequence of sbGPR39-1a contains 423 residues with seven putative transmembrane (TM) domains. On the other hand, sbGPR39-1b contains 284 aa residues with only five putative TM domains. Northern blot analysis confirmed the presence of two GPR39 transcripts in the seabream intestine, stomach, and liver. Apart from seabream, the presence of two GPR39 transcripts was also found to exist in a number of teleosts (zebrafish and pufferfish) and mammals (human and mouse). Analysis of the GPR39 gene structure in different species suggests that the two GPR39 transcripts are generated by alternative splicing. When the seabream receptors were expressed in cultured HEK293 cells, Zn(2)(+) could trigger sbGPR39-1a signaling through the serum response element pathway, but no such functionality could be detected for the sbGPR39-1b receptor. The two receptors were found to be differentially expressed in seabream tissues. sbGPR39-1a is predominantly expressed in the gastrointestinal tract. On the other hand, sbGPR39-1b is widely expressed in most central and peripheral tissues except muscle and ovary. The expression of sbGPR39-1a in the intestine and the expression of sbGPR39-1b in the hypothalamus were decreased significantly during food deprivation in seabream. On the contrary, the expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream. The reciprocal regulatory patterns of expression of these two genes suggest that both of them are involved in controlling the physiological response of the organism during starvation. PMID:18815158

  8. Functional characterization of alternatively spliced human SECISBP2 transcript variants.

    PubMed

    Papp, Laura V; Wang, Junning; Kennedy, Derek; Boucher, Didier; Zhang, Yan; Gladyshev, Vadim N; Singh, Ravindra N; Khanna, Kum Kum

    2008-12-01

    Synthesis of selenoproteins depends on decoding of the UGA stop codon as the amino acid selenocysteine (Sec). This process requires the presence of a Sec insertion sequence element (SECIS) in the 3'-untranslated region of selenoprotein mRNAs and its interaction with the SECIS binding protein 2 (SBP2). In humans, mutations in the SBP2-encoding gene Sec insertion sequence binding protein 2 (SECISBP2) that alter the amino acid sequence or cause splicing defects lead to abnormal thyroid hormone metabolism. Herein, we present the first in silico and in vivo functional characterization of alternative splicing of SECISBP2. We report a complex splicing pattern in the 5'-region of human SECISBP2, wherein at least eight splice variants encode five isoforms with varying N-terminal sequence. One of the isoforms, mtSBP2, contains a mitochondrial targeting sequence and localizes to mitochondria. Using a minigene-based in vivo splicing assay we characterized the splicing efficiency of several alternative transcripts, and show that the splicing event that creates mtSBP2 can be modulated by antisense oligonucleotides. Moreover, we show that full-length SBP2 and some alternatively spliced variants are subject to a coordinated transcriptional and translational regulation in response to ultraviolet type A irradiation-induced stress. Overall, our data broadens the functional scope of a housekeeping protein essential to selenium metabolism. PMID:19004874

  9. De-regulation of gene expression and alternative splicing affects distinct cellular pathways in the aging hippocampus

    PubMed Central

    Stilling, Roman M.; Benito, Eva; Gertig, Michael; Barth, Jonas; Capece, Vincenzo; Burkhardt, Susanne; Bonn, Stefan; Fischer, Andre

    2014-01-01

    Aging is accompanied by gradually increasing impairment of cognitive abilities and constitutes the main risk factor of neurodegenerative conditions like Alzheimer's disease (AD). The underlying mechanisms are however not well understood. Here we analyze the hippocampal transcriptome of young adult mice and two groups of mice at advanced age using RNA sequencing. This approach enabled us to test differential expression of coding and non-coding transcripts, as well as differential splicing and RNA editing. We report a specific age-associated gene expression signature that is associated with major genetic risk factors for late-onset AD (LOAD). This signature is dominated by neuroinflammatory processes, specifically activation of the complement system at the level of increased gene expression, while de-regulation of neuronal plasticity appears to be mediated by compromised RNA splicing. PMID:25431548

  10. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

    USGS Publications Warehouse

    Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

    2009-01-01

    The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

  11. Characterization of the fumonisin biosynthetic regulatory gene FUM21 and multiple alternative splice forms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumonisins are a family of mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in synthesis of mycotoxins and other s...

  12. Organization, regulatory sequences, and alternatively spliced transcripts of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) gene

    SciTech Connect

    Sampaio, S.O.; Mei, C.; Butcher, E.C.

    1995-09-01

    The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type I transmembrane adhesion molecule comprising two distal Ig domains involved in {alpha}4{beta}7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal Ig domain homologous to IgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three Ig domains are encoded by a distinct exon, whereas the transmembrane, cytoplasmic tail, and 3{prime}-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third Ig domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/IgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support {alpha}4{beta}7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5{prime} of the transcription start site include tandem nuclear factor-KB sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs. {alpha}4{beta}7-dependent adhesive events in lymphocyte trafficking. 46 refs., 6 figs., 1 tab.

  13. Genomic architecture and transcriptional activation of the mouse and human tumor susceptibility gene TSG101: common types of shorter transcripts are true alternative splice variants.

    PubMed

    Wagner, K U; Dierisseau, P; Rucker, E B; Robinson, G W; Hennighausen, L

    1998-11-26

    The functional inactivation of the tumor susceptibility gene tsg101 in mouse NIH3T3 cells leads to cell transformation and the formation of metastatic tumors in nude mice. We cloned, mapped and sequenced the mouse tsg101 gene and further identified a processed pseudogene that is 98% identical to the tsg101 cDNA. Based on Northern blot analysis, tsg101 is expressed ubiquitously in mouse tissues. A comparison of the coding region of the mouse tsg101 gene with the human TSG101 cDNA revealed that both the mouse and human gene encode ten additional highly conserved amino acids at the N-terminus. Based on the mouse tsg101 genomic structure, we predicted four additional introns within the human TSG101 gene. Their location was confirmed using PCR and sequencing analysis. The presence of these so far unidentified introns now explains published data on aberrantly spliced mRNA products that were frequently observed in primary breast tumors. We show that a majority of shorter TSG101 transcripts are not the result of aberrant splicing events, but represent a fraction of true alternative splice variants. Finally, we examined tsg101 expression patterns during different stages of mammary gland development and in different transgenic mouse models for breast tumorigenesis. PMID:9840940

  14. The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

    PubMed Central

    Wieczorek, D F; Smith, C W; Nadal-Ginard, B

    1988-01-01

    Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing. Images PMID:3352602

  15. The evolutionary landscape of alternative splicing in vertebrate species.

    PubMed

    Barbosa-Morais, Nuno L; Irimia, Manuel; Pan, Qun; Xiong, Hui Y; Gueroussov, Serge; Lee, Leo J; Slobodeniuc, Valentina; Kutter, Claudia; Watt, Stephen; Colak, Recep; Kim, TaeHyung; Misquitta-Ali, Christine M; Wilson, Michael D; Kim, Philip M; Odom, Duncan T; Frey, Brendan J; Blencowe, Benjamin J

    2012-12-21

    How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species. PMID:23258890

  16. Dysfunctional Gene Splicing as a Potential Contributor to Neuropsychiatric Disorders

    PubMed Central

    Glatt, Stephen J.; Cohen, Ori S.; Faraone, Stephen V.; Tsuang, Ming T.

    2011-01-01

    Alternative pre-mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of gene-focused epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders. PMID:21438146

  17. Function of ERBB4 is determined by alternative splicing.

    TOXLINE Toxicology Bibliographic Information

    Veikkolainen V; Vaparanta K; Halkilahti K; Iljin K; Sundvall M; Elenius K

    2011-08-15

    Alternative splicing is a central tool? of evolution that significantly increases the size of transcriptomes and generates functional specification. Within the human ERBB receptor gene family, only ERBB4 is known to produce functionally distinct isoforms as a result of alternative splicing. While ErbB4 signaling has been demonstrated to regulate cellular processes involved in embryogenesis, carcinogenesis and cardiovascular and psychiatric diseases, relatively little is known about the contribution of the individual isoforms in the different biological contexts. Here, we review recent findings as well as provide novel data about the distribution and functions of the ERBB4 splice variants. These observations represent an example of how minor alterations in the transcripts of a single gene can result in even antagonistic cellular responses. The observations also underline the significance of understanding the unique functions of isoforms of a potential drug target gene.

  18. Function of ERBB4 is determined by alternative splicing.

    PubMed

    Veikkolainen, Ville; Vaparanta, Katri; Halkilahti, Kalle; Iljin, Kristiina; Sundvall, Maria; Elenius, Klaus

    2011-08-15

    Alternative splicing is a central tool? of evolution that significantly increases the size of transcriptomes and generates functional specification. Within the human ERBB receptor gene family, only ERBB4 is known to produce functionally distinct isoforms as a result of alternative splicing. While ErbB4 signaling has been demonstrated to regulate cellular processes involved in embryogenesis, carcinogenesis and cardiovascular and psychiatric diseases, relatively little is known about the contribution of the individual isoforms in the different biological contexts. Here, we review recent findings as well as provide novel data about the distribution and functions of the ERBB4 splice variants. These observations represent an example of how minor alterations in the transcripts of a single gene can result in even antagonistic cellular responses. The observations also underline the significance of understanding the unique functions of isoforms of a potential drug target gene. PMID:21811097

  19. miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL.

    PubMed

    Zhang, Bo-Wen; Cai, Han-Fang; Wei, Xue-Feng; Sun, Jia-Jie; Lan, Xian-Yong; Lei, Chu-Zhao; Lin, Feng-Peng; Qi, Xing-Lei; Plath, Martin; Chen, Hong

    2016-01-01

    MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development. PMID:26840300

  20. miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL

    PubMed Central

    Zhang, Bo-Wen; Cai, Han-Fang; Wei, Xue-Feng; Sun, Jia-Jie; Lan, Xian-Yong; Lei, Chu-Zhao; Lin, Feng-Peng; Qi, Xing-Lei; Plath, Martin; Chen, Hong

    2016-01-01

    MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development. PMID:26840300

  1. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz. PMID:25800735

  2. Sxl-Dependent, tra/tra2-Independent Alternative Splicing of the Drosophila melanogaster X-Linked Gene found in neurons.

    PubMed

    Sun, Xia; Yang, Haiwang; Sturgill, David; Oliver, Brian; Rabinow, Leonard; Samson, Marie-Laure

    2015-01-01

    Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA-binding protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA-binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA, and TRA2 are thought to not function in males. To better understand sex-specific regulation of gene expression, we analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. Our data uncouple the effects of Sxl and tra/tra2 in females in the-sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. We show that FNE protein levels are downregulated by Sxl in female heads, also independently of tra/tra2. We argue that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function. PMID:26511498

  3. Sxl-Dependent, tra/tra2-Independent Alternative Splicing of the Drosophila melanogaster X-Linked Gene found in neurons

    PubMed Central

    Sun, Xia; Yang, Haiwang; Sturgill, David; Oliver, Brian; Rabinow, Leonard; Samson, Marie-Laure

    2015-01-01

    Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA-binding protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA-binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA, and TRA2 are thought to not function in males. To better understand sex-specific regulation of gene expression, we analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. Our data uncouple the effects of Sxl and tra/tra2 in females in the-sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. We show that FNE protein levels are downregulated by Sxl in female heads, also independently of tra/tra2. We argue that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function. PMID:26511498

  4. Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

    PubMed Central

    Dietrich, R C; Shukla, G C; Fuller, J D; Padgett, R A

    2001-01-01

    Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo. PMID:11680842

  5. Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR

    PubMed Central

    Estrada, April D.; Freese, Nowlan H.; Blakley, Ivory C.

    2015-01-01

    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. PMID:25945312

  6. Evolutionary Character of Alternative Splicing in Plants

    PubMed Central

    Zhang, Chengjun; Yang, Hong; Yang, Huizhao

    2015-01-01

    Alternative splicing (AS) is one of the most important ways to enhance the functional diversity of genes. Huge amounts of data have been produced by microarray, expressed sequence tag, and RNA-seq, and plenty of methods have been developed specifically for this task. The most frequently asked questions in previous research were as follows. What is the content rate of AS genes among the whole gene set? How many AS types are presented in the genome, and which type is dominant? How about the conservation ability of AS among different species? Which kinds of isoforms from some genes have the environmental response to help individual adaptation? Based on this background, we collected analysis results from 17 species to try to map out the landscape of AS studies in plants. We have noted the shortages of previous results, and we appeal to all scientists working in the AS field to make a standard protocol so that analyses between different projects are comparable. PMID:26819552

  7. Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

    PubMed

    Laporta, J; Driver, A; Khatib, H

    2011-08-01

    Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. PMID:21787958

  8. Transcriptome Bioinformatical Analysis of Vertebrate Stages of Schistosoma japonicum Reveals Alternative Splicing Events

    PubMed Central

    Wang, Xinye; Xu, Xindong; Lu, Xingyu; Zhang, Yuanbin; Pan, Weiqing

    2015-01-01

    Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type. According to intron boundary analysis, the parasite possesses same intron boundaries as other organisms, namely the classic GT-AG rule. And in alternative spliced introns or exons, this rule is less strict. And we have attempted to detect alternative splicing events in genes encoding proteins with signal peptides and transmembrane helices, suggesting that alternative splicing could change subcellular locations of specific gene products. Our results indicate that alternative splicing is prevalent in this parasitic worm, and that the worm is close to its hosts. The revealed secretome involved in alternative splicing implies new perspective into understanding interaction between the parasite and its host. PMID:26407301

  9. Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer

    PubMed Central

    Sette, Claudio

    2013-01-01

    Alternative splicing (AS) is one of the key processes involved in the regulation of gene expression in eukaryotic cells. AS catalyzes the removal of intronic sequences and the joining of selected exons, thus ensuring the correct processing of the primary transcript into the mature mRNA. The combinatorial nature of AS allows a great expansion of the genome coding potential, as multiple splice-variants encoding for different proteins may arise from a single gene. Splicing is mediated by a large macromolecular complex, the spliceosome, whose activity needs a fine regulation exerted by cis-acting RNA sequence elements and trans-acting RNA binding proteins (RBP). The activity of both core spliceosomal components and accessory splicing factors is modulated by their reversible phosphorylation. The kinases and phosphatases involved in these posttranslational modifications significantly contribute to AS regulation and to its integration in the complex regulative network that controls gene expression in eukaryotic cells. Herein, we will review the major canonical and noncanonical splicing factor kinases and phosphatases, focusing on those whose activity has been implicated in the aberrant splicing events that characterize neoplastic transformation. PMID:24069033

  10. Phosphorylation-mediated regulation of alternative splicing in cancer.

    PubMed

    Naro, Chiara; Sette, Claudio

    2013-01-01

    Alternative splicing (AS) is one of the key processes involved in the regulation of gene expression in eukaryotic cells. AS catalyzes the removal of intronic sequences and the joining of selected exons, thus ensuring the correct processing of the primary transcript into the mature mRNA. The combinatorial nature of AS allows a great expansion of the genome coding potential, as multiple splice-variants encoding for different proteins may arise from a single gene. Splicing is mediated by a large macromolecular complex, the spliceosome, whose activity needs a fine regulation exerted by cis-acting RNA sequence elements and trans-acting RNA binding proteins (RBP). The activity of both core spliceosomal components and accessory splicing factors is modulated by their reversible phosphorylation. The kinases and phosphatases involved in these posttranslational modifications significantly contribute to AS regulation and to its integration in the complex regulative network that controls gene expression in eukaryotic cells. Herein, we will review the major canonical and noncanonical splicing factor kinases and phosphatases, focusing on those whose activity has been implicated in the aberrant splicing events that characterize neoplastic transformation. PMID:24069033

  11. Comprehensive analysis of mutually exclusive alternative splicing in C. elegans

    PubMed Central

    Kuroyanagi, Hidehito; Takei, Satomi; Suzuki, Yutaka

    2014-01-01

    Mutually exclusive selection of one exon in a cluster of exons is a rare form of alternative pre-mRNA splicing, yet suggests strict regulation. However, the repertoires of regulation mechanisms for the mutually exclusive (ME) splicing in vivo are still unknown. Here, we experimentally explore putative ME exons in C. elegans to demonstrate that 29 ME exon clusters in 27 genes are actually selected in a mutually exclusive manner. Twenty-two of the clusters consist of homologous ME exons. Five clusters have too short intervening introns to be excised between the ME exons. Fidelity of ME splicing relies at least in part on nonsense-mediated mRNA decay for 14 clusters. These results thus characterize all the repertoires of ME splicing in this organism. PMID:25254147

  12. Alternative splicing of the androgen receptor in polycystic ovary syndrome

    PubMed Central

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C. K.; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ?62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS. PMID:25825716

  13. Alternative splicing of the androgen receptor in polycystic ovary syndrome.

    PubMed

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C K; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-04-14

    Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ?62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS. PMID:25825716

  14. SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes.

    PubMed

    Kim, Jung-Hyun; Baddoo, Melody C; Park, Eun Young; Stone, Joshua K; Park, Hyeonsoo; Butler, Thomas W; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M; Tan, Ming; Flemington, Erik K; Lim, Ssang-Taek; Ahn, Eun-Young Erin

    2016-03-17

    Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

  15. Modulators of alternative splicing as novel therapeutics in cancer

    PubMed Central

    Oltean, Sebastian

    2015-01-01

    Alternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g., pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics. PMID:26468443

  16. Regulation of alternative splicing by the core spliceosomal machinery

    PubMed Central

    Saltzman, Arneet L.; Pan, Qun; Blencowe, Benjamin J.

    2011-01-01

    Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B? self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B? in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. PMID:21325135

  17. Core clock, SUB1, and ABAR genes mediate flooding and drought responses via alternative splicing in soybean.

    PubMed

    Syed, Naeem H; Prince, Silvas J; Mutava, Raymond N; Patil, Gunvant; Li, Song; Chen, Wei; Babu, Valliyodan; Joshi, Trupti; Khan, Saad; Nguyen, Henry T

    2015-12-01

    Circadian clocks are a great evolutionary innovation and provide competitive advantage during the day/night cycle and under changing environmental conditions. The circadian clock mediates expression of a large proportion of genes in plants, achieving a harmonious relationship between energy metabolism, photosynthesis, and biotic and abiotic stress responses. Here it is shown that multiple paralogues of clock genes are present in soybean (Glycine max) and mediate flooding and drought responses. Differential expression of many clock and SUB1 genes was found under flooding and drought conditions. Furthermore, natural variation in the amplitude and phase shifts in PRR7 and TOC1 genes was also discovered under drought and flooding conditions, respectively. PRR3 exhibited flooding- and drought-specific splicing patterns and may work in concert with PRR7 and TOC1 to achieve energy homeostasis under flooding and drought conditions. Higher expression of TOC1 also coincides with elevated levels of abscisic acid (ABA) and variation in glucose levels in the morning and afternoon, indicating that this response to abiotic stress is mediated by ABA, endogenous sugar levels, and the circadian clock to fine-tune photosynthesis and energy utilization under stress conditions. It is proposed that the presence of multiple clock gene paralogues with variation in DNA sequence, phase, and period could be used to screen exotic germplasm to find sources for drought and flooding tolerance. Furthermore, fine tuning of multiple clock gene paralogues (via a genetic engineering approach) should also facilitate the development of flooding- and drought-tolerant soybean varieties. PMID:26314767

  18. Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy

    SciTech Connect

    Laporte, J.; Hu, Ling-Jia; Kretz, C.

    1997-05-01

    We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at least seven exons, of which two are non-coding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28. 15 refs., 3 figs., 1 tab.

  19. G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

    SciTech Connect

    Willing, M.; Deschenes, S.

    1994-09-01

    We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exon 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.

  20. Inference of alternative splicing from RNA-Seq data with probabilistic splice graphs

    PubMed Central

    LeGault, Laura H.; Dewey, Colin N.

    2013-01-01

    Motivation: Alternative splicing and other processes that allow for different transcripts to be derived from the same gene are significant forces in the eukaryotic cell. RNA-Seq is a promising technology for analyzing alternative transcripts, as it does not require prior knowledge of transcript structures or genome sequences. However, analysis of RNA-Seq data in the presence of genes with large numbers of alternative transcripts is currently challenging due to efficiency, identifiability and representation issues. Results: We present RNA-Seq models and associated inference algorithms based on the concept of probabilistic splice graphs, which alleviate these issues. We prove that our models are often identifiable and demonstrate that our inference methods for quantification and differential processing detection are efficient and accurate. Availability: Software implementing our methods is available at http://deweylab.biostat.wisc.edu/psginfer. Contact: cdewey@biostat.wisc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23846746

  1. Splicing patterns of the Chinese hamster MT-II gene

    SciTech Connect

    Grady, D.L.; Hildebrand, C.E.; Jackson, P.J.; Ratliff, R.L.; Walters, R.A.; Moyzis, R.K.

