Science.gov

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

    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 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

  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. 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

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2015-05-19

    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

  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. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels.

    PubMed

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

    2002-07-01

    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, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. Alternative splicing of human and mouse NPFF2 receptor genes: Implications to receptor expression.

    PubMed

    Ankö, Minna-Liisa; Ostergård, Maria; Lintunen, Minnamaija; Panula, Pertti

    2006-12-22

    Alternative splicing has an important role in the tissue-specific regulation of gene expression. Here we report that similar to the human NPFF2 receptor, the mouse NPFF2 receptor is alternatively spliced. In human the presence of three alternatively spliced receptor variants were verified, whereas two NPFF2 receptor variants were identified in mouse. The alternative splicing affected the 5' untranslated region of the mouse receptor and the variants in mouse were differently distributed. The mouse NPFF system may also have species-specific features since the NPFF2 receptor mRNA expression differs from that reported for rat. PMID:17157836

  1. 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

  2. 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

  3. A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis

    PubMed Central

    Pimentel, Harold; Parra, Marilyn; Gee, Sherry; Ghanem, Dana; An, Xiuli; Li, Jie; Mohandas, Narla; Pachter, Lior; Conboy, John G.

    2014-01-01

    Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells. PMID:24442673

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

    PubMed

    Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

    2016-02-01

    Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-d-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-d-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels. PMID:26788957

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

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

    PubMed

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

    2014-02-01

    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

  16. 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.

  17. 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

  18. Novel alternative splicing of human faciogenital dysplasia 1 gene.

    PubMed

    Yanagi, Kumiko; Kaname, Tadashi; Chinen, Yasutsugu; Naritomi, Kenji

    2004-09-01

    The human faciogenital dysplasia 1 (FGD1) gene product plays an important role in morphogenesis. Its dysfunction causes Aarskog-Scott syndrome (MIM musical sharp 305400). To characterize the FGD1, we investigated its expression by RT-PCR and Southern blot analysis in normal tissues. We found novel alternative forms of the FGD1. One has a novel exon located in intron 8, named exon 8B (8B FDG1) and the other has an exon in intron 7, exon 7B (7B FGD1). The 8B FDG1 is expressed strongly in the brain, testis, spinal cord, trachea and stomach, and weakly in the thymus and lymphocytes. However, expression of the 7B FGD1 is weak and restricted in the testis and salivary gland. Insertion of each novel exon results in production of a premature termination codon, respectively, and the predicted proteins generated from them have only a proline-rich domain and an incomplete DH domain which potentially compete with the wild type of FGD1. PMID:15327482

  19. Regulation of Alternative Splicing by Histone Modifications

    PubMed Central

    Luco, Reini F.; Pan, Qun; Tominaga, Kaoru; Blencowe, Benjamin J.; Pereira-Smith, Olivia M.; Misteli, Tom

    2010-01-01

    Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery. PMID:20133523

  20. 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

  1. A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

    PubMed Central

    Skandalis, Adonis; Uribe, Elke

    2004-01-01

    Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase β (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40–60%), whereas the frequency of HPRT splice variants is considerably lower (∼1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed. PMID:15601998

  2. 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

  3. 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

  4. 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. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer’s 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

  5. 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

  6. 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

  7. Analysis of the human neurexin genes: alternative splicing and the generation of protein diversity.

    PubMed

    Rowen, Lee; Young, Janet; Birditt, Brian; Kaur, Amardeep; Madan, Anup; Philipps, Dana L; Qin, Shizhen; Minx, Patrick; Wilson, Richard K; Hood, Leroy; Graveley, Brenton R

    2002-04-01

    The neurexins are neuronal proteins that function as cell adhesion molecules during synaptogenesis and in intercellular signaling. Although mammalian genomes contain only three neurexin genes, thousands of neurexin isoforms may be expressed through the use of two alternative promoters and alternative splicing at up to five different positions in the pre-mRNA. To begin understanding how the expression of the neurexin genes is regulated, we have determined the complete nucleotide sequence of all three human neurexin genes: NRXN1, NRXN2, and NRXN3. Unexpectedly, two of these, NRXN1 ( approximately 1.1 Mb) and NRXN3 ( approximately 1.7 Mb), are among the largest known human genes. In addition, we have identified several conserved intronic sequence elements that may participate in the regulation of alternative splicing. The sequences of these genes provide insight into the mechanisms used to generate the diversity of neurexin protein isoforms and raise several interesting questions regarding the expression mechanism of large genes. PMID:11944992