    1986-05-01

    Heavy metal induction of the synthesis of metallothioneins (MTs) provides an ideal model system for basic mechanistic studies of gene expression. Previous work from their laboratory has shown that a cell line designated 20 OT1, resistant to 200 ..mu..M CdCl/sub 2/, has coordinately amplified the MT-I and MT-II genes 10-20 fold. A region encompassing 14 kilobases (Kb) of overlapping DNA clones isolated from this cell line includes the structural genes for Chinese hamster MT-I and MT-II proteins. The MT-I gene is located 6 Kb downstream from the MT-II gene and is in the same transcriptional orientation. Analysis of the complete nucleotide sequence of the MT-II gene uncovered an alternative splice site in the first intron of this gene. To determine whether splicing occurs in vivo at this alternative splice site, oligonucleotide probes were synthesized. These probes consisted of 30 nucleotides bracketing either the normal or alternative splice sites. Northern blot analysis indicated that the alternative site is rarely used in the normal processing of the primary MT-II transcript. They suggest that efficient splicing does not occur at this alternative site due to inaccessibility of the proximal lariat site. Intron specific probes have been used to determine if there is a specific order to intron removal for this gene transcript. These studies indicate that the second intron is preferentially removed before the first intron.

  2. Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens

    PubMed Central

    2014-01-01

    Background Light is one of the most important factors regulating plant growth and development. Light-sensing photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although many levels of gene expression are modulated by photoreceptors, regulation at the mRNA splicing step remains unclear. Results We performed high-throughput mRNA sequencing to analyze light-responsive changes in alternative splicing in the moss Physcomitrella patens, and found that a large number of alternative splicing events were induced by light in the moss protonema. Light-responsive intron retention preferentially occurred in transcripts involved in photosynthesis and translation. Many of the alternatively spliced transcripts were expressed from genes with a function relating to splicing or light signaling, suggesting a potential impact on pre-mRNA splicing and photomorphogenic gene regulation in response to light. Moreover, most light-regulated intron retention was induced immediately upon light exposure, while motif analysis identified a repetitive GAA motif that may function as an exonic regulatory cis element in light-mediated alternative splicing. Further analysis in gene-disrupted mutants was consistent with a function for multiple red-light photoreceptors in the upstream regulation of light-responsive alternative splicing. Conclusions Our results indicate that intensive alternative splicing occurs in non-vascular plants and that, during photomorphogenesis, light regulates alternative splicing with transcript selectivity. We further suggest that alternative splicing is rapidly fine-tuned by light to modulate gene expression and reorganize metabolic processes, and that pre-mRNA cis elements are involved in photoreceptor-mediated splicing regulation. PMID:24398233

  3. Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene

    SciTech Connect

    Miki, T.; Bottaro, D.P.; Fleming, T.P.; Smith, C.L.; Chan, A.M.L.; Aaronson, S.A. ); Burgess, W.H. )

    1992-01-01

    Expression cDNA cloning and structural analysis of the human keratinocyte growth factor receptor (KGFR) revealed identity with one of the fibroblast growth factor (FGF) receptors encoded by the bek gene (FGFR-2), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript. Thus, two growth factor receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.

  4. Regulation of Chemoresistance Via Alternative Messenger RNA Splicing

    PubMed Central

    Eblen, Scott T.

    2012-01-01

    The acquisition of drug resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that have were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cells response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype. PMID:22248731

  5. TCGASpliceSeq a compendium of alternative mRNA splicing in cancer.

    PubMed

    Ryan, Michael; Wong, Wing Chung; Brown, Robert; Akbani, Rehan; Su, Xiaoping; Broom, Bradley; Melott, James; Weinstein, John

    2016-01-01

    TCGA's RNASeq data represent one of the largest collections of cancer transcriptomes ever assembled. RNASeq technology, combined with computational tools like our SpliceSeq package, provides a comprehensive, detailed view of alternative mRNA splicing. Aberrant splicing patterns in cancers have been implicated in such processes as carcinogenesis, de-differentiation and metastasis. TCGA SpliceSeq (http://bioinformatics.mdanderson.org/TCGASpliceSeq) is a web-based resource that provides a quick, user-friendly, highly visual interface for exploring the alternative splicing patterns of TCGA tumors. Percent Spliced In (PSI) values for splice events on samples from 33 different tumor types, including available adjacent normal samples, have been loaded into TCGA SpliceSeq. Investigators can interrogate genes of interest, search for the genes that show the strongest variation between or among selected tumor types, or explore splicing pattern changes between tumor and adjacent normal samples. The interface presents intuitive graphical representations of splicing patterns, read counts and various statistical summaries, including percent spliced in. Splicing data can also be downloaded for inclusion in integrative analyses. TCGA SpliceSeq is freely available for academic, government or commercial use. PMID:26602693

  6. TCGASpliceSeq a compendium of alternative mRNA splicing in cancer

    PubMed Central

    Ryan, Michael; Wong, Wing Chung; Brown, Robert; Akbani, Rehan; Su, Xiaoping; Broom, Bradley; Melott, James; Weinstein, John

    2016-01-01

    TCGA's RNASeq data represent one of the largest collections of cancer transcriptomes ever assembled. RNASeq technology, combined with computational tools like our SpliceSeq package, provides a comprehensive, detailed view of alternative mRNA splicing. Aberrant splicing patterns in cancers have been implicated in such processes as carcinogenesis, de-differentiation and metastasis. TCGA SpliceSeq (http://bioinformatics.mdanderson.org/TCGASpliceSeq) is a web-based resource that provides a quick, user-friendly, highly visual interface for exploring the alternative splicing patterns of TCGA tumors. Percent Spliced In (PSI) values for splice events on samples from 33 different tumor types, including available adjacent normal samples, have been loaded into TCGA SpliceSeq. Investigators can interrogate genes of interest, search for the genes that show the strongest variation between or among selected tumor types, or explore splicing pattern changes between tumor and adjacent normal samples. The interface presents intuitive graphical representations of splicing patterns, read counts and various statistical summaries, including percent spliced in. Splicing data can also be downloaded for inclusion in integrative analyses. TCGA SpliceSeq is freely available for academic, government or commercial use. PMID:26602693

  7. A Novel CDX2 Isoform Regulates Alternative Splicing

    PubMed Central

    Witek, Matthew E.; Snook, Adam E.; Lin, Jieru E.; Blomain, Erik S.; Xiang, Bo; Magee, Michael; Waldman, Scott A.

    2014-01-01

    Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-?1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS). PMID:25101906

  8. Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes

    PubMed Central

    2013-01-01

    Background The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease. Results We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3? and 5? alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products. Conclusions To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina. PMID:23865674

  9. Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs

    PubMed Central

    Wan, Jun; Oliver, Verity F.; Zhu, Heng; Zack, Donald J.; Qian, Jiang; Merbs, Shannath L.

    2013-01-01

    The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation. PMID:23887936

  10. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

    PubMed Central

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J.; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene. PMID:26985665

  11. Oligophrenin-1 (OPHN1), a Gene Involved in X-Linked Intellectual Disability, Undergoes RNA Editing and Alternative Splicing during Human Brain Development

    PubMed Central

    Athanasiadis, Alekos; Galeano, Federica; Locatelli, Franco; Bertini, Enrico; Zanni, Ginevra; Gallo, Angela

    2014-01-01

    Oligophrenin-1 (OPHN1) encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development. PMID:24637888

  12. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation. PMID:26407519

  13. FULL-GENOME ANALYSIS OF ALTERNATIVE SPLICING IN MOUSE LIVER AFTER HEPATOTOXICANT EXPOSURE

    EPA Science Inventory

    Alternative splicing plays a role in determining gene function and protein diversity. We have employed whole genome exon profiling using Affymetrix Mouse Exon 1.0 ST arrays to understand the significance of alternative splicing on a genome-wide scale in response to multiple toxic...

  14. Human epigenome data reveal increased CpG methylation in alternatively spliced sites and putative exonic splicing enhancers.

    PubMed

    Anastasiadou, Christina; Malousi, Andigoni; Maglaveras, Nicos; Kouidou, Sofia

    2011-05-01

    The role of gene body methylation, which represents a major part of methylation in DNA, remains mostly unknown. Evidence based on the CpG distribution associates its presence with nucleosome positioning and alternative splicing. Recently, it was also shown that cytosine methylation influences splicing. However, to date, there is no methylation-based data on the association of methylation with alternative splicing and the distribution in exonic splicing enhancers (ESEs). We presently report that, based on the computational analysis of the Human Epigenome Project data, CpG hypermethylation (>80%) is frequent in alternatively spliced sites (particularly in noncanonical) but not in alternate promoters. The methylation frequency increases in sequences containing multiple putative ESEs. However, significant differences in the extent of methylation are observed among different ESEs. Specifically, moderate levels of methylation, ranging from 20% to 80%, are frequent in SRp55-binding elements, which are associated with response to extracellular conditions, but not in SF2/ASF, primarily responsible for alternative splicing, or in CpG islands. Finally, methylation is more frequent in the presence of AT repeats and CpGs separated by 10 nucleotides and lower in adjacent CpGs, probably indicating its dependence on helical formations and on the presence of nucleosome positioning-related sequences. In conclusion, our results show the regulation of methylation in ESEs and support its involvement in alternative splicing. PMID:21545276

  15. The AP2-Like Gene OitaAP2 Is Alternatively Spliced and Differentially Expressed in Inflorescence and Vegetative Tissues of the Orchid Orchis italica

    PubMed Central

    Salemme, Marinella; Sica, Maria; Iazzetti, Giovanni; Gaudio, Luciano; Aceto, Serena

    2013-01-01

    The AP2/ERF proteins are plant-specific transcription factors involved in multiple regulatory pathways, from plant organ development to response to various environmental stresses. One of the mechanisms that regulates the AP2-like genes involves the microRNA miR172, which controls their activity at the post-transcriptional level. Extensive studies on AP2-like genes are available in many different species; however, in orchids, one of the largest plant families, studies are restricted to a few species, all belonging to the Epidendroideae subfamily. In the present study, we report the isolation of an AP2-like gene in the Mediterranean orchid Orchis italica (Orchidoideae). The OitaAP2 locus includes 10 exons and 9 introns, and its transcript is alternatively spliced, resulting in the long OitaAP2 and the short OitaAP2_ISO isoforms, with the latter skipping exon 9. Both isoforms contain the conserved target site for miR172, whose action is demonstrated by the presence of cleaved OitaAP2 mRNA. The OitaAP2 and OitaAP2_ISO mRNAs are present in the tepals and lip before and after anthesis at different expression levels. In addition, the OitaAP2_ISO isoform is expressed in the ovary before pollination and in the root and stem. The isoform-specific expression pattern suggests a functional differentiation of the OitaAP2 alternatively spliced transcripts. The expression profile of miR172 is complementary to that of the OitaAP2 isoforms in inflorescence tissues before anthesis, whereas after anthesis and in ovary tissue before and after pollination, this relationship disappears, suggesting the existence of OitaAP2 inhibitory mechanisms in these tissues that differ from that involving miR172. PMID:24204832

  16. WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo

    SciTech Connect

    Markus, M. Andrea; Heinrich, Bettina; Raitskin, Oleg; Adams, David J.; Mangs, Helena; Goy, Christine; Ladomery, Michael; Sperling, Ruth; Stamm, Stefan; Morris, Brian J. . E-mail: brianm@medsci.usyd.edu.au

    2006-10-15

    Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

  17. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage.

    PubMed

    Alsafadi, Samar; Houy, Alexandre; Battistella, Aude; Popova, Tatiana; Wassef, Michel; Henry, Emilie; Tirode, Franck; Constantinou, Angelos; Piperno-Neumann, Sophie; Roman-Roman, Sergio; Dutertre, Martin; Stern, Marc-Henri

    2016-01-01

    Hotspot mutations in the spliceosome gene SF3B1 are reported in ?20% of uveal melanomas. SF3B1 is involved in 3'-splice site (3'ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1(R625/K666) mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3'ss. Modelling the differential junctions in SF3B1(WT) and SF3B1(R625/K666) cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1(WT) knockdown or overexpression do not reproduce the SF3B1(R625/K666) splice pattern, qualifying SF3B1(R625/K666) as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1(R625/K666)-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease. PMID:26842708

  18. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

    PubMed Central

    Alsafadi, Samar; Houy, Alexandre; Battistella, Aude; Popova, Tatiana; Wassef, Michel; Henry, Emilie; Tirode, Franck; Constantinou, Angelos; Piperno-Neumann, Sophie; Roman-Roman, Sergio; Dutertre, Martin; Stern, Marc-Henri

    2016-01-01

    Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss. Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease. PMID:26842708

  19. Characterization of the Wilson disease gene: Genomic organization; alternative splicing; structure/function predictions; and population frequencies of disease-specific mutations

    SciTech Connect

    Petrukhin, K.; Chernov, I.; Ross, B.M.

    1994-09-01

    The Wilson disease (WD) gene has recently been identified as a putative copper-transporting ATPase with high amino acid similarity with the Menkes disease (MNK) gene. We have further characterized the WD gene by extending the 5{prime}-coding and non-coding DNA sequence and elucidating the intron/exon structure and genomic organization. Analysis of RNA transcripts from liver, brain, kidney and placenta reveals extensive alternative splicing which may provide a mechanism to regulate the quantity of functional protein product. Comparative sequence analysis shows that WD and MNK belong to the sub-family of heavy metal-transporting ATPases with several characterizing features which include unique amino acid motifs and distinct N-terminal and C-terminal transmembrane structure. Our data indicate that the 600 amino acid metal binding portion of the WD and MNK proteins was formed by gene duplication events and splicing of the 6 metal binding domain segment to a common ancestral protein. We have raised a WD-specific anti-peptide antibody to the N-terminal region and are beginning to explore the cellular and intracellular location of the WD protein. The metal-binding segment of the WD protein has been expressed in E. coli and metal binding assays are underway to characterize this aspect of the protein`s function. We have identified numerous disease-specific mutations and developed a rapid {open_quotes}reverse dot blot{close_quotes} screening protocol to determine mutation frequencies in different populations. The most common mutation disrupts the characteristic SEHP motif and accounts for more than 40% of WD cases in North American, Russian, and Swedish populations. This mutation has not been observed in our limited Sicilian sample.

  20. Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

    PubMed

    Semlow, Daniel R; Blanco, Mario R; Walter, Nils G; Staley, Jonathan P

    2016-02-25

    During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, thespliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilizealternative branch sites and 3' splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3' to 5' translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome. PMID:26919433

  1. Regulation of alternative splicing by the circadian clock and food related cues

    PubMed Central

    2012-01-01

    Background The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Results Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. Conclusions The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation. PMID:22721557

  2. Model-based detection of alternative splicing signals

    PubMed Central

    Barash, Yoseph; Blencowe, Benjamin J.; Frey, Brendan J.

    2010-01-01

    Motivation: Transcripts from ?95% of human multi-exon genes are subject to alternative splicing (AS). The growing interest in AS is propelled by its prominent contribution to transcriptome and proteome complexity and the role of aberrant AS in numerous diseases. Recent technological advances enable thousands of exons to be simultaneously profiled across diverse cell types and cellular conditions, but require accurate identification of condition-specific splicing changes. It is necessary to accurately identify such splicing changes to elucidate the underlying regulatory programs or link the splicing changes to specific diseases. Results: We present a probabilistic model tailored for high-throughput AS data, where observed isoform levels are explained as combinations of condition-specific AS signals. According to our formulation, given an AS dataset our tasks are to detect common signals in the data and identify the exons relevant to each signal. Our model can incorporate prior knowledge about underlying AS signals, measurement quality and gene expression level effects. Using a large-scale multi-tissue AS dataset, we demonstrate the advantage of our method over standard alternative approaches. In addition, we describe newly found tissue-specific AS signals which were verified experimentally, and discuss associated regulatory features. Contact: yoseph@psi.utoronto.ca; frey@psi.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20529924

  3. Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors.

    PubMed Central

    Grotewold, E; Athma, P; Peterson, T

    1991-01-01

    The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E. D. & Ceska, O. (1977) Can. J. Genet. Cytol. 19, 289-302]. We have characterized two P transcripts that are alternatively spliced at their 3' ends. One message of 1802 nucleotides encodes a 43.7-kDa protein with an N-terminal region showing approximately 40% homology to the DNA-binding domain of several members of the myb family of protooncogene proteins. A second message of 945 nucleotides encodes a 17.3-kDa protein that contains most of the myb-homologous domain but differs from the first protein at the C terminus. The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1. Additionally, the P and C1 genes are structurally similar in the sizes and positions of the first and second exons and first intron. We show that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone. Images PMID:2052542

  4. Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames

    SciTech Connect

    Wechsler, S.L.; Nesburn, A.B.; Watson, R.; Slanina, S.M.; Ghiasi, H.

    1988-11-01

    The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICP0 and is transcribed in the opposite direction from ICP0, producing LR-RNAs that are complementary (antisense) to ICP0 mRNA. The LR gene is thought to be involved in HSV-1 latency. The authors report here the time mapping and partial sequence analysis of this HSV-1 LR gene. /sup 32/P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LF-RNA 2, respectively. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.

  5. Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig.

    PubMed

    Zhang, Liang; Tao, Lin; Ye, Lin; He, Ling; Zhu, Yuan-Zhong; Zhu, Yue-Dong; Zhou, Yan

    2007-02-01

    Domestic pig (Sus scrofa domestica) is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags (ESTs) have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different non-normalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account. PMID:17572361

  6. Control of Alternative Splicing in Immune Responses: Many Regulators, Many Predictions, Much Still to Learn

    PubMed Central

    Martinez, Nicole M.; Lynch, Kristen W.

    2013-01-01

    Summary Most mammalian pre-mRNAs are alternatively spliced in a manner that alters the resulting open reading frame. Consequently, alternative pre-mRNA splicing provides an important RNA-based layer of protein regulation and cellular function. The ubiquitous nature of alternative splicing coupled with the advent of technologies that allow global interrogation of the transcriptome have led to an increasing awareness of the possibility that widespread changes in splicing patterns may contribute to lymphocyte function during an immune response. Indeed, a few notable examples of alternative splicing have clearly been demonstrated to regulate T-cell responses to antigen. Moreover, several proteins key to the regulation of splicing in T cells have recently been identified. However, much remains to be done to truly identify the spectrum of genes that are regulated at the level of splicing in immune cells and to determine how many of these are controlled by currently known factors and pathways versus unknown mechanisms. Here we describe the proteins, pathways, and mechanisms that have been shown to regulate alternative splicing in human T cells and discuss what is and is not known about the genes regulated by such factors. Finally, we highlight unifying themes with regards to the mechanisms and consequences of alternative splicing in the adaptive immune system and give our view of important directions for future studies. PMID:23550649

  7. Sudemycin E influences alternative splicing and changes chromatin modifications.

    PubMed

    Convertini, Paolo; Shen, Manli; Potter, Philip M; Palacios, Gustavo; Lagisetti, Chandraiah; de la Grange, Pierre; Horbinski, Craig; Fondufe-Mittendorf, Yvonne N; Webb, Thomas R; Stamm, Stefan

    2014-04-01

    Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death. PMID:24623796

  8. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  9. Recursive splicing in long vertebrate genes

    PubMed Central

    Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Toma; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-01-01

    It is generally believed that splicing removes introns as single units from pre-mRNA transcripts. However, some long D. melanogaster introns contain a cryptic site, called a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing1,2. The extent to which recursive splicing occurs in other species and its mechanistic basis remain unclear. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of a RS-exon that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform due to competition with a reconstituted 5? splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic exons or promoters that are prevalent in long introns, but which fail to reconstitute an efficient 5? splice site. Most RS-exons contain a premature stop codon such that their inclusion may decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling inclusion of cryptic elements with RS-exons. PMID:25970246

  10. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Toma; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons. PMID:25970246

  11. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    PubMed Central

    Kroll, Jose E.; Kim, Jihoon; Ohno-Machado, Lucila

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background. Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files) using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events. Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress. PMID:26618088

  12. Molecular Characterization of the ?-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

    PubMed Central

    Lind, Ulrika; Alm Rosenblad, Magnus; Wrange, Anna-Lisa; Sundell, Kristina S.; Jonsson, Per R.; Andr, Carl; Havenhand, Jonathan; Blomberg, Anders

    2013-01-01

    The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na+/K+ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions. PMID:24130836

  13. An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing

    PubMed Central

    2010-01-01

    Background Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each gene isoform termed long, short, super-short and truncated super-short. Many species across the vertebrate lineage contain multiple splice variants of each gene type, which are characterized by length and amino termini. Results A phylogenetic approach was used to visualize splice variant form genesis and identify conserved splice variants (genome conservation with EST support) across the vertebrate taxa. Bayesian and maximum likelihood phylogenetic inference indicated PDE4 gene duplication occurred at the base of the vertebrate lineage and reveals additional gene duplications specific to the teleost lineage. Phylogenetic inference and PDE4 splice variant presence, or absence as determined by EST screens, were further supported by the genomic analysis of select vertebrate taxa. Two conserved PDE4 long form splice variants were found in each of the PDE4A, PDE4B, and PDE4C genes, and eight conserved long forms from the PDE4 D gene. Conserved short and super-short splice variants were found from each of the PDE4A, PDE4B, and PDE4 D genes, while truncated super-short variants were found from the PDE4C and PDE4 D genes. PDE4 long form splice variants were found in all taxa sampled (invertebrate through mammals); short, super-short, and truncated super-short are detected primarily in tetrapods and mammals, indicating an increasing complexity in both alternative splicing and cAMP metabolism through vertebrate evolution. Conclusions There was a progressive independent incorporation of multiple PDE4 splice variant forms and amino termini, increasing PDE4 proteome complexity from primitive vertebrates to humans. While PDE4 gene isoform duplicates with limited alternative splicing were found in teleosts, an expansion of both PDE4 splice variant forms, and alternatively spliced amino termini predominantly occurs in mammals. Since amino termini have been linked to intracellular targeting of the PDE4 enzymes, the conservation of amino termini in PDE4 splice variants in evolution highlights the importance of compartmentalization of PDE4-mediated cAMP hydrolysis. PMID:20701803

  14. Leveraging transcript quantification for fast computation of alternative splicing profiles.

    PubMed

    Alamancos, Gael P; Pags, Amads; Trincado, Juan L; Bellora, Nicols; Eyras, Eduardo

    2015-09-01

    Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. PMID:26179515

  15. Leveraging transcript quantification for fast computation of alternative splicing profiles

    PubMed Central

    Alamancos, Gael P.; Pags, Amads; Trincado, Juan L.; Bellora, Nicols; Eyras, Eduardo

    2015-01-01

    Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. PMID:26179515

  16. Identification of two suites of cyclotide precursor genes from metallophyte Viola baoshanensis: cDNA sequence variation, alternative RNA splicing and potential cyclotide diversity.

    PubMed

    Zhang, Jun; Liao, Bin; Craik, David J; Li, Jin-Tian; Hu, Min; Shu, Wen-Sheng

    2009-02-15

    Cyclotides are a novel family of plant-derived defense peptides that are biosynthetically produced via the processing of cyclotide precursor (CP) proteins containing one, two or three cyclotide domains. By screening a cDNA library of Viola baoshanensis roots and using RACE and RT-PCR methods, 23 cDNA clones were identified and then used to deduce full CP proteins containing one (VbCP1S-5), two (VbCP6S), or three (VbCP7S) cyclotide domains. RT-PCR and sequence analyses suggested that VbCP6S were resulted from the alternative splicing of VbCP7S RNA. The significance of VbCP7S RNA splicing is that it provides a mechanism for increasing the diversity of cyclotide expression via the recombination of N-terminal repeat (NTR) regions and cyclotide domains. After analyzing the full endoplasmic reticulum (ER) signals of known and novel CPs associated with RT-PCR tests, three primers encoding the conserved sequence ALVLIATFA, AAFALPA-LA and AAFALPA-AFA were proposed to be more efficient in cloning CP genes than the well-applied primer encoding AAFALPA. Cyclotide sequence analyses indicated that the cDNA clones encoded a variety of Mbius and bracelet cyclotides, which were likely involved in the known bioactivities of cyclotides, and also might play a previously unreported role in mediating the metal tolerance of V. baoshanensis. Overall, this study shows that CP genes are varied in V. baoshanensis and cyclotide expression is subject to transcriptional and post-transcriptional regulation in this plant. PMID:19071200

  17. Deciphering the Plant Splicing Code: Experimental and Computational Approaches for Predicting Alternative Splicing and Splicing Regulatory Elements

    PubMed Central

    Reddy, Anireddy S. N.; Rogers, Mark F.; Richardson, Dale N.; Hamilton, Michael; Ben-Hur, Asa

    2012-01-01

    Extensive alternative splicing (AS) of precursor mRNAs (pre-mRNAs) in multicellular eukaryotes increases the protein-coding capacity of a genome and allows novel ways to regulate gene expression. In flowering plants, up to 48% of intron-containing genes exhibit AS. However, the full extent of AS in plants is not yet known, as only a few high-throughput RNA-Seq studies have been performed. As the cost of obtaining RNA-Seq reads continues to fall, it is anticipated that huge amounts of plant sequence data will accumulate and help in obtaining a more complete picture of AS in plants. Although it is not an onerous task to obtain hundreds of millions of reads using high-throughput sequencing technologies, computational tools to accurately predict and visualize AS are still being developed and refined. This review will discuss the tools to predict and visualize transcriptome-wide AS in plants using short-reads and highlight their limitations. Comparative studies of AS events between plants and animals have revealed that there are major differences in the most prevalent types of AS events, suggesting that plants and animals differ in the way they recognize exons and introns. Extensive studies have been performed in animals to identify cis-elements involved in regulating AS, especially in exon skipping. However, few such studies have been carried out in plants. Here, we review the current state of research on splicing regulatory elements (SREs) and briefly discuss emerging experimental and computational tools to identify cis-elements involved in regulation of AS in plants. The availability of curated alternative splice forms in plants makes it possible to use computational tools to predict SREs involved in AS regulation, which can then be verified experimentally. Such studies will permit identification of plant-specific features involved in AS regulation and contribute to deciphering the splicing code in plants. PMID:22645572

  18. AB227. RNA-seq analysis of prostate cancer in the Chinese population identifies recurrent gene fusions, cancer-associated long noncoding RNAs and aberrant alternative splicings

    PubMed Central

    Ren, S; Peng, Z; Mao, JH; Yu, Y; Yin, C; Gao, X; Cui, Z; Zhang, J; Yi, K; Xu, W; Chen, C; Wang, F; Guo, X; Lu, J; Yang, J; Wei, M; Tian, Z; Guan, Y; Tang, L; Xu, C; Wang, L; Gao, X; Tian, W; Wang, J; Yang, H; Wang, J; Sun, Y

    2014-01-01

    There are remarkable disparities among patients of different races with prostate cancer; however, the mechanism underlying this difference remains unclear. Here, we present a comprehensive landscape of the transcriptome profiles of 14 primary prostate cancers and their paired normal counterparts from the Chinese population using RNA-seq, revealing tremendous diversity across prostate cancer transcriptomes with respect to gene fusions, long noncoding RNAs (long ncRNA), alternative splicing and somatic mutations. Three of the 14 tumors (21.4%) harbored a TMPRSS2-ERG fusion, and the low prevalence of this fusion in Chinese patients was further confirmed in an additional tumor set (10/54 =18.5%). Notably, two novel gene fusions, CTAGE5-KHDRBS3 (20/54 =37%) and USP9Y-TTTY15 (19/54 =35.2%), occurred frequently in our patient cohort. Further systematic transcriptional profiling identified numerous long ncRNAs that were differentially expressed in the tumors. An analysis of the correlation between expression of long ncRNA and genes suggested that long ncRNAs may have functions beyond transcriptional regulation. This study yielded new insights into the pathogenesis of prostate cancer in the Chinese population.

  19. Nova1 is a master regulator of alternative splicing in pancreatic beta cells

    PubMed Central

    Villate, Olatz; Turatsinze, Jean-Valery; Mascali, Loriana G.; Grieco, Fabio A.; Nogueira, Tatiane C.; Cunha, Daniel A.; Nardelli, Tarlliza R.; Sammeth, Michael; Salunkhe, VishalA.; Esguerra, JonathanL.S.; Eliasson, Lena; Marselli, Lorella; Marchetti, Piero; Eizirik, Decio L.

    2014-01-01

    Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the neuron-specific Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1. PMID:25249621

  20. Alternative splicing matters: N-type calcium channels in nociceptors.

    PubMed

    Lipscombe, Diane; Raingo, Jesica

    2007-01-01

    How many different calcium channels does it take to make a nervous system? The answer: more than any of us predicted. In 1975 Hagiwara and colleagues published the first evidence that functionally different calcium channels are expressed in cells. By 1999, the calcium channel family could boast ten members, each member defined by a unique set of attributes to support their cellular functions and by unique amino acid sequences. Although nine of these genes are expressed in the nervous system, that number still seemed insufficient to support the wide spectrum of neuronal functions controlled by voltage-gated calcium channels. This discrepancy is probably explained by alternative pre-messenger RNA splicing which substantially expands the number of protein activities available from a limited number of genes. Like many other ion channel genes, each Ca(V)alpha(1) gene has the capacity to generate perhaps thousands of unique splice isoforms with unique functional properties. The high level of conservation among alternatively spliced exons in Ca(V)2.2 genes of different species and in some cases closely related genes implies biological importance. A number of Ca(V)alpha(1) isoforms have been identified from neural tissue but until recently we lacked direct evidence linking a specific splice site in a calcium channel gene to a specific function in an identified neuron population. Our recent studies show that alternative pre-mRNA splicing of a pair of 32 amino acid encoding exons in the C-terminus of Ca(V)2.2, e37a and e37b, underlie the expression of two mutually exclusive N-type channel isoforms. The inclusion of e37a creates a module that couples the N-type channel to a powerful form of G protein-dependent inhibition. The inhibitory pathway that works through e37a is voltage-independent, requires G(i/o) and tyrosine kinase activation, and is used by mu opioid and GABA(B) receptors to downregulate N-type channel activity. Combined with our previous studies that show enrichment of e37a in nociceptors, our data suggest a molecular basis for the high susceptibility of N-type currents in sensory neurons to voltage-independent inhibition following G protein activation. PMID:18708749

  1. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

    PubMed Central

    Selvanathan, Saravana P.; Erkizan, Hayriye V.; Dirksen, Uta; Natarajan, Thanemozhi G.; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T.; Ljungman, Mats E.; Wu, Cathy H.; Lawlor, Elizabeth R.; ren, Aykut; Toretsky, Jeffrey A.

    2015-01-01

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intronexon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of ?-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. PMID:25737553

  2. Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize1[OPEN

    PubMed Central

    Thatcher, Shawn R.; Meng, Xin; Beatty, Mary; Zastrow-Hayes, Gina; Harris, Charlotte; Habben, Jeffrey; Li, Bailin

    2016-01-01

    Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage- or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition. PMID:26582726

  3. Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize.

    PubMed

    Thatcher, Shawn R; Danilevskaya, Olga N; Meng, Xin; Beatty, Mary; Zastrow-Hayes, Gina; Harris, Charlotte; Van Allen, Brandon; Habben, Jeffrey; Li, Bailin

    2016-01-01

    Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage- or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition. PMID:26582726

  4. Characterization of Alternative Spliceoforms and the RNA Splicing Machinery in Pancreatic Cancer

    PubMed Central

    Carrigan, Patricia E.; Bingham, Jonathan L.; Srinvasan, Subha; Brentnall, Teresa A.; Miller, Laurence J.

    2010-01-01

    Objectives and Methods Alternative splicing provides proteomic diversity that can have profound effects. The extent, pattern, and roles of alternative splicing in pancreatic cancer have not been systematically investigated. We have utilized a spliceoform-specific microarray and polymerase chain reaction to evaluate all known splice variants in human pancreatic cancer cell lines representing a spectrum of differentiation, from near-normal HPDE6 to Capan-1 and poorly differentiated MiaPaCa2 cells. Validation of altered spliceoforms was verified in primary cancer specimens and normal pancreatic ductal cells. Additionally, expression of 92 spliceosomal genes were examined to better understand the mechanism for observed differences in mRNA splicing. Results A statistically significant reduction in alternative splicing was found in the pancreatic cancer cell lines compared to HPDE6 cells. Many splice variants identified in Capan-1 and MiaPaCa2 cells were observed in Grade 3 and Grade 4 tumors. Analysis of genes encoding spliceosomal proteins revealed that 28 of 92 genes had significantly decreased expression in cancer compared to normal pancreas. Conclusion Pancreatic cancer has reduced alternative splicing diversity compared to normal pancreas. This is demonstrated in both cell lines and primary tumors, with the loss in splicing diversity correlated with relative reduction in expression of spliceosomal genes. PMID:21178653

  5. High-throughput alternative splicing detection using dually constrained correspondence analysis (DCCA).

    PubMed

    Baty, Florent; Klingbiel, Dirk; Zappa, Francesco; Brutsche, Martin

    2015-12-01

    Alternative splicing is an important component of tumorigenesis. Recent advent of exon array technology enables the detection of alternative splicing at a genome-wide scale. The analysis of high-throughput alternative splicing is not yet standard and methodological developments are still needed. We propose a novel statistical approach-Dually Constrained Correspondence Analysis-for the detection of splicing changes in exon array data. Using this methodology, we investigated the genome-wide alteration of alternative splicing in patients with non-small cell lung cancer treated by bevacizumab/erlotinib. Splicing candidates reveal a series of genes related to carcinogenesis (SFTPB), cell adhesion (STAB2, PCDH15, HABP2), tumor aggressiveness (ARNTL2), apoptosis, proliferation and differentiation (PDE4D, FLT3, IL1R2), cell invasion (ETV1), as well as tumor growth (OLFM4, FGF14), tumor necrosis (AFF3) or tumor suppression (TUSC3, CSMD1, RHOBTB2, SERPINB5), with indication of known alternative splicing in a majority of genes. DCCA facilitates the identification of putative biologically relevant alternative splicing events in high-throughput exon array data. PMID:26483173

  6. Alternative Splicing Mediates Responses of the Arabidopsis Circadian Clock to Temperature Changes[W

    PubMed Central

    James, Allan B.; Syed, Naeem Hasan; Bordage, Simon; Marshall, Jacqueline; Nimmo, Gillian A.; Jenkins, Gareth I.; Herzyk, Pawel; Brown, John W.S.; Nimmo, Hugh G.

    2012-01-01

    Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and PSEUDO RESPONSE REGULATOR7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock. PMID:22408072

  7. Comprehensive Analysis of Alternative Splicing and Functionality in Neuronal Differentiation of P19 Cells

    PubMed Central

    Suzuki, Hitoshi; Osaki, Ken; Sano, Kaori; Alam, A. H. M. Khurshid; Nakamura, Yuichiro; Ishigaki, Yasuhito; Kawahara, Kozo; Tsukahara, Toshifumi

    2011-01-01

    Background Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. Methodology/Principal Findings The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. Conclusions/Significance Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation. PMID:21365003

  8. Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.

    PubMed Central

    Murtagh, J J; Eddy, R; Shows, T B; Moss, J; Vaughan, M

    1991-01-01

    Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes. Images PMID:1899283

  9. Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-? Signaling Pathway Identified in Thoracic Aortic Aneurysms

    PubMed Central

    Kurtovic, Sanela; Paloschi, Valentina; Folkersen, Lasse; Gottfries, Johan; Franco-Cereceda, Anders; Eriksson, Per

    2011-01-01

    Impaired regulation of the transforming growth factor-? (TGF?) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGF? pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGF? pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGF? pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcriptionpolymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGF? pathway. PMID:21448509

  10. The splicing fate of plant SPO11 genes

    PubMed Central

    Sprink, Thorben; Hartung, Frank

    2014-01-01

    Toward the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in Arabidopsis thaliana are essential for the initiation of double strand breaks (DSBs) during the meiotic prophase. In nearly all eukaryotic organisms DSB induction during prophase I by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO) as physical connections between the homologous chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa, and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of alternative splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non-functional as they either showed intron retention (IR) or shortened exons. However, the positional distribution and number of alternative splicing events vary strongly between the different plants. The cDNAs showed in most cases premature termination codons (PTCs) due to frameshift. Nevertheless, in some cases we found alternatively spliced but functional cDNAs. These findings let us suggest that alternative splicing of SPO11 depends on the respective gene sequence and on the plant species. Therefore, this conserved mechanism might play a role concerning regulation of SPO11. PMID:25018755

  11. Non-coding functions of alternative pre-mRNA splicing in development

    PubMed Central

    Mockenhaupt, Stefan; Makeyev, Eugene V.

    2015-01-01

    A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization. PMID:26493705

  12. Chorismate mutase: an alternatively spliced parasitism gene and a diagnostic marker for three important Globodera nematode species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chorismate mutase gene is widely distributed in both cyst and root-knot nematode species and believed to play a critical role in nematode parasitism. In this study, we cloned a new chorismate mutase gene (Gt-cm-1) from Globodera tabacum and further characterized the gene structure in both G. tab...

  13. Extensive relationship between antisense transcription and alternative splicing in the human genome.

    PubMed

    Morrissy, A Sorana; Griffith, Malachi; Marra, Marco A

    2011-08-01

    To analyze the relationship between antisense transcription and alternative splicing, we developed a computational approach for the detection of antisense-correlated exon splicing events using Affymetrix exon array data. Our analysis of expression data from 176 lymphoblastoid cell lines revealed that the majority of expressed sense-antisense genes exhibited alternative splicing events that were correlated to the expression of the antisense gene. Most of these events occurred in areas of sense-antisense (SAS) gene overlap, which were significantly enriched in both exons and nucleosome occupancy levels relative to nonoverlapping regions of the same genes. Nucleosome occupancy was highly correlated with Pol II abundance across overlapping regions and with concomitant increases in local alternative exon usage. These results are consistent with an antisense transcription-mediated mechanism of splicing regulation in normal human cells. A comparison of the prevalence of antisense-correlated splicing events between individuals of Mormon versus African descent revealed population-specific events that may indicate the continued evolution of new SAS loci. Furthermore, the presence of antisense transcription was correlated to alternative splicing across multiple metazoan species, suggesting that it may be a conserved mechanism contributing to splicing regulation. PMID:21719572

  14. Incorporating alternative splicing and mRNA editing into the genetic analysis of complex traits

    PubMed Central

    Hassan, Musa A.; Saeij, Jeroen P.J.

    2014-01-01

    The nomination of candidate genes underlying complex traits is often focused on genetic variations that alter mRNA abundance or result in non-conservative changes in amino acids. Although inconspicuous in complex trait analysis, genetic variants that affect splicing or RNA editing can also generate proteomic diversity and impact genetic traits. Indeed it is known that splicing and RNA editing modulate several traits in humans and model organisms. Using high-throughput RNA sequencing (RNA-seq) analysis, it is now possible to integrate the genetics of transcript abundance, alternative splicing and editing with the analysis of complex traits. We recently demonstrated that both alternative splicing and mRNA editing are modulated by genetic and environmental factors, and potentially engender phenotypic diversity in a genetically segregating mouse population. Therefore, the analysis of splicing and RNA editing will expand not only the regulatory landscape of transcriptome and proteome complexity, but also the repertoire of candidate genes for complex traits. PMID:25171292

  15. Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera dorsalis

    PubMed Central

    Yang, Wen-Jia; Xu, Kang-Kang; Cong, Lin; Wang, Jin-Jun

    2013-01-01

    Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5?- and 3?-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E. PMID:23569438

  16. Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

    PubMed

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  17. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis.

    PubMed

    Yang, Wen-Jia; Xu, Kang-Kang; Cong, Lin; Wang, Jin-Jun

    2013-01-01

    Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5'- and 3'-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E. PMID:23569438

  18. Molecular Characteristics, mRNA Expression, and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  19. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  20. Evolution of peroxisome proliferator-activated receptor gamma alternative splicing.

    PubMed

    Dou, Tonghai; Xu, Jiaxi; Gao, Yuan; Gu, Jianlei; Ji, Chaoneng; Xie, Yi; Zhou, Yan

    2010-01-01

    Peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in the control of energy balance and lipid and glucose homeostasis. Different transcript variants were investigated not only in human but also in other vertebrates. To look into the evolutionary changes of these variants, we analyzed the genomic sequences of PPAR gamma genes from several vertebrate species, as well as their mRNA and EST data. Several potential alternative splicing exons at the 5'-end of the PPAR gamma gene were identified. The 5'-end of the PPAR gamma gene is discovered to be evolutionarily active and recruits new exons via different strategies. Moreover, it is shown that the only coding alternative exon (exon B) processes much higher Ka/Ks compared with its constitutive counterparts. In addition, its Ka/Ks is greater than 1 in the rat, mouse, and rabbit, indicating adaptive evolution and possible energy storage related gain-of-function for the exon. PMID:20515805

  1. A General Definition and Nomenclature for Alternative Splicing Events

    PubMed Central

    Guig, Roderic

    2008-01-01

    Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific AS code to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial partin human more than a quarterof the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS. PMID:18688268

  2. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  3. nagnag: Identification and quantification of NAGNAG alternative splicing using RNA-Seq data.