  8. Mouse pseudouridine synthase 1: gene structure and alternative splicing of pre-mRNA.

    PubMed Central

    Chen, J; Patton, J R

    2000-01-01

    Evidence for the alternative splicing of the message for mouse pseudouridine synthase 1 (mPus1p) was found when several expressed sequence tag clones were completely sequenced. The genomic DNA for the MPUS1 gene (6.9 kb) was cloned from a mouse genomic library; the gene contains seven exons, of which three are alternatively spliced. In addition, one of the internal exons (exon VI) is unusually large. RNase protection analysis confirmed that several alternatively spliced messages were present in mouse tissues and cells in culture. A Western blot of total cellular protein from mouse tissues and cultured cells was reacted with an antibody specific for mPus1p; at least three proteins were detected. One protein corresponds to the predicted molecular mass of mPus1p (44 kDa) and is the most abundant. The two other isoforms, one 2 kDa larger and one 7 kDa smaller than mPus1p, were differentially expressed. The cDNA species for the three isoforms were cloned into expression plasmids; the proteins were synthesized in vitro and tested for pseudouridine synthase activity. The two isoforms, one containing an insert of 18 amino acids in a region of the enzyme assumed to be critical for activity, and the other, which has a deletion of the protein coding potential of two exons, were both inactive on tRNA substrates that mPus1p modifies. PMID:11085940

  9. 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

  10. 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

  11. Alternative splicing of the tuberous sclerosis 2 (TSC2) gene in human and mouse tissues

    SciTech Connect

    Xu, Lin; Sterner, C.; Maheshwar, M.M.

    1995-06-10

    The recently isolated gene for tuberous sclerosis 2 (TSC2) encodes a 5.5.kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. We have detected a novel variant of the TSC2 mRNA lacking 129 nucleotides, predicting an in-frame deletion of 43 amino acids spanning codons 946-988 of tuberin. This 129-bp deletion precisely corresponds to exon 25 of the TSC2 gene suggesting that alternative splicing leads to production of two forms of transcripts designated isoforms 1 and 2. Further molecular analysis revealed a third isoform exhibiting a deletion of 44 amino acids spanning codons 946-989 of tuberin. Amino acid 989 is a Ser residue encoded by the first codon of exon 26. The two isoforms also exist in newborn and adult mouse tissues, reinforcing the potential functional importance of these alternatively spliced products. These alternative isoforms should have implications for efforts aimed at identifying mutations in TSC patients. The distinct polypeptides encoded by the TSC2 gene may have different targets as well as functions involved in the regulation of cell growth. 26 refs., 4 figs.

  12. Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

    PubMed

    Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

    2016-05-27

    Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus-specific mechanism of gene expression regulation on RNA levels, and might play a role in epigenetic regulation of the link between the circadian clock and ambient temperature fluctuation in Medicago. PMID:27086112

  13. Divergent functions through alternative splicing: the Drosophila CRMP gene in pyrimidine metabolism, brain, and behavior.

    PubMed

    Morris, Deanna H; Dubnau, Josh; Park, Jae H; Rawls, John M

    2012-08-01

    DHP and CRMP proteins comprise a family of structurally similar proteins that perform divergent functions, DHP in pyrimidine catabolism in most organisms and CRMP in neuronal dynamics in animals. In vertebrates, one DHP and five CRMP proteins are products of six genes; however, Drosophila melanogaster has a single CRMP gene that encodes one DHP and one CRMP protein through tissue-specific, alternative splicing of a pair of paralogous exons. The proteins derived from the fly gene are identical over 90% of their lengths, suggesting that unique, novel functions of these proteins derive from the segment corresponding to the paralogous exons. Functional homologies of the Drosophila and mammalian CRMP proteins are revealed by several types of evidence. Loss-of-function CRMP mutation modifies both Ras and Rac misexpression phenotypes during fly eye development in a manner that is consistent with the roles of CRMP in Ras and Rac signaling pathways in mammalian neurons. In both mice and flies, CRMP mutation impairs learning and memory. CRMP mutant flies are defective in circadian activity rhythm. Thus, DHP and CRMP proteins are derived by different processes in flies (tissue-specific, alternative splicing of paralogous exons of a single gene) and vertebrates (tissue-specific expression of different genes), indicating that diverse genetic mechanisms have mediated the evolution of this protein family in animals. PMID:22649077

  14. 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

  15. Divergent Functions Through Alternative Splicing: The Drosophila CRMP Gene in Pyrimidine Metabolism, Brain, and Behavior

    PubMed Central

    Morris, Deanna H.; Dubnau, Josh; Park, Jae H.; Rawls, John M.

    2012-01-01

    DHP and CRMP proteins comprise a family of structurally similar proteins that perform divergent functions, DHP in pyrimidine catabolism in most organisms and CRMP in neuronal dynamics in animals. In vertebrates, one DHP and five CRMP proteins are products of six genes; however, Drosophila melanogaster has a single CRMP gene that encodes one DHP and one CRMP protein through tissue-specific, alternative splicing of a pair of paralogous exons. The proteins derived from the fly gene are identical over 90% of their lengths, suggesting that unique, novel functions of these proteins derive from the segment corresponding to the paralogous exons. Functional homologies of the Drosophila and mammalian CRMP proteins are revealed by several types of evidence. Loss-of-function CRMP mutation modifies both Ras and Rac misexpression phenotypes during fly eye development in a manner that is consistent with the roles of CRMP in Ras and Rac signaling pathways in mammalian neurons. In both mice and flies, CRMP mutation impairs learning and memory. CRMP mutant flies are defective in circadian activity rhythm. Thus, DHP and CRMP proteins are derived by different processes in flies (tissue-specific, alternative splicing of paralogous exons of a single gene) and vertebrates (tissue-specific expression of different genes), indicating that diverse genetic mechanisms have mediated the evolution of this protein family in animals. PMID:22649077

  16. 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

  17. Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia.