    PubMed

    Yan, Xiaoyan; Sablok, Gaurav; Feng, Gang; Ma, Jiaxin; Zhao, Hongwei; Sun, Xiaoyong

    2015-07-01

    Regulation of proteome diversity by alternative splicing has been widely demonstrated in plants and animals. NAGNAG splicing, which was recently defined as a tissue specific event, results in the production of two distinct isoforms that are distinguished by three nucleotides (NAG) as a consequence of the intron proximal or distal to the splice site. Since the NAGNAG mechanism is not well characterized, tools for the identification and quantification of NAGNAG splicing events remain under-developed. Here we report nagnag, an R-based NAGNAG splicing detection tool, which accurately identifies and quantifies NAGNAG splicing events using RNA-Seq. Overall, nagnag produces user-friendly visualization reports and highlights differences between the DNA/RNA/protein across the identified isoforms of the reported gene. The package is available on https://sourceforge.net/projects/nagnag/files/; or http://genome.sdau.edu.cn/research/software/nagnag.html. PMID:26028313

  4. Alternative splicing the Neurofibromatosis type I pre-mRNA

    PubMed Central

    Barron, Victoria A.; Lou, Hua

    2013-01-01

    Synopsis Neurofibromatosis type I (NF1) is a common genetic disease that affects 1 in 3,500 individuals. The disease is completely penetrant but shows variable phenotypic expression in patients. NF1 is a large gene, and its pre-mRNA undergoes alternative splicing. The NF1 protein, neurofibromin, has a myriad of molecular functions, one of the best characterized being its function as a Ras-GAP. NF1 exon 23a is an alternative exon, which lies within the GAP-related domain of neurofibromin. This exon is predominantly included in most tissues, and it is skipped in central nervous system neurons. The isoform in which exon 23a is skipped has ten times higher Ras-GAP activity than the isoform in which exon 23a is included. Exon 23a inclusion is tightly regulated by at least three different families of RNA-binding proteins: CELF, Hu, and TIA-1/TIAR. The CELF and Hu proteins promote exon 23a skipping, while the TIA-1/TIAR proteins promote its inclusion. The widespread clinical variability that is observed among NF1 patients cannot be explained by NF1 mutations alone, and it is believed that modifier genes may have a role in the variability. We suggest that the regulation of alternative splicing may act as a modifier to contribute to the variable expression in NF1 patients. PMID:22115364

  5. BIPASS: BioInformatics Pipeline Alternative Splicing Services

    PubMed Central

    Lacroix, Zo; Legendre, Christophe; Raschid, Louiqa; Snyder, Ben

    2007-01-01

    BioInformatics Pipeline Alternative Splicing Services (BIPASS) offer support to scientists interested in gathering information related to alternative splicing (AS) events. The service BIPASSpliceDB provides access to AS information that has been extracted a priori from various public databases and stored in a data warehouse. In contrast, the BIPASAlign&Splice service allows scientists to submit their own sequences and genome to compute AS analysis results. BIPAS services offer various user-friendly ways to navigate through the results. AS results are organized at different conceptual levels (clusters and sequences), and are displayed in graphs or summarized in tables that can be downloaded in XML or text format. The two BIPAS services SpliceDB and Align&Splice are available online at http://bip.umiacs.umd.edu:8080/. PMID:17584795

  6. Detection of alternative splicing during epithelial-mesenchymal transition.

    PubMed

    Huang, Huilin; Xu, Yilin; Cheng, Chonghui

    2014-01-01

    Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes. PMID:25350517

  7. The alternative role of DNA methylation in splicing regulation.

    PubMed

    Lev Maor, Galit; Yearim, Ahuvi; Ast, Gil

    2015-05-01

    Although DNA methylation was originally thought to only affect transcription, emerging evidence shows that it also regulates alternative splicing. Exons, and especially splice sites, have higher levels of DNA methylation than flanking introns, and the splicing of about 22% of alternative exons is regulated by DNA methylation. Two different mechanisms convey DNA methylation information into the regulation of alternative splicing. The first involves modulation of the elongation rate of RNA polymerase II (Pol II) by CCCTC-binding factor (CTCF) and methyl-CpG binding protein 2 (MeCP2); the second involves the formation of a protein bridge by heterochromatin protein 1 (HP1) that recruits splicing factors onto transcribed alternative exons. These two mechanisms, however, regulate only a fraction of such events, implying that more underlying mechanisms remain to be found. PMID:25837375

  8. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  9. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  10. An Alternative Splicing Network Links Cell Cycle Control to Apoptosis

    PubMed Central

    Moore, Michael J.; Wang, Qingqing; Kennedy, Caleb J.; Silver, Pamela A.

    2010-01-01

    Summary Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2-family apoptosis factors in a genome-wide siRNA screen. The screen identified many novel regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promoted pro-apoptotic splicing of Bcl-x, Mcl1, and caspase-9, and altered splicing of other apoptotic transcripts. This response preceded mitotic arrest, indicating coordinated upregulation of pro-death splice variants that promotes apoptosis in arrested cells. These shifts corresponded to post-translational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell cycle control to apoptosis. PMID:20705336

  11. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

    PubMed Central

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  12. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  13. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  14. A functional alternative splicing mutation in human tryptophan hydroxylase-2.

    PubMed

    Zhang, X; Nicholls, P J; Laje, G; Sotnikova, T D; Gainetdinov, R R; Albert, P R; Rajkowska, G; Stockmeier, C A; Speer, M C; Steffens, D C; Austin, M C; McMahon, F J; Krishnan, K R R; Garcia-Blanco, M A; Caron, M G

    2011-12-01

    The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences. PMID:20856248

  15. Genome-wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron-based splicing and its possible function through network analysis.

    PubMed

    Panahi, Bahman; Mohammadi, Seyed Abolghasem; Ebrahimi Khaksefidi, Reyhaneh; Fallah Mehrabadi, Jalil; Ebrahimie, Esmaeil

    2015-11-30

    In this study, using homology mapping of assembled expressed sequence tags against the genomic data, we identified alternative splicing events in barley. Results demonstrated that intron retention is frequently associated with specific abiotic stresses. Network analysis resulted in discovery of some specific sub-networks between miRNAs and transcription factors in genes with high number of alternative splicing, such as cross talk between SPL2, SPL10 and SPL11 regulated by miR156 and miR157 families. To confirm the alternative splicing events, elongation factor protein (MLOC_3412) was selected followed by experimental verification of the predicted splice variants by Semi quantitative Reverse Transcription PCR (qRT-PCR). Our novel integrative approach opens a new avenue for functional annotation of alternative splicing through regulatory-based network discovery. PMID:26454178

  16. RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

    PubMed Central

    Khan, Dilshad H.; Gonzalez, Carolina; Cooper, Charlton; Sun, Jian-Min; Chen, Hou Yu; Healy, Shannon; Xu, Wayne; Smith, Karen T.; Workman, Jerry L.; Leygue, Etienne; Davie, James R.

    2014-01-01

    Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA. PMID:24234443

  17. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    SciTech Connect

    Lin, D.; Miller, W.L. ); Chang, Y.J.; Strauss, J.F. III )

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  18. Molecular cloning of the sex-related gene PSI in Bemisia tabaci and its alternative splicing properties.

    PubMed

    Liu, Yating; Xie, Wen; Yang, Xin; Guo, Litao; Wang, Shaoli; Wu, Qingjun; Yang, Zezhong; Zhou, Xuguo; Zhang, Youjun

    2016-04-15

    The P-element somatic inhibitor (PSI) is gene known to regulate the transcription of doublesex (dsx) when transformer (tra) is absent in Bombyx mori. In this study, we identified and characterized a PSI homolog in Bemisia tabaci (BtPSI). BtPSI cDNA had a total length of 5700bp and contained a predicted open reading frame (ORF) of 2208 nucleotides encoding for 735 amino acids. Multiple sequence alignments of the common regions of PSI proteins from B. tabaci and five other insect species revealed a high degree of sequence conservation. BtPSI is expressed in all stages of B. tabaci development, and expression did not significantly differ between female and male adult. A total of 92 BtPSI isoforms (78 in female and 22 in male) were identified, and a marker indicating the female-specific form was found. These results increase the understanding of genes that may determine sex in B. tabaci and provide a foundation for research on the sex determination mechanism in this insect. PMID:26773355

  19. Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: Evidence for the alternative splicing of a single-copy gene

    SciTech Connect

    Thiede, M.A.; Strewler, G.J.; Nissenson, R.A.; Rosenblatt, M.; Rodan, G.A. )

    1988-07-01

    A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study the authors obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3{prime} untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A){sup +} RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3{prime} untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.

  20. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    PubMed

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothe; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig.1). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70% of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies. PMID:242223

  1. Splice factor mutations and alternative splicing as drivers of hematopoietic malignancy.

    PubMed

    Hahn, Christopher N; Venugopal, Parvathy; Scott, Hamish S; Hiwase, Devendra K

    2015-01-01

    Differential splicing contributes to the vast complexity of mRNA transcripts and protein isoforms that are necessary for cellular homeostasis and response to developmental cues and external signals. The hematopoietic system provides an exquisite example of this. Recently, discovery of mutations in components of the spliceosome in various hematopoietic malignancies (HMs) has led to an explosion in knowledge of the role of splicing and splice factors in HMs and other cancers. A better understanding of the mechanisms by which alternative splicing and aberrant splicing contributes to the leukemogenic process will enable more efficacious targeted approaches to tackle these often difficult to treat diseases. The clinical implications are only just starting to be realized with novel drug targets and therapeutic strategies open to exploitation for patient benefit. PMID:25510282

  2. Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms

    PubMed Central

    Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

    2014-01-01

    The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

  3. Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms.

    PubMed

    Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

    2014-01-01

    The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

  4. SRSF10 Plays a Role in Myoblast Differentiation and Glucose Production via Regulation of Alternative Splicing.

    PubMed

    Wei, Ning; Cheng, Yuanming; Wang, Zhijia; Liu, Yuguo; Luo, Chunling; Liu, Lina; Chen, Linlin; Xie, Zhiqin; Lu, Yun; Feng, Ying

    2015-11-24

    Alternative splicing is a major mechanism of controlling gene expression and protein diversity in higher eukaryotes. We report that the splicing factor SRSF10 functions during striated muscle development, myoblast differentiation, and glucose production both in cells and in mice. A combination of RNA-sequencing and molecular analysis allowed us to identify muscle-specific splicing events controlled by SRSF10 that are critically involved in striated muscle development. Inclusion of alternative exons 16 and 17 of Lrrfip1 is a muscle-specific event that is activated by SRSF10 and essential for myoblast differentiation. On the other hand, in mouse primary hepatocytes, PGC1? is a key target of SRSF10 that regulates glucose production by fasting. SRSF10 represses inclusion of PGC1? exon 7a and facilitates the production of functional protein. The results highlight the biological significance of SRSF10 and regulated alternative splicing invivo. PMID:26586428

  5. Alternative Splicing Regulation of Cancer-Related Pathways in Caenorhabditis elegans: An In Vivo Model System with a Powerful Reverse Genetics Toolbox

    PubMed Central

    Barberán-Soler, Sergio; Ragle, James Matthew

    2013-01-01

    Alternative splicing allows for the generation of protein diversity and fine-tunes gene expression. Several model systems have been used for the in vivo study of alternative splicing. Here we review the use of the nematode Caenorhabditis elegans to study splicing regulation in vivo. Recent studies have shown that close to 25% of genes in the worm genome undergo alternative splicing. A big proportion of these events are functional, conserved, and under strict regulation either across development or other conditions. Several techniques like genome-wide RNAi screens and bichromatic reporters are available for the study of alternative splicing in worms. In this review, we focus, first, on the main studies that have been performed to dissect alternative splicing in this system and later on examples from genes that have human homologs that are implicated in cancer. The significant advancement towards understanding the regulation of alternative splicing and cancer that the C. elegans system has offered is discussed. PMID:24069034

  6. Alternative splicing: Functional diversity among voltage-gated calcium channels and behavioral consequences?

    PubMed Central

    Lipscombe, Diane; Andrade, Arturo; Allen, Summer E.

    2012-01-01

    Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal CaV channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinsons disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of CaV channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of CaV channel structures and functions. The precise composition of CaV channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of CaV splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of CaV pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels. PMID:23022282

  7. A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype

    PubMed Central

    2014-01-01

    Background Alternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes. Results We used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event. Conclusions As described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures. PMID:24739459

  8. Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing.

    PubMed

    Younis, Ihab; Berg, Michael; Kaida, Daisuke; Dittmar, Kimberly; Wang, Congli; Dreyfuss, Gideon

    2010-04-01

    Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns. PMID:20123975

  9. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    SciTech Connect

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  10. Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy

    PubMed Central

    Wong, Mandy S.; Chen, Ling; Foster, Christopher; Kainthla, Radhika; Shay, Jerry W.; Wright, Woodring E.

    2013-01-01

    SUMMARY Telomerase is present in human cancer cells but absent in most somatic tissues. The mRNA of human telomerase (hTERT) is alternatively spliced into mostly non-functional products. We sought to understand splicing so we could decrease functional splice isoforms to reduce telomerase activity to complement direct enzyme inhibition. Unexpectedly, minigenes containing hTERT exons 5–10 flanked by 150–300bp intronic sequences did not produce alternative splicing. A 1.1kb region of 38bp repeats ~2kb from the exon 6/intron junction restored exclusion of exons 7/8. An element within intron 8, also >1kb from intron/exon junctions, modulated this effect. Transducing an oligonucleotide complementary to this second element increased non-functional hTERT mRNA from endogenous telomerase. These results demonstrate the potential of manipulating hTERT splicing for both chemotherapy and regenerative medicine, and provide the first specific sequences deep within introns that regulate alternative splicing in mammalian cells by mechanisms other than introducing cryptic splice sites. PMID:23562158

  11. Developmental expression of p107 mRNA and evidence for alternative splicing of the p107 (RBL1) gene product

    SciTech Connect

    Kim, Kyung Keun; Soonpaa, M.H.; Wang, He

    1995-08-10

    Expression of p107, a protein with homology to the retinoblastoma tumor suppressor (pRB), was monitored during murine development. Northern blot tissue surveys identified two transcripts of 4.9 and 2.4 kb that hybridized to a p107 cDNA clone. Expression of both transcripts was detected in fetal tissues, with particularly high levels in the liver and heart. In contrast, p107 transcripts were markedly decreased in most adult tissues examined. Molecular cloning analyses revealed that the 4.9- and 2.4-kb transcripts encoded proteins with deduced molecular masses of 119 and 68 kDa, respectively. Genetic mapping studies suggested that the two p107 transcripts arose by alternative splicing of a common precursor. The protein encoded by the 2.4-kb transcript lacks the spacer and B motif of the {open_quotes}pocket domain,{close_quotes} a region of homology between p107 and pRB that is required for binding to cell cycle regulatory proteins. Structural modifications resulting from alternative splicing may this confer functional diversity upon the 119- and 68-kDa proteins. 52 refs., 4 figs., 1 tab.

  12. The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing

    PubMed Central

    Du, Chen; Ma, Xinran; Meruvu, Sunitha; Hugendubler, Lynne; Mueller, Elisabetta

    2014-01-01

    Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPAR? in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638s interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms. PMID:25024404

  13. C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

    PubMed Central

    Sumanasekera, Chiranthani; Kelemen, Olga; Beullens, Monique; Aubol, Brandon E.; Adams, Joseph A.; Sunkara, Manjula; Morris, Andrew; Bollen, Mathieu; Andreadis, Athena; Stamm, Stefan

    2012-01-01

    Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity. PMID:22210893

  14. Identifying Alternative Hyper-Splicing Signatures in MG-Thymoma by Exon Arrays

    PubMed Central

    Soreq, Lilach; Gilboa-Geffen, Adi; Berrih-Aknin, Sonia; Lacoste, Paul; Darvasi, Ariel; Soreq, Eyal; Bergman, Hagai; Soreq, Hermona

    2008-01-01

    Background The vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. Methodology/Principal Findings We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. Conclusions/Significance Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatability gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches. PMID:18545673

  15. Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq

    PubMed Central

    Gan, Qiang; Chepelev, Iouri; Wei, Gang; Tarayrah, Lama; Cui, Kairong; Zhao, Keji; Chen, Xin

    2010-01-01

    Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understandings of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (wt) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic down-regulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated-cell enriched bag of marbles (bam) mutant testis, but down-regulated upon differentiation in wt testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in wt testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are co-enriched in undifferentiated cells in testis, suggesting these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs. terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960. PMID:20440302

  16. Expression of splice variants of mts1 gene in normal and neoplastic human tissues

    SciTech Connect

    Ambartsumyan, N.S. |; Grigorian, M.S.; Lukanidin, E.M.

    1995-09-01

    Data on cloning of cDNA corresponding to human mts1 gene transcripts are presented. By comparing nucleotide sequences of the genomic DNA clone and cDNA of mts1, it was shown that human osteosarcoma OHS cells contain two alternative splice variants of mts1 transcripts. Alternative splicing occurs in the 5{prime}-untranslated region of the mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1(var), demonstrate similar stability in the cells, and each contains one open reading frame for the MTS1 protein. However, the two types of transcripts are translated with different effectiveness. The level of transcription of mts1 splice variants in different normal and neoplastic tissues and cell lines varies significantly. The role of alternative splicing as the mechanism responsible for posttranscriptional regulation of mts1 gene expression is discussed. 31 refs., 5 figs.

  17. A Pan-Cancer Analysis of Alternative Splicing Events Reveals Novel Tumor-Associated Splice Variants of Matriptase

    PubMed Central

    Dargahi, Daryanaz; Swayze, Richard D; Yee, Leanna; Bergqvist, Peter J; Hedberg, Bradley J; Heravi-Moussavi, Alireza; Dullaghan, Edie M; Dercho, Ryan; An, Jianghong; Babcook, John S; Jones, Steven JM

    2014-01-01

    High-throughput transcriptome sequencing allows identification of cancer-related changes that occur at the stages of transcription, pre-messenger RNA (mRNA), and splicing. In the current study, we devised a pipeline to predict novel alternative splicing (AS) variants from high-throughput transcriptome sequencing data and applied it to large sets of tumor transcriptomes from The Cancer Genome Atlas (TCGA). We identified two novel tumor-associated splice variants of matriptase, a known cancer-associated gene, in the transcriptome data from epithelial-derived tumors but not normal tissue. Most notably, these variants were found in 69% of lung squamous cell carcinoma (LUSC) samples studied. We confirmed the expression of matriptase AS transcripts using quantitative reverse transcription PCR (qRT-PCR) in an orthogonal panel of tumor tissues and cell lines. Furthermore, flow cytometric analysis confirmed surface expression of matriptase splice variants in chinese hamster ovary (CHO) cells transiently transfected with cDNA encoding the novel transcripts. Our findings further implicate matriptase in contributing to oncogenic processes and suggest potential novel therapeutic uses for matriptase splice variants. PMID:25506199

  18. CAG repeats mimic CUG repeats in the misregulation of alternative splicing

    PubMed Central

    Mykowska, Agnieszka; Sobczak, Krzysztof; Wojciechowska, Marzena; Kozlowski, Piotr; Krzyzosiak, Wlodzimierz J.

    2011-01-01

    Mutant transcripts containing expanded CUG repeats in the untranslated region are a pathogenic factor in myotonic dystrophy type 1 (DM1). The mutant RNA sequesters the muscleblind-like 1 (MBNL1) splicing factor and causes misregulation of the alternative splicing of multiple genes that are linked to clinical symptoms of the disease. In this study, we show that either long untranslated CAG repeat RNA or short synthetic CAG repeats induce splicing aberrations typical of DM1. Alternative splicing defects are also caused by translated CAG repeats in normal cells transfected with a mutant ATXN3 gene construct and in cells derived from spinocerebellar ataxia type 3 and Huntington's disease patients. Splicing misregulation is unlikely to be caused by traces of antisense transcripts with CUG repeats, and the possible trigger of this misregulation may be sequestration of the MBNL1 protein with nuclear RNA inclusions containing expanded CAG repeat transcripts. We propose that alternative splicing misregulation by mutant CAG repeats may contribute to the pathological features of polyglutamine disorders. PMID:21795378

  19. Gene splice sites correlate with nucleosome positions.

    PubMed

    Kogan, Simon; Trifonov, Edward N

    2005-06-01

    Gene sequences in the vicinity of splice sites are found to possess dinucleotide periodicities, especially RR and YY, with the period close to the pitch of nucleosome DNA. This confirms previously reported findings about preferential positioning of splice junctions within the nucleosomes. The RR and YY dinucleotides oscillate counter-phase, i.e., their respective preferred positions are shifted about half-period from one another, as it was observed earlier for AA and TT dinucleotides. Species specificity of nucleosome positioning DNA pattern is indicated by the predominant use of the periodical GG(CC) dinucleotides in human and mouse genes, as opposed to predominant AA(TT) dinucleotides in Arabidopsis and C. elegans. PMID:15862762

  20. Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site.

    PubMed

    Tacke, R; Goridis, C

    1991-08-01

    Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5'-splice site is an important element for regulation. Replacing it with a corresponding sequence from the alpha-globin gene resulted in constitutive use of the optional exon. When placed in the alpha-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5'-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells. PMID:1869048

  1. PIntron: a fast method for detecting the gene structure due to alternative splicing via maximal pairings of a pattern and a text

    PubMed Central

    2012-01-01

    Background A challenging issue in designing computational methods for predicting the gene structure into exons and introns from a cluster of transcript (EST, mRNA) sequences, is guaranteeing accuracy as well as efficiency in time and space, when large clusters of more than 20,000 ESTs and genes longer than 1 Mb are processed. Traditionally, the problem has been faced by combining different tools, not specifically designed for this task. Results We propose a fast method based on ad hoc procedures for solving the problem. Our method combines two ideas: a novel algorithm of proved small time complexity for computing spliced alignments of a transcript against a genome, and an efficient algorithm that exploits the inherent redundancy of information in a cluster of transcripts to select, among all possible factorizations of EST sequences, those allowing to infer splice site junctions that are largely confirmed by the input data. The EST alignment procedure is based on the construction of maximal embeddings, that are sequences obtained from paths of a graph structure, called embedding graph, whose vertices are the maximal pairings of a genomic sequence T and an EST P. The procedure runs in time linear in the length of P and T and in the size of the output. The method was implemented into the PIntron package. PIntron requires as input a genomic sequence or region and a set of EST and/or mRNA sequences. Besides the prediction of the full-length transcript isoforms potentially expressed by the gene, the PIntron package includes a module for the CDS annotation of the predicted transcripts. Conclusions PIntron, the software tool implementing our methodology, is available at http://www.algolab.eu/PIntron under GNU AGPL. PIntron has been shown to outperform state-of-the-art methods, and to quickly process some critical genes. At the same time, PIntron exhibits high accuracy (sensitivity and specificity) when benchmarked with ENCODE annotations. PMID:22537006

  2. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin.

    PubMed

    Laustriat, Delphine; Gide, Jacqueline; Barrault, Laetitia; Chautard, Emilie; Benoit, Clara; Auboeuf, Didier; Boland, Anne; Battail, Christophe; Artiguenave, François; Deleuze, Jean-François; Bénit, Paule; Rustin, Pierre; Franc, Sylvia; Charpentier, Guillaume; Furling, Denis; Bassez, Guillaume; Nissan, Xavier; Martinat, Cécile; Peschanski, Marc; Baghdoyan, Sandrine

    2015-01-01

    Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing. PMID:26528939

  3. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

    PubMed Central

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  4. Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations

    PubMed Central

    Kalari, Krishna R.; Rossell, David; Necela, Brian M.; Asmann, Yan W.; Nair, Asha; Baheti, Saurabh; Kachergus, Jennifer M.; Younkin, Curtis S.; Baker, Tiffany; Carr, Jennifer M.; Tang, Xiaojia; Walsh, Michael P.; Chai, High-Seng; Sun, Zhifu; Hart, Steven N.; Leontovich, Alexey A.; Hossain, Asif; Kocher, Jean-Pierre; Perez, Edith A.; Reisman, David N.; Fields, Alan P.; Thompson, E. Aubrey

    2012-01-01

    KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NF?B, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on genegene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NF?B, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPAR? signaling pathways, suggesting that targeted PPAR? antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations. PMID:22655260

  5. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    PubMed Central

    2010-01-01

    Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level. PMID:20920313

  6. Role of tissue specific alternative pre-mRNA splicing in the differentiation of the erythrocyte membrane.

    PubMed

    Benz, E J; Huang, S C

    1997-01-01

    Regulated alternative pre-mRNA splicing is neither as widely appreciated as a fundamental aspect of controlled gene expression nor as thoroughly studied as transcriptional regulation. However, as exemplified by the phenomena cited in this review, alternative splicing is a fundamentally important mechanism used in the eukaryotic world to enhance the range, versatility and plasticity of the structural information contained within a gene, and to create additional strategies by which the net quantitative output of a given gene product can be controlled. Regulation of RNA splicing gives genes a modularity that adds flexibility, and, therefore, selective advantage, to eukaryotes. It is likely, though unproven, that this opportunity for refined regulation and diversification provides at least one basis for the existence of the tandem exon-intron-exon structure found in the vast majority of eukaryotic genes and many viral genes. Many examples of alternative splicing are known, but, for the majority, no obvious biological impact of the alternatively spliced proteins on known cellular functions can be appreciated. Examples by which selectively regulated splicing pathways alter both the physiology and pathology of a major cellular event, such as differentiation and mechanical function of the red cell membrane, are thus relatively rare. The protein 4.1 gene and mRNA products thus provide an instructive and unusual system in which to explore the broader issue of the role of these regulatory mechanisms in the overall scheme of gene regulation and adaptation. The fact that hereditary hemolytic anemias result from mutations that directly or indirectly disrupt the splicing system emphasized the relevance of these mechanisms to molecular medicine. The features of splicing that we have reviewed in this paper, and the specific impact that regulated splicing exerts on differentiating red cells have, we hope, convinced the reader that RNA splicing is an important, fascinating, and potentially fruitful area for future study of human disease processes. PMID:9108669

  7. Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing

    PubMed Central

    Selman, Mohammed; Dankar, Samar K; Forbes, Nicole E; Jia, Jian-Jun; Brown, Earl G

    2012-01-01

    Little is known about the processes that enable influenza A viruses to jump into new host species. Here we show that the non-structural protein1 nucleotide substitution, A374G, encoding the D125G(GAT?GGT) mutation, which evolved during the adaptation of a human virus within a mouse host, activates a novel donor splice site in the non-structural gene, hence producing a novel influenza A viral protein, NS3. Using synonymous 125G mutations that do not activate the novel donor splice site, NS3 was shown to provide replicative gain-of-function. The protein sequence of NS3 is similar to NS1 protein but with an internal deletion of a motif comprised of three antiparallel ?-strands spanning codons 126 to 168 in NS1. The NS1-125G(GGT) codon was also found in 33 natural influenza A viruses that were strongly associated with switching from avian to mammalian hosts, including human, swine and canine populations. In addition to the experimental human to mouse switch, the NS1-125G(GGT) codon was selected on avian to human transmission of the 1997 H5N1 and 1999 H9N2 lineages, as well as the avian to swine jump of 1979 H1N1 Eurasian swine influenza viruses, linking the NS1 125G(GGT) codon with host adaptation and switching among multiple species. PMID:26038410

  8. CUG-BP1 regulates RyR1 ASI alternative splicing in skeletal muscle atrophy.

    PubMed

    Tang, Yinglong; Wang, Huiwen; Wei, Bin; Guo, Yuting; Gu, Lei; Yang, Zhiguang; Zhang, Qing; Wu, Yanyun; Yuan, Qi; Zhao, Gang; Ji, Guangju

    2015-01-01

    RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease. PMID:26531141

  9. CUG-BP1 regulates RyR1 ASI alternative splicing in skeletal muscle atrophy

    PubMed Central

    Tang, Yinglong; Wang, Huiwen; Wei, Bin; Guo, Yuting; Gu, Lei; Yang, Zhiguang; Zhang, Qing; Wu, Yanyun; Yuan, Qi; Zhao, Gang; Ji, Guangju

    2015-01-01

    RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease. PMID:26531141

  10. Regulation of alternative splicing of CD44 in cancer.

    PubMed

    Prochazka, Lubomir; Tesarik, Radek; Turanek, Jaroslav

    2014-10-01

    CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor microenvironment. CD44 contains two variable regions encoded by variable exons. Alternative splicing, which is often deregulated in cancer, can produce various isoforms of CD44 with properties that may have different tissue specific effects and therefore even diverse effects on cancer progression. This review summarizes and puts together all major regulators of alternative splicing of CD44 in cancer that have been documented so far and that have an experimentally proved effect on CD44 isoform switching. It is important to better understand the mechanisms of alternative splicing of CD44, where all the variability of CD44 originates, to be able to explain the isoform switching and occurrence of variant isoforms of CD44 (CD44v) in cancer. PMID:25025570

  11. Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

    PubMed Central

    Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S.; Kretzler, Matthias; Guan, Yuanfang

    2013-01-01

    Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ground-truth functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the responsible isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the responsible isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions. PMID:24244129

  12. Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

    PubMed

    Mameli, Eva; Lepori, Maria Barbara; Chiappe, Francesca; Ranucci, Giusy; Di Dato, Fabiola; Iorio, Raffaele; Loudianos, Georgios

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease. PMID:26031236

  13. Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation.

    PubMed

    Zhou, Xuexia; Wu, Wenwu; Li, Huang; Cheng, Yuanming; Wei, Ning; Zong, Jie; Feng, Xiaoyan; Xie, Zhiqin; Chen, Dai; Manley, James L; Wang, Hui; Feng, Ying

    2014-04-01

    Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions. PMID:24442672

  14. QUANTIFYING ALTERNATIVE SPLICING FROM PAIRED-END RNA-SEQUENCING DATA.

    PubMed

    Rossell, David; Stephan-Otto Attolini, Camille; Kroiss, Manuel; Stöcker, Almond

    2014-03-01

    RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence sub-optimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a non-parametric, highly flexible manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream analyses. We found a several fold improvement in estimation mean square error compared popular approaches in simulations, and substantially higher consistency between replicates in experimental data. Our findings indicate the need for adjusting the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper. PMID:24795787

  15. QUANTIFYING ALTERNATIVE SPLICING FROM PAIRED-END RNA-SEQUENCING DATA

    PubMed Central

    Rossell, David; Stephan-Otto Attolini, Camille; Kroiss, Manuel; Stöcker, Almond

    2014-01-01

    RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence sub-optimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a non-parametric, highly flexible manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream analyses. We found a several fold improvement in estimation mean square error compared popular approaches in simulations, and substantially higher consistency between replicates in experimental data. Our findings indicate the need for adjusting the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper* PMID:24795787

  16. ALTERNATE PATCHED SPLICE FORMS ARE EXPRESSED IN A TISSUE SPECIFIC MANNER DURING EARLY EMBRYONIC DEVELOPMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The Hedgehog (Hh) pathway is critical for embryonic patterning of nearly every organ system in the developing fetus and is highly conserved across phylogeny. We have previously characterized three alternate splice forms of the Ptc gene, including a novel Exon 1C isoform in the mouse, but...

  17. Microenvironment Changes (in pH) Affect VEGF Alternative Splicing

    PubMed Central

    Elias, Ana Paula

    2008-01-01

    Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p?splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process. Electronic supplementary material The online version of this article (doi:10.1007/s12307-008-0013-4) contains supplementary material, which is available to authorized users. PMID:19308691

  18. Ancient nature of alternative splicing and functions of introns

    SciTech Connect

    Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

    2011-03-21

    Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

  19. The human decorin gene: Intron-exon organization, discovery of two alternatively spliced exons in the 5[prime] untralsated region, and mapping of the gene to chromosome 12q23

    SciTech Connect

    Danielson, K.G.; Fazzio, A.; Cohen, I.; Cannizzaro, L.A.; Eichstetter, I.; Iozzo, R.V. )

    1993-01-01

    Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5[prime] untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and lb are found in the 5[prime]untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of [approx]1.6 and [approx]1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This sturdy provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene. 57 refs., 11 figs., 3 tabs.

  20. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

  1. Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

    SciTech Connect

    Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

    2006-06-15

    A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

  2. Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

    PubMed Central

    Song, Jingwen

    2015-01-01

    The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur. PMID:25776557

  3. Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development

    PubMed Central

    Kim, Kee K.; Nam, Joseph

    2013-01-01

    Alternative premRNA splicing is a major mechanism to generate diversity of gene products. However, the biological roles of alternative splicing during development remain elusive. Here, we focus on a neuron-specific RNA-binding protein, Rbfox3, recently identified as the antigen of the widely used anti-NeuN antibody. siRNA-mediated loss-of-function studies using the developing chicken spinal cord revealed that Rbfox3 is required to promote neuronal differentiation of postmitotic neurons. Numb premRNA encoding a signaling adaptor protein was found to be a target of Rbfox3 action, and Rbfox3 repressed the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform reproduced similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform was sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development. PMID:23420872

  4. Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis.

    PubMed

    Song, Jingwen; Richard, Stéphane

    2015-06-01

    The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur. PMID:25776557

  5. Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation

    PubMed Central

    Amelio, Antonio L; Caputi, Massimo; Conkright, Michael D

    2009-01-01

    The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1–MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity. PMID:19644446

  6. Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data.

    PubMed

    Misra, Ashish; Ou, Jianhong; Zhu, Lihua Julie; Green, Michael R

    2015-12-01

    Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392. PMID:26697379

  7. Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data

    PubMed Central

    Misra, Ashish; Ou, Jianhong; Zhu, Lihua Julie; Green, Michael R.

    2015-01-01

    Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392.

  8. Gene and splicing therapies for neuromuscular diseases.

    PubMed

    Benchaouir, Rachid; Robin, Valerie; Goyenvalle, Aurelie

    2015-01-01

    Neuromuscular disorders (NMD) are heterogeneous group of genetic diseases characterized by muscle weakness and wasting. Duchenne Muscular dystrophy (DMD) and Spinal muscular atrophy (SMA) are two of the most common and severe forms in humans and although the molecular mechanisms of these diseases have been extensively investigated, there is currently no effective treatment. However, new gene-based therapies have recently emerged with particular noted advances in using conventional gene replacement strategies and RNA-based technology. Whilst proof of principle have been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense mediated exon skipping has shown encouraging results and hold promise for the treatment of dystrophic muscle. In this review, we summarize the recent progress of therapeutic approaches to neuromuscular diseases, with an emphasis on gene therapy and splicing modulation for DMD and SMA, focusing on the advantages offered by these technologies but also their challenges. PMID:25961553

  9. Alternatively spliced isoforms of the human constitutive androstane receptor

    PubMed Central

    Auerbach, Scott S.; Ramsden, Richard; Stoner, Matthew A.; Verlinde, Christophe; Hassett, Christopher; Omiecinski, Curtis J.

    2003-01-01

    The nuclear receptor CAR (NR1I3) regulates transcription of genes encoding xenobiotic- and steroid-metabolizing enzymes. Regulatory processes that are mediated by CAR are modulated by a structurally diverse array of chemicals including common pharmaceutical and environmental agents. Here we describe four in-frame splice variants of the human CAR receptor gene. The variant mRNA splice transcripts were expressed in all human livers evaluated. Molecular modeling of the splice variant proteins predicts that the structural effects are localized within the receptors ligand-binding domain. Assays to assess function indicate that the variant proteins, when compared with the reference protein isoform, exhibit compromised activities with respect to DNA binding, transcriptional activation and coactivator recruitment. PMID:12799447

  10. Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility

    PubMed Central

    Wang, Xiuge; Yang, Chunhong; Sun, Yan; Jiang, Qiang; Wang, Fei; Li, Mengjiao; Zhong, Jifeng; Huang, Jinming

    2015-01-01

    Neutrophil cytosolic factor 4 (NCF4) is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice variants and functional single-nucleotide polymorphism (SNP) of NCF4 were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as NCF4-TV and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the NCF4-reference main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether NCF4-TV could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant NCF4 g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of NCF4 was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced NCF4-TV with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows. NCF4 expression was also regulated by alternative splicing. This study proposes that NCF4 splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows. PMID:26600390

  11. Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells

    PubMed Central

    Gonalves, Vnia; Henriques, Andreia; Pereira, Joana; Neves Costa, Ana; Moyer, Mary Pat; Moita, Lus Ferreira; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, Peter

    2014-01-01

    The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3?, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3? or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival. PMID:24550521

  12. A long noncoding way to alternative splicing in plant development.

    PubMed

    Kornblihtt, Alberto R

    2014-07-28

    In this issue of Developmental Cell, Bardou et al. (2014) elucidate how long, highly structured noncoding RNAs control alternative splicing regulators that specifically mediate the action of the hormone auxin in the promotion of lateral root growth in Arabidopsis. PMID:25073153

  13. Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses[OPEN

    PubMed Central

    Staiger, Dorothee; Brown, John W.S.

    2013-01-01

    High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely. PMID:24179132

  14. Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11-48K and U11/U12-65K genes.

    PubMed

    Niemel, Elina H; Verbeeren, Jens; Singha, Prosanta; Nurmi, Visa; Frilander, Mikko J

    2015-11-01

    Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5' splice sites (5'ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50nt downstream from the target 3' splice site (3'ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3'ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5'ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5'ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions. PMID:26479860

  15. RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing

    PubMed Central

    2014-01-01

    RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs. PMID:24485098

  16. Neuronal cell typespecific alternative splicing is regulated by the KH domain protein SLM1

    PubMed Central

    Iijima, Takatoshi; Iijima, Yoko; Witte, Harald

    2014-01-01

    The unique functional properties and molecular identity of neuronal cell populations rely on cell typespecific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domaincontaining splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell typespecific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo. PMID:24469635

  17. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Lngst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3? end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ?15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exonexon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  18. Alternative splicing and exon duplication generates 10 unique porcine 5-HT 4 receptor splice variants including a functional homofusion variant.

    PubMed

    De Maeyer, Joris H; Aerssens, Jeroen; Verhasselt, Peter; Lefebvre, Romain A

    2008-06-12

    5-HT(4) receptors are present in human and porcine atrial myocytes while they are absent from the hearts of small laboratory animals. The pig is therefore the only available nonprimate animal model in which to study cardiac 5-HT(4) receptor function under physiological conditions. While several human splice variants of the 5-HT(4) receptor have been described, the splicing behavior of this receptor in porcine tissue is currently unknown. Here we report on the identification of nine novel COOH-terminal splice variants of the porcine 5-HT(4) receptor, which were named 5-HT(4(b2, j, k, l, m, o, p, q, r)). The internal h-variant was found in combination with several COOH-terminal exons. In addition, splice variants were found that comprised duplicated exons fused to the common region of the 5-HT(4) receptor, thereby providing evidence for a duplication of the porcine HTR4 gene. One of these variants putatively encoded a nine transmembrane-spanning domain homofusion receptor, 5-HT(4(9TM)); also the other variants with a duplicated region might translate into functional, transcriptionally fused dimeric 5-HT(4) receptor variants. The elucidation of the genomic context confirmed that the variants were not genomic artefacts but originated from alternative splicing. This was further corroborated by a functional analysis of the variants 5-HT(4(a)), 5-HT(4(r)), and 5-HT(4(9TM)). To our knowledge, our data are the first to report on a functional GPCR with more than seven predicted transmembrane domains. These findings urge for caution when interpreting data on 5-HT(4) receptor-related pharmacology obtained in the pig; validation at the molecular level might be needed before extrapolating results to human. PMID:18430808

  19. Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

    PubMed

    g, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsbet

    2015-12-01

    Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing. PMID:26514742

  20. An alternative splicing event amplifies evolutionary differences between vertebrates.

    PubMed

    Gueroussov, Serge; Gonatopoulos-Pournatzis, Thomas; Irimia, Manuel; Raj, Bushra; Lin, Zhen-Yuan; Gingras, Anne-Claude; Blencowe, Benjamin J

    2015-08-21

    Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here we show that mammalian-specific skipping of polypyrimidine tract-binding protein 1 (PTBP1) exon 9 alters the splicing regulatory activities of PTBP1 and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon-skipping event in an RNA binding regulator directs numerous AS changes between species. Our results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems. PMID:26293963

  1. Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

    PubMed

    Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

    2015-10-01

    Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. PMID:26190308

  2. Alternative Splicing Shapes the Phenotype of a Mutation in BBS8 To Cause Nonsyndromic Retinitis Pigmentosa

    PubMed Central

    Murphy, Daniel; Singh, Ratnesh; Kolandaivelu, Saravanan

    2015-01-01

    Bardet-Biedl syndrome (BBS) is a genetic disorder affecting multiple systems and organs in the body. Several mutations in genes associated with BBS affect only photoreceptor cells and cause nonsyndromic retinitis pigmentosa (RP), raising the issue of why certain mutations manifest as a systemic disorder whereas other changes in the same gene affect only a specific cell type. Here, we show that cell-type-specific alternative splicing is responsible for confining the phenotype of the A-to-G substitution in the 3? splice site of BBS8 exon 2A (IVS1-2A>G mutation) in the BBS8 gene to photoreceptor cells. The IVS1-2A>G mutation leads to missplicing of BBS8 exon 2A, producing a frameshift in the BBS8 reading frame and thus eliminating the protein specifically in photoreceptor cells. Cell types other than photoreceptors skip exon 2A from the mature BBS8 transcript, which renders them immune to the mutation. We also show that the splicing of Bbs8 exon 2A in photoreceptors is directed exclusively by redundant splicing enhancers located in the adjacent introns. These intronic sequences are sufficient for photoreceptor-cell-specific splicing of heterologous exons, including an exon with a randomized sequence. PMID:25776555

  3. Dynamic integration of splicing within gene regulatory pathways

    PubMed Central

    Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

    2013-01-01

    Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

  4. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

    PubMed Central

    Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

    2014-01-01

    Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

  5. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

    PubMed

    Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

    2014-04-01

    Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

  6. Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN

    PubMed Central

    Reddy, Anireddy S.N.; Marquez, Yamile; Kalyna, Maria; Barta, Andrea

    2013-01-01

    Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation. PMID:24179125

  7. Evidence of Extensive Alternative Splicing in Post Mortem Human Brain HTT Transcription by mRNA Sequencing

    PubMed Central

    Labadorf, Adam T.; Myers, Richard H.