    PubMed Central

    Miyoshi, H; Ohira, M; Shimizu, K; Mitani, K; Hirai, H; Imai, T; Yokoyama, K; Soeda, E; Ohki, M

    1995-01-01

    We previously isolated the AML1 gene, which is rearranged by the t(8;21) translocation in acute myeloid leukemia. The AML1 gene is highly homologous to the Drosophila segmentation gene runt and the mouse transcription factor PEBP2 alpha subunit gene. This region of homology, called the Runt domain, is responsible for DNA-binding and protein--protein interaction. In this study, we isolated and characterized various forms of AML1 cDNAs which reflect a complex pattern of mRNA species. Analysis of these cDNAs has led to the identification of two distinct AML1 proteins, designated AML1b (453 amino acids) and AML1c (480 amino acids), which differ markedly from the previously reported AML1a (250 amino acids) with regard to their C-terminal regions, although all three contain the Runt domain. The large C-terminal region common to AML1b and AML1c is suggested to be a transcriptional activation domain. AML1c differs from AML1b by only 32 amino acids in the N-terminal. Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA. Northern blot analysis demonstrated the presence of six major transcripts, encoding AML1b or AML1c, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites. A minor transcript encoding AML1a which results from alternative splicing of a separate exon can be detected only by reverse transcription-polymerase chain reaction amplification. The distinct proteins encoded by the AML1 gene may have different functions, which could contribute to regulating cell growth and/or differentiation through transcriptional regulation of a specific subset of target genes. Images PMID:7651838

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Diversity and complexity of the mu opioid receptor gene: alternative pre-mRNA splicing and promoters.

    PubMed

    Pan, Ying-Xian

    2005-11-01

    Mu opioid receptors play an important role in mediating the actions of a class of opioids including morphine and heroin. Binding and pharmacological studies have proposed several mu opioid receptor subtypes: mu(1), mu(2), and morphine-6beta-glucuronide (M6G). The cloning of a mu opioid receptor, MOR-1, has provided an invaluable tool to explore pharmacological and physiological functions of mu opioid receptors at the molecular level. However, only one mu opioid receptor (Oprm) gene has been isolated. Alternative pre-mRNA splicing has been proposed as a molecular explanation for the existence of pharmacologically identified subtypes. In recent years, we have extensively investigated alternative splicing of the Oprm gene, particularly of the mouse Oprm gene. So far we have identified 25 splice variants from the mouse Oprm gene, which are controlled by two diverse promoters, eight splice variants from the rat Oprm gene, and 11 splice variants from the human Oprm gene. Diversity and complexity of the Oprm gene was further demonstrated by functional differences in agonist-induced G protein activation, adenylyl cyclase activity, and receptor internalization among carboxyl terminal variants. This review summarizes these recent results and provides a new perspective on understanding and exploring complex opioid actions in animals and humans. PMID:16274294

  4. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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...

  15. 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

  16. Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

    PubMed Central

    Münk, Carsten; Beck, Thomas; Zielonka, Jörg; Hotz-Wagenblatt, Agnes; Chareza, Sarah; Battenberg, Marion; Thielebein, Jens; Cichutek, Klaus; Bravo, Ignacio G; O'Brien, Stephen J; Lochelt, Martin; Yuhki, Naoya

    2008-01-01

    Background Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Results Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Conclusion Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher. PMID:18315870

  17. 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

  18. Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

    PubMed Central

    Du, Hongqing; Cline, Melissa S.; Osborne, Robert J.; Tuttle, Daniel L.; Clark, Tyson A.; Donohue, John Paul; Hall, Megan P.; Shiue, Lily; Swanson, Maurice S.; Thornton, Charles A.; Ares, Manuel

    2009-01-01

    Myotonic dystrophy (DM1) is associated with expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. Strong correlation in splicing changes for ~100 new Mbnl1-regulated exons indicates loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA level changes can be attributed to loss of Mbnl1, indicating CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix (ECM) proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function, disrupting splicing, and loss of another function that disrupts ECM mRNA regulation, possibly mediated by MBNL2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies. PMID:20098426

  19. 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

  20. 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...

  1. 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

  2. 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.

  3. Sequence analysis and in vivo expression show that alternative splicing of ED-B and ED-A regions of the human fibronectin gene are independent events.

    PubMed Central

    Paolella, G; Henchcliffe, C; Sebastio, G; Baralle, F E

    1988-01-01

    The structure of two alternatively spliced regions. ED-A and ED-B, of human fibronectin gene, was determined, in order to show whether any similarity was present between the two. Although some interesting features are present in each, no obvious common structure or sequence homology was found. Functional analysis of the alternative splicing events was carried out by transient expression in Hela cells. A hybrid gene was constructed by inserting the ED-B region into the third exon of the human alpha 1-globin gene. The transfected hybrid gene is expressed and produces, in Hela cells, two alternatively spliced RNAs, showing a pattern very similar to that observed for the endogenous fibronectin gene in fibroblasts. Cotransfection of this gene with a similar gene containing the ED-A region, shows that no interference is present between the two alternative splicing processes. Images PMID:3375063

  4. Alternative Splicing in Bone Following Mechanical Loading

    PubMed Central

    Mantila Roosa, Sara M.; Liu, Yunlong; Turner, Charles H.

    2010-01-01

    It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 minutes, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnae were sampled at 11 time points, from 4 hours to 32 days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/β-catenin and TGF-β signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading. PMID:21095247

  5. 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

  6. 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

  7. Identification of alternative splicing regulators by RNA interference in Drosophila

    PubMed Central

    Park, Jung W.; Parisky, Katherine; Celotto, Alicia M.; Reenan, Robert A.; Graveley, Brenton R.

    2004-01-01

    Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. PMID:15492211

  8. 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

  9. Gene regulation, alternative splicing, and posttranslational modification of troponin subunits in cardiac development and adaptation: a focused review

    PubMed Central

    Sheng, Juan-Juan; Jin, Jian-Ping

    2014-01-01

    Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases. PMID:24817852

  10. 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

  11. 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

  12. 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

  13. Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

    PubMed Central

    Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

    1999-01-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed. PMID:10085246

  14. Population genetics of duplicated alternatively spliced exons of the Dscam gene in Daphnia and Drosophila.

    PubMed

    Brites, Daniela; Encinas-Viso, Francisco; Ebert, Dieter; Du Pasquier, Louis; Haag, Christoph R

    2011-01-01

    In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam. PMID:22174757

  15. Population Genetics of Duplicated Alternatively Spliced Exons of the Dscam Gene in Daphnia and Drosophila

    PubMed Central

    Brites, Daniela; Encinas-Viso, Francisco; Ebert, Dieter; Du Pasquier, Louis; Haag, Christoph R.

    2011-01-01

    In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam. PMID:22174757

  16. MAp19, the alternative splice product of the MASP2 gene.

    PubMed

    Degn, Søren E; Thiel, Steffen; Nielsen, Ole; Hansen, Annette G; Steffensen, Rudi; Jensenius, Jens C

    2011-10-28

    The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent. PMID:21871896

  17. 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 sites—GYNNGYs (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

  18. 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

  19. 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

  20. 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

  1. 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 parasite’s 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 parasite’s transcriptome. PMID:21245033

  2. 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

  3. 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

  4. 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

  5. Differential Expressions of the Alternatively Spliced Variant mRNAs of the µ 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 µ 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 µ 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

  6. Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome.

    PubMed

    Shao, Wei; Zhao, Qiong-Yi; Wang, Xiu-Ye; Xu, Xin-Yan; Tang, Qing; Li, Muwang; Li, Xuan; Xu, Yong-Zhen

    2012-07-01

    Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5'-gene with 46 newly identified alternative 3'-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F(1) hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies. PMID:22627775

  7. 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

  8. Expression and alternative splicing of classical and nonclassical MHCI genes in the hippocampus and neuromuscular junction.

    PubMed

    Tetruashvily, Mazell M; Melson, John W; Park, Joseph J; Peng, Xiaoyu; Boulanger, Lisa M

    2016-04-01

    The major histocompatibility complex class I (MHCI) is a large gene family, with over 20 members in mouse. Some MHCIs are well-known for their critical roles in the immune response. Studies in mice which lack stable cell-surface expression of many MHCI proteins suggest that one or more MHCIs also play unexpected, essential roles in the establishment, function, and modification of neuronal synapses. However, there is little information about which genes mediate MHCI's effects in neurons. In this study, RT-PCR was used to simultaneously assess transcription of many MHCI genes in regions of the central and peripheral nervous system where MHCI has a known or suspected role. In the hippocampus, a part of the CNS where MHCI regulates synapse density, synaptic transmission, and plasticity, we found that more than a dozen MHCI genes are transcribed. Single-cell RT-PCR revealed that individual hippocampal neurons can express more than one MHCI gene, and that the MHCI gene expression profile of CA1 pyramidal neurons differs significantly from that of CA3 pyramidal neurons or granule cells of the dentate gyrus. MHCI gene expression was also assessed at the neuromuscular junction (NMJ), a part of the peripheral nervous system (PNS) where MHCI plays a role in developmental synapse elimination, aging-related synapse loss, and neuronal regeneration. Four MHCI genes are expressed at the NMJ at an age when synapse elimination is occurring in three different muscles. Several MHCI mRNA splice variants were detected in hippocampus, but not at the NMJ. Together, these results establish the first profile of MHCI gene expression at the developing NMJ, and demonstrate that MHCI gene expression is under tight spatial and temporal regulation in the nervous system. They also identify more than a dozen MHCIs that could play important roles in regulating synaptic transmission and plasticity in the central and peripheral nervous systems. PMID:26802536