    2015-01-01

    Despite 20 years since its discovery, the gene responsible for Huntington’s Disease, HTT, has still not had its function or transcriptional profile completely characterized. In response to a recent report by Ruzo et al. of several novel splice forms of HTT in human embryonic stem cell lines, we have analyzed a set of mRNA sequencing datasets from post mortem human brain from Huntington’s disease, Parkinson’s disease, and neurologically normal control subjects to evaluate support for previously observed and to identify novel splice patterns. A custom analysis pipeline produced supporting evidence for some of the results reported by two previous studies of alternative isoforms as well as identifying previously unreported splice patterns. All of the alternative splice patterns were of relatively low abundance compared to the canonical splice form. PMID:26496077

  8. Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

    SciTech Connect

    O'Malley, K.L.; Anhalt, M.J.; Martin, B.M.; Kelsoe, J.R.; Winfield, S.L.; Ginns, E.I.

    1987-11-03

    A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.

  9. The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA.

    PubMed Central

    Hodges, D; Cripps, R M; O'Connor, M E; Bernstein, S I

    1999-01-01

    Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle. PMID:9872965

  10. Statistical and Computational Methods for High-Throughput Sequencing Data Analysis of Alternative Splicing

    PubMed Central

    2013-01-01

    The burgeoning field of high-throughput sequencing significantly improves our ability to understand the complexity of transcriptomes. Alternative splicing, as one of the most important driving forces for transcriptome diversity, can now be studied at an unprecedent resolution. Efficient and powerful computational and statistical methods are in urgent need to facilitate the characterization and quantification of alternative splicing events. Here we discuss methods in splice junction read mapping, and methods in exon-centric or isoform-centric quantification of alternative splicing. In addition, we discuss HITS-CLIP and splicing QTL analyses which are novel high-throughput sequencing based approaches in the dissection of splicing regulation. PMID:24058384

  11. Analyzing alternative splicing data of splice junction arrays from Parkinson patients' leukocytes before and after deep brain stimulation as compared with control donors.

    PubMed

    Soreq, Lilach; Salomonis, Nathan; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2015-09-01

    Few studies so far examined alternative splicing alterations in blood cells of neurodegenerative disease patients, particularly Parkinson's disease (PD). Prototype junction microarrays interrogate known human genome junctions and enable characterization of alternative splicing events; however, the analysis is not straightforward and different methods can be used to estimate junction-specific alternative splicing events (some of which can also be applied for analyzing RNA sequencing junction-level data). In this study, we characterized alternative splicing changes in blood leukocyte samples from Parkinson's patients prior to, and following deep brain stimulation (DBS) treatment; both on stimulation and following 1 h off electrical stimulation. Here, we describe in detail analysis approaches for junction microarrays and provide suggestions for further analyses to delineate transcript level effects of the observed alterations as well as detection of microRNA binding sites and protein domains in the alternatively spliced target regions spanning across both untranslated and the coding regions of the targets. The raw expression data files are publically available in the Gene Expression Omnibus (GEO) database (accession number: GSE37591) and in Synapse, and can be re-analyzed. The results may be useful for designing of future experiments and cross correlations with other datasets from PD or patients having other neurodegenerative diseases. PMID:26484282

  12. HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements.

    PubMed

    Kabat, Jennifer L; Barberan-Soler, Sergio; Zahler, Alan M

    2009-10-16

    Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence. PMID:19706616

  13. Alternative splicing of EKLF/KLF1 in murine primary erythroid tissues.

    PubMed

    Yien, Yvette Y; Gnanapragasam, Merlin Nithya; Gupta, Ritama; Rivella, Stefano; Bieker, James J

    2015-01-01

    Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non-erythroid Krppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krppel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation. PMID:25283745

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

    PubMed Central

    Hestand, Matthew S.; Zeng, Zheng; Coleman, Stephen J.; Liu, Jinze; MacLeod, James N.

    2015-01-01

    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. PMID:26713731

  15. RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA

    PubMed Central

    Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

    2013-01-01

    Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5? and 3? untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

  16. The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation

    PubMed Central

    Tripathi, Vidisha; Ellis, Jonathan D.; Shen, Zhen; Song, David Y.; Pan, Qun; Watt, Andrew T.; Freier, Susan M.; Bennett, C. Frank; Sharma, Alok; Bubulya, Paula A.; Blencowe, Benjamin J.; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2014-01-01

    SUMMARY Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression. PMID:20797886

  17. [Alternative Splicing Detection as a Biomarker for Cancer Diagnosis: A Novel Progressive Mechanism of Acute Lymphoblastic Leukemia with Alternative Splicing as a Biomarker Candidate].

    PubMed

    Kitamura, Kouichi; Matsushita, Kazuyuki; Kobayashi, Souhei; Ishige, Takayuki; Semba, Toshihisa; Kimura, Asako; Kazami, Takahiro; Ohyama, Masayuki; Itoga, Sakae; Beppu, Minako; Nishimura, Motoi; Satoh, Mamoru; Nomura, Fumio

    2015-09-01

    Alternative splicing is an important mechanism that links to transcription and contributes to protein diversity. Disturbed alternative splicing is frequently observed in cancers, but its precise mechanism remains largely unknown. FUSE-binding protein (FBP) -interacting repressor (FIR) is a transcriptional repressor of the c-myc gene. Previous studies indicated that a splice variant of FIR, FIR?exon2, that lacks exon2 in the transcriptional repressor domain, was increased in colorectal cancers, hepatocellular carcinomas, and leukemia cells. Furthermore, FIR?exon2 activated c-myc transcription by disabling wild-type FIR as a dominant-negative form of FIR. Recently, somatic mutations of the SF3B1 (SAP155) gene, a subunit of the SF3B spliceosome complex, were found in myelodysplastic leukemia. In this study, FIR heterozygous knockout (FIR(+/-)) was established as a dominant-negative model of FIR in the C57BL/6 mouse. FIR(+/-) mice showed an increased c-myc mRNA expression level, particularly in peripheral blood, although FIR(+/-) mice had no apparent pathogenic phenotype. Therefore, an increased c-myc mRNA expression level alone is not enough for leukemogenesis. Nevertheless, FIR(+/-)TP53(-/-) mice generated acute T-cell lymphoblastic leukemia (T-ALL) with increased organ and/or bone marrow invasion. In conclusion, alternative splicing of FIR, generating FIR?exon2, contributes to not only colorectal carcinogenesis but also leukemogenesis independent of the c-Myc activation pathway. Finally, we will discuss our hypothesis that FIR?exon2 interferes with FBW7, that FIR?exon2 inhibits PP1 in the EGFR pathway, and that FIR haploinsufficiency is potentially associated with protein expression through transcriptional and post-transcriptional mechanisms. PMID:26731899

  18. SRSF1-Regulated Alternative Splicing in Breast Cancer.

    PubMed

    Anczukw, Olga; Akerman, Martin; Clry, Antoine; Wu, Jie; Shen, Chen; Shirole, Nitin H; Raimer, Amanda; Sun, Shuying; Jensen, Mads A; Hua, Yimin; Allain, Frdric H-T; Krainer, Adrian R

    2015-10-01

    Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5' splice site generally promoted exon inclusion, whereas binding near the 3' splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development. PMID:26431027

  19. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    PubMed

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation. PMID:26997278

  20. Ras Promotes Growth by Alternative Splicing-Mediated Inactivation of the KLF6 Tumor Suppressor in Hepatocellular Carcinoma

    PubMed Central

    Yea, Steven; Narla, Goutham; Zhao, Xiao; Garg, Rakhi; Tal-Kremer, Sigal; Hod, Eldad; Villanueva, Augusto; Loke, Johnny; Tarocchi, Mirko; Akita, Kunihara; Shirasawa, Senji; Sasazuki, Takehiko; Martignetti, John A; Llovet, Josep M; Friedman, Scott L

    2008-01-01

    Background & Aims HCC is the 5th most prevalent cancer worldwide and the 3rd most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been fully explored. The KLF6 gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers due to increased alternative splicing that yields a dominant negative isoform, KLF6 SV1, which antagonizes full-length KLF6 (KLF6Full)-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown. Methods In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by PI-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2. Results In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to KLF6Full, thereby enhancing proliferation. Abrogation of oncogenic Ras-signaling by siRNA or a farnesyl-transferase inhibitor (FTS) decreases KLF6 SV1 and suppresses growth. Growth inhibition by FTS in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 mRNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation. Conlcusions Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade. PMID:18471523

  1. SRRM2, a Potential Blood Biomarker Revealing High Alternative Splicing in Parkinson's Disease

    PubMed Central

    Shehadeh, Lina A.; Yu, Kristine; Wang, Liyong; Guevara, Alexandra; Singer, Carlos; Vance, Jeffery; Papapetropoulos, Spyridon

    2010-01-01

    Background Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects about five million people worldwide. Diagnosis remains clinical, based on phenotypic patterns. The discovery of laboratory markers that will enhance diagnostic accuracy, allow pre-clinical detection and tracking of disease progression is critically needed. These biomarkers may include transcripts with different isoforms. Methodology/Principal Findings We performed extensive analysis on 3 PD microarray experiments available through GEO and found that the RNA splicing gene SRRM2 (or SRm300), sereine/arginine repetitive matrix 2, was the only gene differentially upregulated among all the three PD experiments. SRRM2 expression was not changed in the blood of other neurological diseased patients versus the healthy controls. Using real-time PCR, we report that the shorter transcript of SRRM2 was 1.7 fold (p?=?0.008) upregulated in the substantia nigra of PDs vs controls while the longer transcript was 0.4 downregulated in both the substantia nigra (p?=?0.03) and amygdala (p?=?0.003). To validate our results and test for the possibility of alternative splicing in PD, we performed independent microarray scans, using Affymetrix Exon_ST1 arrays, from peripheral blood of 28 individuals (17 PDs and 11 Ctrls) and found a significant upregulation of the upstream (5?) exons of SRRM2 and a downregulation of the downstream exons, causing a total of 0.7 fold down regulation (p?=?0.04) of the long isoform. In addition, we report novel information about hundreds of genes with significant alternative splicing (differential exonic expression) in PD blood versus controls. Conclusions/Significance The consistent dysregulation of the RNA splicing factor SRRM2 in two different PD neuronal sources and in PD blood but not in blood of other neurologically diseased patients makes SRRM2 a strong candidate gene for PD and draws attention to the role of RNA splicing in the disease. PMID:20161708

  2. Exon organization and novel alternative splicing of Ank3 in mouse heart.

    PubMed

    Yamankurt, Gokay; Wu, Henry C; McCarthy, Michael; Cunha, Shane R

    2015-01-01

    Ankyrin-G is an adaptor protein that links membrane proteins to the underlying cytoskeletal network. Alternative splicing of the Ank3 gene gives rise to multiple ankyrin-G isoforms in numerous tissues. To date, only one ankyrin-G isoform has been characterized in heart and transcriptional regulation of the Ank3 gene is completely unknown. In this study, we describe the first comprehensive analysis of Ank3 expression in heart. Using a PCR-based screen of cardiac mRNA transcripts, we identify two new exons and 28 alternative splice variants of the Ank3 gene. We measure the relative expression of each splice variant using quantitative real-time PCR and exon-exon boundary spanning primers that specifically amplify individual Ank3 variants. Six variants are rarely expressed (<1%), while the remaining variants display similar expression patterns in three hearts. Of the five first exons in the Ank3 gene, exon 1d is only expressed in heart and skeletal muscle as it was not detected in brain, kidney, cerebellum, and lung. Immunoblot analysis reveals multiple ankyrin-G isoforms in heart, and two ankyrin-G subpopulations are detected in adult cardiomyocytes by immunofluorescence. One population co-localizes with the voltage-gated sodium channel NaV1.5 at the intercalated disc, while the other population expresses at the Z-line. Two of the rare splice variants excise a portion of the ZU5 motif, which encodes the minimal spectrin-binding domain, and these variants lack ?-spectrin binding. Together, these data demonstrate that Ank3 is subject to complex splicing regulation resulting in a diverse population of ankyrin-G isoforms in heart. PMID:26024478

  3. Exon Organization and Novel Alternative Splicing of Ank3 in Mouse Heart

    PubMed Central

    Yamankurt, Gokay; Wu, Henry C.; McCarthy, Michael; Cunha, Shane R.

    2015-01-01

    Ankyrin-G is an adaptor protein that links membrane proteins to the underlying cytoskeletal network. Alternative splicing of the Ank3 gene gives rise to multiple ankyrin-G isoforms in numerous tissues. To date, only one ankyrin-G isoform has been characterized in heart and transcriptional regulation of the Ank3 gene is completely unknown. In this study, we describe the first comprehensive analysis of Ank3 expression in heart. Using a PCR-based screen of cardiac mRNA transcripts, we identify two new exons and 28 alternative splice variants of the Ank3 gene. We measure the relative expression of each splice variant using quantitative real-time PCR and exon-exon boundary spanning primers that specifically amplify individual Ank3 variants. Six variants are rarely expressed (<1%), while the remaining variants display similar expression patterns in three hearts. Of the five first exons in the Ank3 gene, exon 1d is only expressed in heart and skeletal muscle as it was not detected in brain, kidney, cerebellum, and lung. Immunoblot analysis reveals multiple ankyrin-G isoforms in heart, and two ankyrin-G subpopulations are detected in adult cardiomyocytes by immunofluorescence. One population co-localizes with the voltage-gated sodium channel NaV1.5 at the intercalated disc, while the other population expresses at the Z-line. Two of the rare splice variants excise a portion of the ZU5 motif, which encodes the minimal spectrin-binding domain, and these variants lack β-spectrin binding. Together, these data demonstrate that Ank3 is subject to complex splicing regulation resulting in a diverse population of ankyrin-G isoforms in heart. PMID:26024478

  4. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

    PubMed Central

    Holm, Frida; Hellqvist, Eva; Mason, Cayla N.; Ali, Shawn A.; Delos-Santos, Nathaniel; Barrett, Christian L.; Chun, Hye-Jung; Minden, Mark D.; Moore, Richard A.; Marra, Marco A.; Runza, Valeria; Frazer, Kelly A.; Sadarangani, Anil; Jamieson, Catriona H. M.

    2015-01-01

    Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination. PMID:26621726

  5. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal.

    PubMed

    Holm, Frida; Hellqvist, Eva; Mason, Cayla N; Ali, Shawn A; Delos-Santos, Nathaniel; Barrett, Christian L; Chun, Hye-Jung; Minden, Mark D; Moore, Richard A; Marra, Marco A; Runza, Valeria; Frazer, Kelly A; Sadarangani, Anil; Jamieson, Catriona H M

    2015-12-15

    Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination. PMID:26621726

  6. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing

    PubMed Central

    Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

    2010-01-01

    Alternative pre-mRNA splicing predominates in the nervous systems of complex organisms including humans dramatically expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type CaV2.2 calcium channels in nociceptors. Using an exon replacement strategy in mice, we show that mutually exclusive splicing in the CaV2.2 gene modulates N-type channel function in nociceptors leading to a change in morphine analgesia. Exon 37a enhances ?-opioid receptor mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a spinal morphine analgesia is weakened in vivo without influencing the basal response to noxious thermal stimuli. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons. PMID:20852623

  7. Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing

    PubMed Central

    Douglas, Katherine B.; Windels, Daniel C.; Zhao, Jian; Gadeliya, Agnessa V.; Wu, Hui; Kaufman, Kenneth M.; Harley, John B.; Merrill, Joan; Kimberly, Robert P.; Alarcn, Graciela S.; Brown, Elizabeth E.; Edberg, Jeffrey C.; Ramsey-Goldman, Rosalind; Petri, Michelle; Reveille, John D.; Vil, Luis M.; Gaffney, Patrick M.; James, Judith A.; Moser, Kathy L.; Alarcn-Riquelme, Marta E.; Vyse, Timothy J.; Gilkeson, Gary S.; Jacob, Chaim O.; Ziegler, Julie T.; Langefeld, Carl D.; Ulgiati, Daniela; Tsao, Betty P.; Boackle, Susan A.

    2009-01-01

    Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs. 32.6% in controls, P = 0.016, OR = 0.90 [0.82-0.98]). Two of these SNPs are in exon 10, directly 5? of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs as well as a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact. PMID:19387458

  8. Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.

    PubMed Central

    Luneau, C J; Williams, J B; Marshall, J; Levitan, E S; Oliva, C; Smith, J S; Antanavage, J; Folander, K; Stein, R B; Swanson, R

    1991-01-01

    In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA. Images PMID:2023941

  9. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes.

    PubMed

    Paraboschi, Elvezia Maria; Cardamone, Giulia; Rimoldi, Valeria; Gemmati, Donato; Spreafico, Marta; Duga, Stefano; Sold, Giulia; Asselta, Rosanna

    2015-01-01

    Abnormalities in RNA metabolism and alternative splicing (AS) are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS) and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls), followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p=0.0015) by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes. PMID:26437396

  10. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

    PubMed Central

    Paraboschi, Elvezia Maria; Cardamone, Giulia; Rimoldi, Valeria; Gemmati, Donato; Spreafico, Marta; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

    2015-01-01

    Abnormalities in RNA metabolism and alternative splicing (AS) are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS) and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls), followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p = 0.0015) by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes. PMID:26437396

  11. Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons

    PubMed Central

    Toonen, Ruud F.; Verhage, Matthijs

    2015-01-01

    The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions. Furthermore, Munc18-1a is the major splice variant in the Calyx of Held. Synaptic transmission was analyzed in autaptic hippocampal munc18-1 KO neurons re-expressing either Munc18-1a or Munc18-1b. The two splice variants supported synaptic transmission to a similar extent, but Munc18-1b was slightly more potent in sustaining synchronous release during high frequency stimulation. Our data suggest that alternative splicing of Munc18-1 support synaptic transmission to a similar extent, but could modulate presynaptic short-term plasticity. PMID:26407320

  12. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    SciTech Connect

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi . E-mail: hiro@pharm.hokudai.ac.jp; Iguchi-Ariga, Sanae M.M.

    2005-02-15

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF{sup 35}. CIR was found to interact with U2AF{sup 35} through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.

  13. Alternative Splicing of the LIM-Homeodomain Transcription Factor Isl1 in the Mouse Retina

    PubMed Central

    Whitney, Irene E.; Kautzman, Amanda G.; Reese, Benjamin E.