  9. Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells

    PubMed Central

    2014-01-01

    Background The endosteum of the bone marrow provides a specialized hypoxic niche that may serve to preserve the integrity, pluripotency, longevity and stemness of resident mesenchymal stem cells (MSCs). To explore the molecular genetic consequences of such a niche we subjected human (h) MSCs to a pO2 of 4 mmHg and analyzed global gene expression and alternative splicing (AS) by genome-exon microarray and RT-qPCR, and phenotype by western blot and immunostaining. Results Out of 446 genes differentially regulated by >2.5-fold, down-regulated genes outnumbered up-regulated genes by 243:203. Exon analyses revealed 60 hypoxia-regulated AS events with splice indices (SI) >1.0 from 53 genes and a correlation between high SI and degree of transcript regulation. Parallel analyses of a publicly available AS study on human umbilical vein endothelial cells (HUVECs) showed that there was a strong cell-specific component with only 11 genes commonly regulated in hMSCs and HUVECs and 17 common differentially spliced genes. Only 3 genes were differentially responsive to hypoxia at the gene (>2.0) and AS levels in both cell types. Functional assignments revealed unique profiles of gene expression with complex regulation of differentiation, extracellular matrix, intermediate filament and metabolic marker genes. Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated. There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition. Conclusions We conclude that severe hypoxia confers a quiescent phenotype in hMSCs that is reflected by both the transcriptome profile and gene-specific changes of splicosome actions. The results reveal that severe hypoxia imposes markedly different patterns of gene regulation of MSCs compared with more moderate hypoxia. This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state. PMID:24758227

  10. 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

  11. 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

  12. 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

  13. 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

  14. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    PubMed

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution. PMID:26186942

  15. Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes

    PubMed Central

    Kabat, Jennifer L; Barberan-Soler, Sergio; McKenna, Paul; Clawson, Hiram; Farrer, Tracy; Zahler, Alan M

    2006-01-01

    Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene. PMID:16839192

  16. 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.

  17. 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

  18. 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

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

    PubMed

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

    2007-11-15

    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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

    PubMed Central

    Chateigner-Boutin, Anne-Laure; Mila, Isabelle; Frasse, Pierre; Wang, Hua; Audran, Corinne; Roustan, Jean-Paul; Bouzayen, Mondher

    2014-01-01

    Background The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. Results Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5′-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. Conclusion All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner. PMID:24427281

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

    PubMed

    Slavov, D; Gardiner, K

    2002-10-16

    Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events. PMID:12459255

  14. 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

  15. 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

  16. 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

  17. Two of four alternatively spliced isoforms of RUNX2 control osteocalcin gene expression in human osteoblast cells.

    PubMed

    Makita, Naoyuki; Suzuki, Mitsuhiro; Asami, Shiori; Takahata, Rintaroh; Kohzaki, Daika; Kobayashi, Sho; Hakamazuka, Takashi; Hozumi, Nobumichi

    2008-04-30

    Runx2 is a Runt domain transcription factor that transcriptionally regulates osteoblast differentiation and bone formation. In this study, we show that human chondro- and osteosarcoma cell lines, human mesenchymal stem cells (hMSC) and a human primary chondrocytes (HC), osteoblst cells (HOb) express an intact isoform (RUNX2wt) and 3 alternatively spliced isoforms (RUNX2Delta5, Delta7, and Delta5Delta7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Delta5 and RUNX2Delta5Delta7) did not localize in the nucleus and had lost their DNA binding activity. In cotransfection experiments with an osteocalcin (OC) promoter construct, we confirmed that only RUNX2wt and RUNX2Delta7 could upregulate the OC promoter activity in the osteosarcoma cell line. In addition, the coactivator CBP/p300 enhanced the transcriptional activity of the OC promoter when coexpressed with RUNX2wt or RUNX2Delta7, but not when coexpressed with RUNX2Delta5 or RUNX2Delta5Delta7. In contrast, the corepressor HDAC3 only repressed the activation from the OC promoter when coexpressed with RUNX2wt. These results support the hypothesis that RUNX2 both up- and downregulates its target gene promoters, as exemplified by the OC gene, using various isoforms and context-dependent formation of transcriptional complexes. PMID:18321663

  18. 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

  19. 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...

  20. Splicing: is there an alternative contribution to Parkinson's disease?

    PubMed

    La Cognata, Valentina; D'Agata, Velia; Cavalcanti, Francesca; Cavallaro, Sebastiano

    2015-10-01

    Alternative splicing is a crucial mechanism of gene expression regulation that enormously increases the coding potential of our genome and represents an intermediate step between messenger RNA (mRNA) transcription and protein posttranslational modifications. Alternative splicing occupies a central position in the development and functions of the nervous system. Therefore, its deregulation frequently leads to several neurological human disorders. In the present review, we provide an updated overview on the impact of alternative splicing in Parkinson's disease (PD), the second most common neurodegenerative disorder worldwide. We will describe the alternative splicing of major PD-linked genes by collecting the current evidences about this intricate and not carefully explored aspect. Assessing the role of this mechanism on PD pathobiology may represent a central step toward an improved understanding of this complex disease. PMID:25980689

  1. 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

  2. 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

  3. 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

  4. Zebrafish ftz-f1 gene has two promoters, is alternatively spliced, and is expressed in digestive organs.

    PubMed Central

    Lin, W; Wang, H W; Sum, C; Liu, D; Hew, C L; Chung, B

    2000-01-01

    Fushi-tarazu Factor-1 (FTZ-F1) is a family of nuclear receptors involved in various developmental processes. We have cloned a zebrafish FTZ-F1 gene, termed ff1, which belongs to the fetoprotein transcription factor/liver receptor homologue-1 (FTF/LRH-1) subgroup of the FTZ-F1 family. Four transcripts arise as a result of differential promoter usage and alternative splicing at the 3'-most exons. The longer transcript, form A, encodes a transcriptional activator. The shorter transcript, form B, lacks the activation domain, and hence could not activate transcription. The difference in promoter usage generates FF1 proteins with different N-terminal sequences. All four transcripts appear to be expressed in most of the adult tissues, whereas, during embryo development, the IIA form is the predominant transcript. Reverse transcriptase-PCR and in situ hybridization experiments showed that the ff1 transcript is expressed in the hypothalamus, spinal cord, mandibular arch and digestive organs, including pancreas, liver, and intestine. The expression of ff1 in the digestive organs implies its function in gut development. PMID:10816440

  5. Zebrafish ftz-f1 gene has two promoters, is alternatively spliced, and is expressed in digestive organs.

    PubMed

    Lin, W; Wang, H W; Sum, C; Liu, D; Hew, C L; Chung, B

    2000-06-01

    Fushi-tarazu Factor-1 (FTZ-F1) is a family of nuclear receptors involved in various developmental processes. We have cloned a zebrafish FTZ-F1 gene, termed ff1, which belongs to the fetoprotein transcription factor/liver receptor homologue-1 (FTF/LRH-1) subgroup of the FTZ-F1 family. Four transcripts arise as a result of differential promoter usage and alternative splicing at the 3'-most exons. The longer transcript, form A, encodes a transcriptional activator. The shorter transcript, form B, lacks the activation domain, and hence could not activate transcription. The difference in promoter usage generates FF1 proteins with different N-terminal sequences. All four transcripts appear to be expressed in most of the adult tissues, whereas, during embryo development, the IIA form is the predominant transcript. Reverse transcriptase-PCR and in situ hybridization experiments showed that the ff1 transcript is expressed in the hypothalamus, spinal cord, mandibular arch and digestive organs, including pancreas, liver, and intestine. The expression of ff1 in the digestive organs implies its function in gut development. PMID:10816440

  6. 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.

  7. Alternative splicing networks regulated by signaling in human T cells

    PubMed Central

    Martinez, Nicole M.; Pan, Qun; Cole, Brian S.; Yarosh, Christopher A.; Babcock, Grace A.; Heyd, Florian; Zhu, William; Ajith, Sandya; Blencowe, Benjamin J.; Lynch, Kristen W.

    2012-01-01

    The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway–specific regulation of alternative splicing during T-cell stimulation. PMID:22454538

  8. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations. PMID:26300000

  9. 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

  10. Functional roles of alternative splicing factors in human disease.

    PubMed

    Cieply, Benjamin; Carstens, Russ P

    2015-01-01

    Alternative splicing (AS) is an important mechanism used to generate greater transcriptomic and proteomic diversity from a finite genome. Nearly all human gene transcripts are alternatively spliced and can produce protein isoforms with divergent and even antagonistic properties that impact cell functions. Many AS events are tightly regulated in a cell-type or tissue-specific manner, and at different developmental stages. AS is regulated by RNA-binding proteins, including cell- or tissue-specific splicing factors. In the past few years, technological advances have defined genome-wide programs of AS regulated by increasing numbers of splicing factors. These splicing regulatory networks (SRNs) consist of transcripts that encode proteins that function in coordinated and related processes that impact the development and phenotypes of different cell types. As such, it is increasingly recognized that disruption of normal programs of splicing regulated by different splicing factors can lead to human diseases. We will summarize examples of diseases in which altered expression or function of splicing regulatory proteins has been implicated in human disease pathophysiology. As the role of AS continues to be unveiled in human disease and disease risk, it is hoped that further investigations into the functions of numerous splicing factors and their regulated targets will enable the development of novel therapies that are directed at specific AS events as well as the biological pathways they impact. PMID:25630614

  11. 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

  12. 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.

  13. 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

  14. 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

  15. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.

    PubMed Central

    Xu, R; Teng, J; Cooper, T A

    1993-01-01

    We have characterized a novel positive-acting splicing element within the developmentally regulated alternative exon (exon 5) of the cardiac troponin T (cTNT) gene. The exon splicing element (ESE) is internal to the exon portions of the splice sites and is required for splicing to the 3' splice site but not the 5' splice site flanking the exon. Sequence comparisons between cTNT exon 5 and other exons that contain regions required for splicing reveal a common purine-rich motif. Sequence within cTNT exon 5 or a synthetic purine-rich motif facilitates splicing of heterologous alternative and constitutive splice sites in vivo. Interestingly, the ESE is not required for the preferential inclusion of cTNT exon 5 observed in primary skeletal muscle cultures. Our results strongly suggest that the purine-rich ESE serves as a general splicing element that is recognized by the constitutive splicing machinery. Images PMID:8388541

  16. 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.