    2015-01-01

    Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1β, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1 β expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system. PMID:25752730

  14. Coordinated tissue-specific regulation of adjacent alternative 3? splice sites in C. elegans

    PubMed Central

    Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

    2015-01-01

    Adjacent alternative 3? splice sites, those separated by ?18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3? end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3? splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3? splice site (furthest from the 5? end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3? splice site (closer to the 5? end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3? splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3? splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3? splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

  15. The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels

    PubMed Central

    Allen, Summer E; Darnell, Robert B

    2010-01-01

    Many cellular processes are involved in optimizing protein function for specific neuronal tasks; here we focus on alternative pre-mRNA splicing. Alternative pre-mRNA splicing gives cells the capacity to modify and selectively re-balance their existing pool of transcripts in a coordinated way across multiple mRNAs, thereby effecting relatively rapid and relatively stable changes in protein activity. Here we report on and discuss the coordinated regulation of two sites of alternative splicing, e24a and e31a, in P-type CaV2.1 and N-type CaV2.2 channels. These two exons encode 4 and 2 amino acids, respectively, in the extracellular linker regions between transmembrane spanning segments S3 and S4 in domains III and IV of each CaV2 subunit. Recent genome-wide screens of splicing factor-RNA binding events by Darnell and colleagues show that Nova-2 promotes inclusion of e24a in CaV2.2 mRNAs in brain. We review these studies and show that a homologous e24a is present in the CaV2.1 gene, Cacna1a, and that it is expressed in different regions of the nervous system. Nova-2 enhances inclusion of e24a but represses e31a inclusion in CaV2.1 and CaV2.2 mRNAs in brain. It is likely that coordinated alternative pre-mRNA splicing across related CaV2 genes by common splicing factors allows neurons to orchestrate changes in synaptic protein function while maintaining a balanced and functioning system. PMID:21150296

  16. Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

    PubMed

    Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

    2016-04-15

    Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. PMID:26599481

  17. Novel alternative splicing isoform biomarkers identification from high-throughput plasma proteomics profiling of breast cancer

    PubMed Central

    2013-01-01

    Background In the biopharmaceutical industry, biomarkers define molecular taxonomies of patients and diseases and serve as surrogate endpoints in early-phase drug trials. Molecular biomarkers can be much more sensitive than traditional lab tests. Discriminating disease biomarkers by traditional method such as DNA microarray has proved challenging. Alternative splicing isoform represents a new class of diagnostic biomarkers. Recent scientific evidence is demonstrating that the differentiation and quantification of individual alternative splicing isoforms could improve insights into disease diagnosis and management. Identifying and characterizing alternative splicing isoforms are essential to the study of molecular mechanisms and early detection of complex diseases such as breast cancer. However, there are limitations with traditional methods used for alternative splicing isoform determination such as transcriptome-level, low level of coverage and poor focus on alternative splicing. Results Therefore, we presented a peptidomics approach to searching novel alternative splicing isoforms in clinical proteomics. Our results showed that the approach has significant potential in enabling discovery of new types of high-quality alternative splicing isoform biomarkers. Conclusions We developed a peptidomics approach for the proteomics community to analyze, identify, and characterize alternative splicing isoforms from MS-based proteomics experiments with more coverage and exclusive focus on alternative splicing. The approach can help generate novel hypotheses on molecular risk factors and molecular mechanisms of cancer in early stage, leading to identification of potentially highly specific alternative splicing isoform biomarkers for early detection of cancer. PMID:24565027

  18. Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation

    PubMed Central

    Allemand, Eric; Gattoni, Renata; Bourbon, Henri-Marc; Stevenin, James; Cceres, Javier F.; Soret, Johann; Tazi, Jamal

    2001-01-01

    The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5? alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo. PMID:11158320

  19. MECHANISMS IN ENDOCRINOLOGY: Alternative splicing: the new frontier in diabetes research.

    PubMed

    Juan-Mateu, Jonàs; Villate, Olatz; Eizirik, Décio L

    2016-05-01

    Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic β cells are killed by infiltrating immune cells and by cytokines released by these cells. This takes place in the context of a dysregulated dialogue between invading immune cells and target β cells, but the intracellular signals that decide β cell fate remain to be clarified. Alternative splicing (AS) is a complex post-transcriptional regulatory mechanism affecting gene expression. It regulates the inclusion/exclusion of exons into mature mRNAs, allowing individual genes to produce multiple protein isoforms that expand the proteome diversity. Functionally related transcript populations are co-ordinately spliced by master splicing factors, defining regulatory networks that allow cells to rapidly adapt their transcriptome in response to intra and extracellular cues. There is a growing interest in the role of AS in autoimmune diseases, but little is known regarding its role in T1D. In this review, we discuss recent findings suggesting that splicing events occurring in both immune and pancreatic β cells contribute to the pathogenesis of T1D. Splicing switches in T cells and in lymph node stromal cells are involved in the modulation of the immune response against β cells, while β cells exposed to pro-inflammatory cytokines activate complex splicing networks that modulate β cell viability, expression of neoantigens and susceptibility to immune-induced stress. Unveiling the role of AS in β cell functional loss and death will increase our understanding of T1D pathogenesis and may open new avenues for disease prevention and therapy. PMID:26628584

  20. Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein

    SciTech Connect

    Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. )

    1992-03-03

    The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experiments show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.

  1. Alternative splicing: An important mechanism in stem cell biology

    PubMed Central

    Chen, Kenian; Dai, Xiaojing; Wu, Jiaqian

    2015-01-01

    Alternative splicing (AS) is an essential mechanism in post-transcriptional regulation and leads to protein diversity. It has been shown that AS is prevalent in metazoan genomes, and the splicing pattern is dynamically regulated in different tissues and cell types, including embryonic stem cells. These observations suggest that AS may play critical roles in stem cell biology. Since embryonic stem cells and induced pluripotent stem cells have the ability to give rise to all types of cells and tissues, they hold the promise of future cell-based therapy. Many efforts have been devoted to understanding the mechanisms underlying stem cell self-renewal and differentiation. However, most of the studies focused on the expression of a core set of transcription factors and regulatory RNAs. The role of AS in stem cell differentiation was not clear. Recent advances in high-throughput technologies have allowed the profiling of dynamic splicing patterns and cis-motifs that are responsible for AS at a genome-wide scale, and provided novel insights in a number of studies. In this review, we discuss some recent findings involving AS and stem cells. An emerging picture from these findings is that AS is integrated in the transcriptional and post-transcriptional networks and together they control pluripotency maintenance and differentiation of stem cells. PMID:25621101

  2. Transcriptome-wide Landscape of Pre-mRNA Alternative Splicing Associated with Metastatic Colonization

    PubMed Central

    Lu, Zhi-xiang; Huang, Qin; Park, Juw Won; Shen, Shihao; Lin, Lan; Tokheim, Collin J.; Henry, Michael D.; Xing, Yi

    2014-01-01

    Metastatic colonization is an ominous feature of cancer progression. Recent studies have established the importance of pre-mRNA alternative splicing (AS) in cancer biology. However, little is known about the transcriptome-wide landscape of AS associated with metastatic colonization. Both in vitro and in vivo models of metastatic colonization were utilized to study AS regulation associated with cancer metastasis. Transcriptome profiling of prostate cancer cells and derivatives crossing in vitro or in vivo barriers of metastasis revealed splicing factors with significant gene expression changes associated with metastatic colonization. These include splicing factors known to be differentially regulated in epithelial-mesenchymal transition (ESRP1, ESRP2, RBFOX2), a cellular process critical for cancer metastasis, as well as novel findings (NOVA1, MBNL3). Finally, RNA-seq indicated a large network of AS events regulated by multiple splicing factors with altered gene expression or protein activity. These AS events are enriched for pathways important for cell motility and signaling, and affect key regulators of the invasive phenotype such as CD44 and GRHL1. PMID:25274489

  3. Modeling Alternative Splicing Variants from RNA-Seq Data with Isoform Graphs

    PubMed Central

    Beretta, Stefano; Vedova, Gianluca Della; Pirola, Yuri; Rizzi, Raffaella

    2014-01-01

    Abstract Next-generation sequencing (NGS) technologies need new methodologies for alternative splicing (AS) analysis. Current computational methods for AS analysis from NGS data are mainly based on aligning short reads against a reference genome, while methods that do not need a reference genome are mostly underdeveloped. In this context, the main developed tools for NGS data focus on de novo transcriptome assembly (Grabherr et al., 2011; Schulz et al., 2012). While these tools are extensively applied for biological investigations and often show intrinsic shortcomings from the obtained results, a theoretical investigation of the inherent computational limits of transcriptome analysis from NGS data, when a reference genome is unknown or highly unreliable, is still missing. On the other hand, we still lack methods for computing the gene structures due to AS events under the above assumptions—a problem that we start to tackle with this article. More precisely, based on the notion of isoform graph (Lacroix et al., 2008), we define a compact representation of gene structures—called splicing graph—and investigate the computational problem of building a splicing graph that is (i) compatible with NGS data and (ii) isomorphic to the isoform graph. We characterize when there is only one representative splicing graph compatible with input data, and we propose an efficient algorithmic approach to compute this graph. PMID:24200390

  4. Modeling alternative splicing variants from RNA-Seq data with isoform graphs.

    PubMed

    Beretta, Stefano; Bonizzoni, Paola; Vedova, Gianluca Della; Pirola, Yuri; Rizzi, Raffaella

    2014-01-01

    Next-generation sequencing (NGS) technologies need new methodologies for alternative splicing (AS) analysis. Current computational methods for AS analysis from NGS data are mainly based on aligning short reads against a reference genome, while methods that do not need a reference genome are mostly underdeveloped. In this context, the main developed tools for NGS data focus on de novo transcriptome assembly (Grabherr et al., 2011 ; Schulz et al., 2012). While these tools are extensively applied for biological investigations and often show intrinsic shortcomings from the obtained results, a theoretical investigation of the inherent computational limits of transcriptome analysis from NGS data, when a reference genome is unknown or highly unreliable, is still missing. On the other hand, we still lack methods for computing the gene structures due to AS events under the above assumptions--a problem that we start to tackle with this article. More precisely, based on the notion of isoform graph (Lacroix et al., 2008), we define a compact representation of gene structures--called splicing graph--and investigate the computational problem of building a splicing graph that is (i) compatible with NGS data and (ii) isomorphic to the isoform graph. We characterize when there is only one representative splicing graph compatible with input data, and we propose an efficient algorithmic approach to compute this graph. PMID:24200390

  5. ASPicDB: a database of annotated transcript and protein variants generated by alternative splicing

    PubMed Central

    Martelli, Pier L.; DAntonio, Mattia; Bonizzoni, Paola; Castrignan, Tiziana; DErchia, Anna M.; DOnorio De Meo, Paolo; Fariselli, Piero; Finelli, Michele; Licciulli, Flavio; Mangiulli, Marina; Mignone, Flavio; Pavesi, Giulio; Picardi, Ernesto; Rizzi, Raffaella; Rossi, Ivan; Valletti, Alessio; Zauli, Andrea; Zambelli, Federico; Casadio, Rita; Pesole, Graziano

    2011-01-01

    Alternative splicing is emerging as a major mechanism for the expansion of the transcriptome and proteome diversity, particularly in human and other vertebrates. However, the proportion of alternative transcripts and proteins actually endowed with functional activity is currently highly debated. We present here a new release of ASPicDB which now provides a unique annotation resource of human protein variants generated by alternative splicing. A total of 256?939 protein variants from 17?191 multi-exon genes have been extensively annotated through state of the art machine learning tools providing information of the protein type (globular and transmembrane), localization, presence of PFAM domains, signal peptides, GPI-anchor propeptides, transmembrane and coiled-coil segments. Furthermore, full-length variants can be now specifically selected based on the annotation of CAGE-tags and polyA signal and/or polyA sites, marking transcription initiation and termination sites, respectively. The retrieval can be carried out at gene, transcript, exon, protein or splice site level allowing the selection of data sets fulfilling one or more features settled by the user. The retrieval interface also enables the selection of protein variants showing specific differences in the annotated features. ASPicDB is available at http://www.caspur.it/ASPicDB/. PMID:21051348

  6. Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis.

    PubMed

    Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

    2015-11-01

    Gravitropism is an important growth movement in response to gravity in virtually all higher plants: the roots showing positive gravitropism and the shoots showing negative gravitropism. The gravitropic orientation of plant organs is also influenced by environmental factors, such as light and temperature. It is known that a zinc finger (ZF)-containing transcription factor SHOOT GRAVITROPISM 5/INDETERMINATE DOMAIN 15 (SGR5/IDD15) mediates the early events of gravitropic responses occurring in inflorescence stems. We have recently found that SGR5 gene undergoes alternative splicing to produce 2 protein variants, the full-size SGR5? transcription factor and the truncated SGR5? form lacking functional ZF motifs. The SGR5? form inhibits SGR5? function possibly by forming nonfunctional heterodimers that are excluded from DNA binding. Notably, SGR5 alternative splicing is accelerated at high temperatures, resulting in a high-level accumulation of SGR5? proteins. Accordingly, transgenic plants overexpressing SGR5? exhibit a reduction in the negative gravitropism of inflorescence stems, as observed in the SGR5-defective mutant. It is proposed that the thermos-responsive alternative splicing of SGR5 gene provides an adaptation strategy by which plants protect the shoots from aerial heat frequently occurring in natural habitats. PMID:26452406

  7. Survey of Programs Used to Detect Alternative Splicing Isoforms from Deep Sequencing Data In Silico

    PubMed Central

    Min, Feng; Wang, Sumei; Zhang, Li

    2015-01-01

    Next-generation sequencing techniques have been rapidly emerging. However, the massive sequencing reads hide a great deal of unknown important information. Advances have enabled researchers to discover alternative splicing (AS) sites and isoforms using computational approaches instead of molecular experiments. Given the importance of AS for gene expression and protein diversity in eukaryotes, detecting alternative splicing and isoforms represents a hot topic in systems biology and epigenetics research. The computational methods applied to AS prediction have improved since the emergence of next-generation sequencing. In this study, we introduce state-of-the-art research on AS and then compare the research methods and software tools available for AS based on next-generation sequencing reads. Finally, we discuss the prospects of computational methods related to AS. PMID:26421304

  8. ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization.

    PubMed

    Castrignan, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

    2006-07-01

    Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource-available at http://www.caspur.it/ASPIC/--is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. PMID:16845044

  9. ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization

    PubMed Central

    Castrignan, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

    2006-01-01

    Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resourceavailable at is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. PMID:16845044

  10. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  11. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    PubMed

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. PMID:26320575

  12. Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia?

    PubMed Central

    Hulse, R.P.; Beazley-Long, N.; Hua, J.; Kennedy, H.; Prager, J.; Bevan, H.; Qiu, Y.; Fernandes, E.S.; Gammons, M.V.; Ballmer-Hofer, K.; Gittenberger de Groot, A.C.; Churchill, A.J.; Harper, S.J.; Brain, S.D.; Bates, D.O.; Donaldson, L.F.

    2014-01-01

    Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event leading to the preferential expression of VEGF-A165b over VEGF165a prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. PMID:25151644

  13. Structural and functional analyses of Barth syndrome-causing mutations and alternative splicing in the tafazzin acyltransferase domain

    PubMed Central

    Hijikata, Atsushi; Yura, Kei; Ohara, Osamu; Go, Mitiko

    2015-01-01

    Tafazzin is a mitochondrial phospholipid transacylase, and its mutations cause Barth syndrome (BTHS). Human tafazzin gene produces four distinct alternatively spliced transcripts. To understand the molecular mechanisms of tafazzin deficiency, we performed an atomic resolution analysis of the influence of the BTHS mutations and of alternative splicing on the structure and function of tafazzin. From the three-dimensional (3D) homology modeling of tafazzin, we identified candidate amino acid residues that contribute to cardiolipin binding and to mitochondrial membrane associations that facilitate acyl-transfer reactions. Primate specific exon 5, which is alternatively spliced, is predicted to correspond to an intrinsically unstructured region in the protein. We proposed that this region should change the substrate-binding affinity and/or contribute to primate-specific molecular interactions. Exon 7, another alternatively spliced exon, encodes a region forming a part of the putative substrate-binding cleft, suggesting that the gene products lacking exon 7 will lose their substrate-binding ability. We demonstrate a clear localization of the BTHS mutations at residues responsible for membrane association, substrate binding, and the conformational stability of tafazzin. These findings provide new insights into the function of defective tafazzin and the pathogenesis of BTHS at the level of protein 3D structure and the evolution of alternatively spliced exons in primates. PMID:25941633

  14. Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths.

    PubMed

    Shirk, Paul D; Perera, Omaththage P; Shelby, Kent S; Furlong, Richard B; LoVullo, Eric D; Popham, Holly J R

    2015-12-10

    Chitin is an extracellular biopolymer that contributes to the cuticular structural matrix in arthropods. As a consequence of its rigid structure, the chitinous cuticle must be shed and replaced to accommodate growth. Two chitin synthase genes that encode for chitin synthase A (ChSA), which produces cuticular exoskeleton, and chitin synthase B (ChSB), which produces peritrophic membrane, were characterized in the genomes of two heliothine moths: the corn earworm/cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). In both moths, the two genes were arranged in tandem with the same orientation on the same strand with ChSB located 5' of ChSA. Sequence comparisons showed that the coding sequences were highly conserved with homologues from other species but that the tandem juxtaposed genomic arrangement of the two genes was unique in these insects. The mechanism that has led to this arrangement is unclear but is most likely a recent recombinational event. Transcript mapping of HzChSB and HzChSA in H. zea demonstrated that both transcripts were differentially spliced in various tissues and larval stages. The identification of the HzChSB-E12b alternate spliced transcript is the first report of alternate splicing for the ChSB group. The importance of this splice form is not clear because the protein produced would lack any enzymatic activity but retain the membrane insertion motifs. As for other insects, these genes provide an important target for potential control through RNAi but also provide a subject for broad scale genomic recombinational events. PMID:26253161

  15. The in vivo use of alternate 3'-splice sites in group I introns.

    PubMed Central

    Sellem, C H; Belcour, L

    1994-01-01

    Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used. Images PMID:8165125

  16. Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera).

    PubMed

    Jarosch, Antje; Stolle, Eckart; Crewe, Robin M; Moritz, Robin F A

    2011-09-13

    In eusocial insects the production of daughters is generally restricted to mated queens, and unmated workers are functionally sterile. The evolution of this worker sterility has been plausibly explained by kin selection theory [Hamilton W (1964) J Theor Biol 7:1-52], and many traits have evolved to prevent conflict over reproduction among the females in an insect colony. In honeybees (Apis mellifera), worker reproduction is regulated by the queen, brood pheromones, and worker policing. However, workers of the Cape honeybee, Apis mellifera capensis, can evade this control and establish themselves as social parasites by activating their ovaries, parthenogenetically producing diploid female offspring (thelytoky) and producing queen-like amounts of queen pheromones. All these traits have been shown to be strongly influenced by a single locus on chromosome 13 [Lattorff HMG, et al. (2007) Biol Lett 3:292-295]. We screened this region for candidate genes and found that alternative splicing of a gene homologous to the gemini transcription factor of Drosophila controls worker sterility. Knocking out the critical exon in a series of RNAi experiments resulted in rapid worker ovary activation-one of the traits characteristic of the social parasites. This genetic switch may be controlled by a short intronic splice enhancer motif of nine nucleotides attached to the alternative splice site. The lack of this motif in parasitic Cape honeybee clones suggests that the removal of nine nucleotides from the altruistic worker genome may be sufficient to turn a honeybee from an altruistic worker into a parasite. PMID:21896748

  17. Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera)

    PubMed Central

    Jarosch, Antje; Stolle, Eckart; Crewe, Robin M.; Moritz, Robin F. A.

    2011-01-01

    In eusocial insects the production of daughters is generally restricted to mated queens, and unmated workers are functionally sterile. The evolution of this worker sterility has been plausibly explained by kin selection theory [Hamilton W (1964) J Theor Biol 7:152], and many traits have evolved to prevent conflict over reproduction among the females in an insect colony. In honeybees (Apis mellifera), worker reproduction is regulated by the queen, brood pheromones, and worker policing. However, workers of the Cape honeybee, Apis mellifera capensis, can evade this control and establish themselves as social parasites by activating their ovaries, parthenogenetically producing diploid female offspring (thelytoky) and producing queen-like amounts of queen pheromones. All these traits have been shown to be strongly influenced by a single locus on chromosome 13 [Lattorff HMG, et al. (2007) Biol Lett 3:292295]. We screened this region for candidate genes and found that alternative splicing of a gene homologous to the gemini transcription factor of Drosophila controls worker sterility. Knocking out the critical exon in a series of RNAi experiments resulted in rapid worker ovary activationone of the traits characteristic of the social parasites. This genetic switch may be controlled by a short intronic splice enhancer motif of nine nucleotides attached to the alternative splice site. The lack of this motif in parasitic Cape honeybee clones suggests that the removal of nine nucleotides from the altruistic worker genome may be sufficient to turn a honeybee from an altruistic worker into a parasite. PMID:21896748

  18. Structural determinants for alternative splicing regulation of the MAPT pre-mRNA.

    PubMed

    Lisowiec, Jolanta; Magner, Dorota; Kierzek, Elzbieta; Lenartowicz, Elzbieta; Kierzek, Ryszard

    2015-01-01

    Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms. (1) The presence of mutations, particularly in exon 10 and intron 10-11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data. PMID:25826665

  19. Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb

    PubMed Central

    Ganassi, M.; Badodi, S.; Polacchini, A.; Baruffaldi, F.; Battini, R.; Hughes, S.M.; Hinits, Y.; Molinari, S.

    2014-01-01

    In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 46 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 46), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 456) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity. PMID:24844180

  20. Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

    PubMed

    Kwiatkowski, Stefan; Sviripa, Vitaliy M; Zhang, Zhaiyi; Wendlandt, Alison E; Hbartner, Claudia; Watt, David S; Stamm, Stefan

    2016-02-01

    Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)oxidophosphoryl)oxy)ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing. PMID:26725024

  1. Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

    PubMed

    Wang, Xinping; Deng, Xuliang; Zhang, Xichen

    2012-01-01

    Amelogenin, the major enamel matrix protein in tooth development, has been demonstrated to play a significant role in tooth enamel formation. Previous studies have identified the alternative splicing of amelogenin in many mammalian vertebrates as one mechanism for amelogenin heterogeneous expression in teeth. While amelogenin and its splicing forms in mammalian vertebrates have been cloned and sequenced, the amelogenin gene, especially its splicing forms in non-mammalian species, remains largely unknown. To better understand the mechanism underlying amelogenin evolution, we previously cloned and characterized an amelogenin gene sequence from a squamate, the green iguana. In this study, we employed RT-PCR to amplify the amelogenin gene from the black spiny-tailed iguana Ctenosaura similis teeth, and discovered a novel splicing form of the amelogenin gene. The transcript of the newly identified iguana amelogenin gene (named C. Similis-T2L) is 873 nucleotides long encoding an expected polypeptide of 206 amino acids. The C. Similis-T2L contains a unique exon denominated exon X, which is located between exon 5 and exon 6. The C. Similis-T2L contains 7 exons including exon 1, 2, 3, 5, X, 6, and 7. Analysis of the secondary and tertiary structures of T2L amelogenin protein demonstrated that exon X has a dramatic effect on the amelogenin structures. This is the first report to provide definitive evidence for the amelogenin alternative splicing in non-mammalian vertebrates, revealing a unique exon X and the splicing form of the amelogenin gene transcript in Ctenosaura similis. PMID:23029286

  2. Alternative Splicing Regulates Prdm1/Blimp-1 DNA Binding Activities and Corepressor Interactions

    PubMed Central

    Morgan, Marc A. J.; Mould, Arne W.; Li, Li; Robertson, Elizabeth J.

    2012-01-01

    Prdm1/Blimp-1 is a master regulator of gene expression in diverse tissues of the developing embryo and adult organism. Its C-terminal zinc finger domain mediates nuclear import, DNA binding, and recruitment of the corepressors G9a and HDAC1/2. Alternatively spliced transcripts lacking exon 7 sequences encode a structurally divergent isoform (Blimp-1Δexon7) predicted to have distinct functions. Here we demonstrate that the short Blimp-1Δexon7 isoform lacks DNA binding activity and fails to bind G9a or HDAC1/2 but retains the ability to interact with PRMT5. To investigate functional roles of alternative splicing in vivo, we engineered novel mouse strains via embryonic stem (ES) cell technology. Like null mutants, embryos carrying a targeted deletion of exon 7 and exclusively expressing Blimp-1Δexon7 die at around embryonic day 10.5 (E10.5) due to placental defects. In heterozygous Δexon7 mice, there is no evidence of dominant-negative effects. Mice carrying a knock-in allele with an exon 6-exon 7 fusion express full-length Blimp-1 only, develop normally, are healthy and fertile as adults, and efficiently generate mature plasma cells. These findings strongly suggest that the short Blimp-1Δexon7 isoform is dispensable. We propose that developmentally regulated alternative splicing is influenced by chromatin structure at the locus and fine-tunes Blimp-1's functional capabilities. PMID:22733990

  3. The role played by alternative splicing in antigenic variability in human endo-parasites

    PubMed Central

    2014-01-01

    Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the hosts immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites. PMID:24472559

  4. Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation

    PubMed Central

    Tan, Lit-Yeen; Whitfield, Peter; Llorian, Miriam; Monzon-Casanova, Elisa; Diaz-Munoz, Manuel D.; Turner, Martin; Smith, Christopher W.J.

    2015-01-01

    Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5? end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3. PMID:25940628

  5. Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation.

    PubMed

    Tan, Lit-Yeen; Whitfield, Peter; Llorian, Miriam; Monzon-Casanova, Elisa; Diaz-Munoz, Manuel D; Turner, Martin; Smith, Christopher W J

    2015-06-23

    Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3. PMID:25940628

  6. A broadly applicable high-throughput screening strategy identifies new regulators of Dlg4 (Psd-95) alternative splicing

    PubMed Central

    Zheng, Sika; Damoiseaux, Robert; Chen, Liang; Black, Douglas L.

    2013-01-01

    Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening method that simultaneously identifies multiple positive and negative regulators of a particular exon. Two minigene reporters were constructed: One produces green fluorescent protein (GFP) from the mRNA including an exon, and red fluorescent protein (RFP) from the mRNA lacking the exon; the other switches these fluorescent products of exon inclusion and exclusion. Combining results from these two reporters eliminates many false positives and greatly enriches for true splicing regulators. After extensive optimization of this method, we performed a gain-of-function screen of 15,779 cDNA clones and identified 40 genes affecting exon 18 of Discs large homolog 4 (Dlg4; also known as post-synaptic density protein 95 [Psd-95]). We confirmed that 28 of the 34 recoverable clones alter reporter splicing in RT-PCR assays. Remarkably, 18 of the identified genes encode splicing factors or RNA binding proteins, including PTBP1, a previously identified regulator of this exon. Loss-of-function experiments examining endogenous Dlg4 transcripts validated the effects of five of eight genes tested in independent cell lines, and two genes were further confirmed to regulate Dlg4 splicing in primary neurons. These results identify multiple new regulators of Dlg4 splicing, and validate an approach to isolating splicing regulators for almost any cassette exon from libraries of cDNAs, shRNAs, or small molecules. PMID:23636947

  7. Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens.

    PubMed

    Chang, Chiung-Yun; Lin, Wen-Dar; Tu, Shih-Long

    2014-04-28

    Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes. PMID:24777346

  8. Identification and characterization of the variants of metastasis-associated protein 1 generated following alternative splicing.

    PubMed

    Yaguchi, Masahiro; Wada, Yoriko; Toh, Yasushi; Iguchi, Haruo; Kono, Akira; Matsusue, Kimihiko; Takiguchi, Soichi

    2005-12-30

    The metastasis-associated gene 1 (mta1) was identified initially in rat highly metastatic cancer cell lines and found to be a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. The gene for mouse mta1 was screened and its genomic structure was determined. It consists of 21 exons spanning 40 kb of genomic DNA. The full-length mouse Mta1 cDNA contained a 2145 nucleotide open reading frame encoding 715 amino acids. In addition to the full-length cDNA, several alternative splicing variants were found. Some differences in the splicing variants found were observed among various mouse organs and cells examined by the semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cDNAs of the splicing variants were inserted into green fluorescent protein (GFP) expression vector and the subcellular localization of the GFP-Mta1 fusion proteins were analyzed. Knowledge of the Mta1 gene expression pattern will be useful in better understanding its functional diversity. PMID:16445996

  9. Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2?*

    PubMed Central

    Jiang, Zhihong; Tang, Hao; Havlioglu, Necat; Zhang, Xiaochun; Stamm, Stefan; Yan, Riqiang; Wu, Jane Y.

    2007-01-01

    Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2? protein. The interaction between Tra2? and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2? protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2?, suggesting that Tra2? may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies. PMID:12649279

  10. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    PubMed

    Kraemer, Nadine; Issa-Jahns, Lina; Neubert, Gerda; Ravindran, Ethiraj; Mani, Shyamala; Ninnemann, Olaf; Kaindl, Angela M

    2015-01-01

    Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice. PMID:26322982

  11. Novel Alternative Splice Variants of Mouse Cdk5rap2

    PubMed Central

    Kraemer, Nadine; Issa-Jahns, Lina; Neubert, Gerda; Ravindran, Ethiraj; Mani, Shyamala; Ninnemann, Olaf; Kaindl, Angela M.

    2015-01-01

    Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice. PMID:26322982

  12. A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

    PubMed Central

    Gopalakrishna-Pillai, Sailesh; Iverson, Linda E.

    2011-01-01

    A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs) relative to spontaneously differentiated cells (SDCs). Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide) upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency. PMID:21698279

  13. Comparative cross-species alternative splicing in plants.

    PubMed

    Ner-Gaon, Hadas; Leviatan, Noam; Rubin, Eitan; Fluhr, Robert

    2007-07-01

    Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less AS than animals. An algorithm, based on expressed sequence tag (EST) pairs gapped alignment, was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for AS. EST pairs gapped alignment was first evaluated in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum) for which annotated genome sequence is available and was shown to accurately predict splicing events. The method was then applied to 11 plant species that include 17 cultivars for which enough ESTs are available. The results show a large, 3.7-fold difference in AS rates between plant species with Arabidopsis and rice in the lower range and lettuce (Lactuca sativa) and sorghum (Sorghum bicolor) in the upper range. Hence, compared to higher animals, plants show a much greater degree of variety in their AS rates and in some plant species the rates of animal and plant AS are comparable although the distribution of AS types may differ. In eudicots but not monocots, a correlation between genome size and AS rates was detected, implying that in eudicots the mechanisms that lead to larger genomes are a driving force for the evolution of AS. PMID:17496110

  14. Alternative splicing of BRAF transcripts and characterization of C-terminally truncated B-Raf isoforms in colorectal cancer.

    PubMed

    Hirschi, Benjamin; Kolligs, Frank T

    2013-08-01

    The BRAF proto-oncogene is mutated in a subset of human tumors, including colorectal cancer. A splicing variant lacking exons 14 and 15 (BRAF del E14/15) has been described recently. However, the frequency of the variant, the kinase activity of the protein isoform, its biological function, and which allele it is derived from remains unknown. BRAF mRNA from colorectal cancer cell lines and colonic epithelium was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants and allelic origin of alternatively spliced transcripts were analyzed by DNA sequencing. Kinase activity of the B-Raf isoforms was determined by Western blotting after transfections with expression constructs of the different BRAF variants. Four additional BRAF transcript variants resulting in C-terminal truncation of the gene product were found. Alternative splicing was found at frequencies from 4.7 to 16.7% in normal and neoplastic colorectal cells. Alternative transcripts were shown to be derived from both wild-type and V600E alleles. All nonconsensus B-Raf protein variants were found to be kinase-dead and failed to coactivate full-length B-Raf. In conclusion, we present a highly sensitive method for the detection of aberrantly spliced transcripts. Alternative splicing of exons 14, 15, 15b, 16b and 16c occurs in a considerable fraction of BRAF mRNA in normal colon and colorectal cancer cells and is independent of the V600E mutational status of the parental allele. Splicing of nonfunctional transcripts affects overall cellular B-Raf activity and might represent a mechanism to decrease sensitivity to growth signals. PMID:23354951

  15. Mouse Models of Mutations and Variations in Autism Spectrum Disorder-Associated Genes: Mice Expressing Caps2/Cadps2 Copy Number and Alternative Splicing Variants

    PubMed Central

    Sadakata, Tetsushi; Shinoda, Yo; Sato, Akira; Iguchi, Hirotoshi; Ishii, Chiaki; Matsuo, Makoto; Yamaga, Ryosuke; Furuichi, Teiichi

    2013-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by disturbances in interpersonal relationships and behavior. Although the prevalence of autism is high, effective treatments have not yet been identified. Recently, genome-wide association studies have identified many mutations or variations associated with ASD risk on many chromosome loci and genes. Identification of the biological roles of these mutations or variations is necessary to identify the mechanisms underlying ASD pathogenesis and to develop clinical treatments. At present, mice harboring genetic modifications of ASD-associated gene candidates are the best animal models to analyze hereditary factors involved in autism. In this report, the biological significance of ASD-associated genes is discussed by examining the phenotypes of mouse models with ASD-associated mutations or variations in mouse homologs, with a focus on mice harboring genetic modifications of the Caps2/Cadps2 (Ca2+-dependent activator protein for secretion 2) gene. PMID:24287856

  16. Splice-mediated Variants of Proteins (SpliVaP) – data and characterization of changes in signatures among protein isoforms due to alternative splicing

    PubMed Central

    Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

    2008-01-01

    Background It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. Results (i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. Conclusion (i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms – since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii) Data: The reported resource (at ) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures. PMID:18831736

  17. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform.

    PubMed

    Dichmann, Darwin S; Walentek, Peter; Harland, Richard M

    2015-02-01

    Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism. PMID:25620705

  18. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform

    PubMed Central

    Dichmann, Darwin S; Walentek, Peter; Harland, Richard M

    2014-01-01

    SUMMARY Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA-seq, we identify 142 splice changes, mostly intron retention and exon skipping, of which 89% are not in current annotations. A previously not described isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism. PMID:25620705

  19. Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing

    PubMed Central

    Montes, Marta; Coiras, Mayte; Becerra, Soraya; Moreno-Castro, Cristina; Mateos, Elena; Majuelos, Jara; Oliver, F. Javier; Hernndez-Munain, Cristina; Alcam, Jos; Su, Carlos

    2015-01-01

    Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis. PMID:26462236

  20. Novel alternative splice variants of chicken NPAS3 are expressed in the developing central nervous system.

    PubMed

    Shin, Jiheon; Kim, Jaesang

    2013-11-10

    We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process. PMID:23962688

  1. Clathrin light chain B: gene structure and neuron-specific splicing.

    PubMed Central

    Stamm, S; Casper, D; Dinsmore, J; Kaufmann, C A; Brosius, J; Helfman, D M

    1992-01-01

    The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing. Images PMID:1408826

  2. Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions

    PubMed Central

    Mauger, Oriane; Klinck, Roscoe; Chabot, Benoit; Muchardt, Christian; Allemand, Eric; Batsché, Eric

    2015-01-01

    Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study. PMID:25605796

  3. Qualitative research of alternatively splice variants of fibronectin in different development stage of mice heart

    PubMed Central

    Lu, Feng; Ma, Fang-Fang; Zhang, Wei; Li, Ying; Wei, Fei-Yu

    2015-01-01

    Background Fibronectin (FN) plays vital roles in cell adhesion, differentiation, proliferation and migration. It is involved in the process of embryonic development and is highly conserved during evolution. The EIIIA and EIIIB of FN show a very high degree of homology among vertebrates. Embryos deleting both EIIIA and EIIIB displayed multiple embryonic cardiovascular defects, implying their crucial role during embryogenesis. The correlation of spliced EIIIB, EIIIA, and IIICS of FN to heart development was studied by observing their chronological expression in mice heart. Methods C57 mice embryos at E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, E19.5 days, postnatal day 1 (P1d), and adult male mice (3 months) were used. For each alternatively spliced FN1 domain (EIIIB, EIIIA and IIICS), primer pairs were designed for specific amplification. Total RNA was extracted from the heart tissue, reverse transcripted to cDNA, followed by RT-PCR with specific primers. The PCR amplification was verified by agarose gel electrophoresis, showing specific fragments of the expected sizes. Results In adult mice heart, only alternatively splice variants of EIIIA-, EIIIB-, IIICS+ were expressed. While in embryonic mice, spliced variant of EIIIA+/-, EIIIB+/-, IIICS+ were observed. The expression of EIIIA and EIIIB changed during heart development. Conclusions FN is crucial for the normal development of the embryonic heart by modulating cardiac neural crest (CNC) proliferation and survival, and maintenance of CNC cells. FN1 gene seems to play a significant role by expression of highly conserved EIIIA and EIIIB in embryonic heart development. PMID:26793352

  4. LSD1 Neurospecific Alternative Splicing Controls Neuronal Excitability in Mouse Models of Epilepsy.

    PubMed

    Rusconi, Francesco; Paganini, Leda; Braida, Daniela; Ponzoni, Luisa; Toffolo, Emanuela; Maroli, Annalisa; Landsberger, Nicoletta; Bedogni, Francesco; Turco, Emilia; Pattini, Linda; Altruda, Fiorella; De Biasi, Silvia; Sala, Mariaelvina; Battaglioli, Elena

    2015-09-01

    Alternative splicing in the brain is dynamic and instrumental to adaptive changes in response to stimuli. Lysine-specific demethylase 1 (LSD1/KDM1A) is a ubiquitously expressed histone H3Lys4 demethylase that acts as a transcriptional co-repressor in complex with its molecular partners CoREST and HDAC1/2. In mammalian brain, alternative splicing of LSD1 mini-exon E8a gives rise to neuroLSD1, a neurospecific isoform that, upon phosphorylation, acts as a dominant-negative causing disassembly of the co-repressor complex and de-repression of target genes. Here we show that the LSD1/neuroLSD1 ratio changes in response to neuronal activation and such effect is mediated by neurospecific splicing factors NOVA1 and nSR100/SRRM4 together with a novel cis-silencer. Indeed, we found that, in response to epileptogenic stimuli, downregulation of NOVA1 reduces exon E8a splicing and expression of neuroLSD1. Using behavioral and EEG analyses we observed that neuroLSD1-specific null mice are hypoexcitable and display decreased seizure susceptibility. Conversely, in a mouse model of Rett syndrome characterized by hyperexcitability, we measured higher levels of NOVA1 protein and upregulation of neuroLSD1. In conclusion, we propose that, in the brain, correct ratio between LSD1 and neuroLSD1 contributes to excitability and, when altered, could represent a pathogenic event associated with neurological disorders involving altered E/I. PMID:24735673

  5. Comparative Analysis of Serine/Arginine-Rich Proteins across 27 Eukaryotes: Insights into Sub-Family Classification and Extent of Alternative Splicing

    PubMed Central

    Richardson, Dale N.; Rogers, Mark F.; Labadorf, Adam; Ben-Hur, Asa; Guo, Hui; Paterson, Andrew H.; Reddy, Anireddy S. N.

    2011-01-01

    Alternative splicing (AS) of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs) at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and “basal” eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs) as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes). AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3′ or 5′ splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%–88% of their SR genes experiencing some type of AS compared to the 40%–54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms. PMID:21935421

  6. Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

    SciTech Connect

    Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki; Yagi, Hiroaki; Shigaki-Miyamoto, Kaya; Toyota, Syukichi; Azuma, Yuko; Igarashi, Masayuki; Tani, Tokio

    2014-03-28

    Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

  7. NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML

    PubMed Central

    Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M.; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A.; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P.; Motyckova, Gabriela; Deangelo, Daniel J.; Quackenbush, John; Tenen, Daniel G.; Stone, Richard M.; Griffin, James D.

    2014-01-01

    Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34+ bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics. PMID:24574459

  8. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells

    PubMed Central

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P.; Beato, Miguel

    2015-01-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions. PMID:25589247

  9. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    PubMed

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions. PMID:25589247

  10. Alternative splicing and the steady-state ratios of mRNA isoforms generated by it are under strong stabilizing selection in Caenorhabditis elegans.

    PubMed

    Barberan-Soler, Sergio; Zahler, Alan M

    2008-11-01

    Evolutionary studies indicate that a high proportion of alternative splicing (AS) events are species-specific; just 28% of minor-form alternatively spliced exons are conserved between mice and humans. We employed a splicing-sensitive microarray to study the evolution of allele-specific AS in nematodes. We compared splicing levels among five distinct Caenorhabditis elegans lines. Our results indicate that AS is less variable between natural isolates (NIs) from England, Hawaii, and Australia than when compared with mutation accumulation lines (6% vs. 21%, respectively, vary compared with N2). This suggests that strong stabilizing selection shapes the evolution of the ratios of isoforms generated by AS in C. elegans. When we analyzed some of the splicing changes between the NIs, we found examples of changes in both cis and trans that lead to alterations in gene-specific AS. This indicates that both these mechanisms for changing AS are employed along the path toward speciation in nematodes. PMID:18718918

  11. From single-cell to cell-pool transcriptomes: Stochasticity in gene expression and RNA splicing

    PubMed Central

    Marinov, Georgi K.; Williams, Brian A.; McCue, Ken; Schroth, Gary P.; Gertz, Jason; Myers, Richard M.; Wold, Barbara J.

    2014-01-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30–100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

  12. Recognition of alternatively spliced cassette exons based on a hybrid model.

    PubMed

    Zhang, Xiaokang; Peng, Qinke; Li, Liang; Li, Xintong

    2016-03-11

    Alternative splicing (AS) is an important mechanism of gene regulation that contributes to protein diversity. It is of great significance to recognize different kinds of AS accurately so as to understand the mechanism of gene regulation. Many in silico methods have been applied to detecting AS with vast features, but the result is far from satisfactory. In this paper, we used the features proven to be useful in recognizing AS in previous literature and proposed a hybrid method combining Gene Expression Programming (GEP) and Random Forests (RF) to classify the constitutive exons and cassette exons which is the most common AS phenomenon. GEP will firstly make prediction to the samples of strong signal, and the other samples of weak signal will be distinguished with a more complex classifier based on RF. The experiment result indicates that this method can highly improve the recognition level in this issue. PMID:26869516

  13. Determination of Alternate Splicing Events using the Affymetrix Exon 1.0 ST Arrays

    PubMed Central

    Subbaram, Sita; Kuentzel, Marcy; Frank, David; DiPersio, C. Michael; Chittur, Sridar V.

    2015-01-01

    Alternative splicing plays an important role in regulation of normal cellular function. Alternative splicing of pre-mRNA leads to the diversity of downstream protein products in the cell. The Affymetrix Exon arrays allow for a high throughput evaluation of the differences in spliced mRNA expressed in a biological system. In this study, we describe a method using this technology to study the generation of alternative mRNA transcripts in breast cancer cells that differ in the levels of a particular integrin, ?3?1. PMID:20217571