  17. 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

  18. 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

  19. 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

  20. Transcriptome assembly and alternative splicing analysis.

    PubMed

    Bonizzoni, Paola; Della Vedova, Gianluca; Pesole, Graziano; Picardi, Ernesto; Pirola, Yuri; Rizzi, Raffaella

    2015-01-01

    Alternative Splicing (AS) is the molecular phenomenon whereby multiple transcripts are produced from the same gene locus. As a consequence, it is responsible for the expansion of eukaryotic transcriptomes. Aberrant AS is involved in the onset and progression of several human diseases. Therefore, the characterization of exon-intron structure of a gene and the detection of corresponding transcript isoforms is an extremely relevant biological task. Nonetheless, the computational prediction of AS events and the repertoire of alternative transcripts is yet a challenging issue. Hereafter we introduce PIntron, a software package to predict the exon-intron structure and the full-length isoforms of a gene given a genomic region and a set of transcripts (ESTs and/or mRNAs). The software is open source and available at http://pintron.algolab.eu. PIntron has been designed for (and extensively tested on) a standard workstation without requiring dedicated expensive hardware. It easily manages large genomic regions and more than 20,000 ESTs, achieving good accuracy as shown in an experimental evaluation performed on 112 well-annotated genes selected from the ENCODE human regions used as training set in the EGASP competition. PMID:25577379

  1. Cloning of the gene encoding the murine clathrin-associated adaptor medium chain mu 2: gene organization, alternative splicing and chromosomal assignment.

    PubMed

    Ohno, H; Poy, G; Bonifacino, J S

    1998-04-14

    The mu 2 chain of the clathrin-associated adaptor complex AP-2 is a member of the adaptor medium chain family, a group of proteins involved in the sorting of integral membrane proteins in endocytic/exocytic pathways. Here, we report the cloning of the (MMU)CLAPM1 gene encoding the murine mu 2 chain, the first member of the family for which this information has become available. The mu 2 gene is approximately 8.5 kb long and is organized into 12 exons and 11 introns. Two transcripts are generated by alternative splicing of exon 5, a mini-exon of only six nucleotides. Proteins encoded by both transcripts are capable of interacting with tyrosine-based sorting signals, suggesting that they are functionally equivalent. The mu 2 gene is localized to the proximal region of mouse chromosome 16, which is syntenic to the proximal region of human chromosome 3. The isolation and characterization of the mu 2 gene should be instrumental for future studies of the genetics and physiological role of the adaptor medium chains in mammals. PMID:9573360

  2. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    PubMed

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells. PMID:15363843

  3. 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

  4. Genetic Variation of Pre-mRNA Alternative Splicing in Human Populations

    PubMed Central

    Lu, Zhi-xiang; Jiang, Peng; Xing, Yi

    2011-01-01

    The precise splicing outcome of a transcribed gene is controlled by complex interactions between cis regulatory splicing signals and trans-acting regulators. In higher eukaryotes, alternative splicing is a prevalent mechanism for generating transcriptome and proteome diversity. Alternative splicing can modulate gene function, affect organismal phenotype and cause disease. Common genetic variation that affects splicing regulation can lead to differences in alternative splicing between human individuals and consequently impact expression level or protein function. In several well-documented examples, such natural variation of alternative splicing has indeed been shown to influence disease susceptibility and drug response. With new microarray- and sequencing-based genomic technologies that can analyze eukaryotic transcriptomes at the exon- or nucleotide-level, it has become possible to globally compare the alternative splicing profiles across human individuals in any tissue or cell type of interest. Recent large-scale transcriptome studies using high-density splicing-sensitive microarray and deep RNA sequencing (RNA-Seq) have revealed widespread genetic variation of alternative splicing in humans. In the future, an extensive catalogue of alternative splicing variation in human populations will help elucidate the molecular underpinnings of complex traits and human diseases, and shed light on the mechanisms of splicing regulation in human cells. PMID:22095823

  5. 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

    2015-12-01

    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

  6. 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

  7. Variable number tandem repeat in exon/intron border of the cystathionine beta-synthase gene: a single nucleotide substitution in the second repeat prevents multiple alternate splicing.

    PubMed

    Yang, F; Hanson, N Q; Schwichtenberg, K; Tsai, M Y

    2000-12-11

    We studied a large number of individuals with respect to the 31-bp variable number tandem repeat (VNTR) in the cystathionine beta-synthase (CBS) gene. The number of repeats varies from 15-20, with 17 repeats the most common allele. Significantly, we found that the first repeat of the 31-bp VNTR originates 12 bp from the 5' end of exon 13 and extends 19 bp into intron 13. Since this VNTR spans across the exon-intron border, it can theoretically create multiple alternate splice sites. However, a substitution of g-->a at the exon-intron border is uniquely present in the second repeat, preventing alternate splicing at that site. While the g-->a substitution is absent from all subsequent 31-bp repeats, alternate splicing probably does not occur at those distal sites due to the lack of exon 13 sequences not contained in the repeats but needed for the binding of spliceosomes. Investigation of five individuals with normal plasma total homocysteine (tHcy) and five individuals with mild hyper-homocysteinemia shows that all have the g-->a substitution in the second repeat. Nonetheless, we speculate that the absence of this substitution may be found in rare individuals with normal CBS cDNA and unexplained hyperhomocysteinemia. Gene scanning and direct nucleotide sequencing were used to characterize the VNTR in 398 patients with premature coronary artery disease and 137 controls. Five alleles and 10 genotypes were found; 17/17 is the most prevalent genotype in our study population. The two other prevalent genotypes, 16/17 and 17/18, are associated with significantly decreased tHcy levels as compared to the 17/ 17 genotype, suggesting that the 16 and 18 repeats haplotype may be in linkage disequilibrium with regulatory elements which upregulate CBS gene transcription. PMID:11186895

  8. 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 T; 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

  9. 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

  10. 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

  11. 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

  12. Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing*

    PubMed Central

    Rappe, Ulrike; Schlechter, Tanja; Aschoff, Moritz; Hotz-Wagenblatt, Agnes; Hofmann, Ilse

    2014-01-01

    The armadillo repeat protein ARVCF is a component of adherens junctions. Similar to related proteins, such as p120-catenin and β-catenin, with known signaling functions, localization studies indicate a cytoplasmic and a nuclear pool of ARVCF. We find that ARVCF interacts with different proteins involved in mRNA-processing: the splicing factor SRSF1 (SF2/ASF), the RNA helicase p68 (DDX5), and the heterogeneous nuclear ribonucleoprotein hnRNP H2. All three proteins bind to ARVCF in an RNA-independent manner. Furthermore, ARVCF occurs in large RNA-containing complexes that contain both spliced and unspliced mRNAs of housekeeping genes. By domain analysis, we show that interactions occur via the ARVCF C terminus. Overexpression of ARVCF, p68, SRSF1, and hnRNP H2 induces a significant increase in splicing activity of a reporter mRNA. Upon depletion of ARVCF followed by RNA sequence analysis, several alternatively spliced transcripts are significantly changed. Therefore, we conclude that nuclear ARVCF influences splicing of pre-mRNAs. We hypothesize that ARVCF is involved in alternative splicing, generating proteomic diversity, and its deregulation may contribute to diseased states, such as cancer and neurological disorders. PMID:24644279

  13. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  14. A chloroplast retrograde signal regulates nuclear alternative splicing.

    PubMed

    Petrillo, Ezequiel; Godoy Herz, Micaela A; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J; Simpson, Craig; Brown, John W S; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R

    2014-04-25

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action, we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  15. 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

  16. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  17. 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

  18. Mutations in the unc-52 gene responsible for body wall muscle defects in adult Caenorhabditis elegans are located in alternatively spliced exons

    SciTech Connect

    Rogalski, T.M.; Gilchrist, E.J.; Mullen, G.P.

    1995-01-01

    The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping. 38 refs., 6 figs., 2 tabs.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  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. 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

  6. 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

  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. Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2.

    PubMed

    Santoro, Massimo; Masciullo, Marcella; Bonvissuto, Davide; Bianchi, Maria Laura Ester; Michetti, Fabrizio; Silvestri, Gabriella

    2013-08-01

    INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies. PMID:23666741

  9. Generation of structural and functional diversity in furin-like proteins in Drosophila melanogaster by alternative splicing of the Dfur1 gene.

    PubMed Central

    Roebroek, A J; Creemers, J W; Pauli, I G; Bogaert, T; Van de Ven, W J

    1993-01-01

    To investigate whether or not alternative splicing might be a mechanism by which in Drosophila melanogaster diversity is generated in endoproteases of the novel eukaryotic family of subtilisin-like proprotein processing enzymes, we determined structural and functional characteristics of the Dfur1 gene. Northern blot analysis revealed Dfur1 transcripts of 7.6, 6.5, 4.5 and 4.0 kb. By comparative nucleotide sequence analysis of Dfur1 genomic and cDNA clones, 10 coding exons were identified and, together with Northern blot analysis using exon-specific probes, evidence was obtained that the four transcripts are generated by alternative splicing and polyadenylation. The apparently complete open reading frames of three Dfur1 cDNAs revealed that these coded for three furin-like proteins, Dfurin1 (892 residues), Dfurin1-CRR (1101 residues) and Dfurin1-X (1269 residues), which possessed common but also unique structural domains. These various isoforms of furin in Drosophila were characterized in gene transfer studies using immunoprecipitation analysis. Differential expression of Dfur1 transcripts was found in Northern blot analysis of RNA from various developmental stages of Drosophila. RNA in situ hybridization experiments revealed that the Dfurin1-X and Dfurin1-CRR isoforms are expressed in non-overlapping sets of tissues during Drosophila embryogenesis. In gene transfer experiments in which the Dfurin1, Dfurin1-CRR and Dfurin1-X proteins were expressed at high levels together with the precursor of the beta A-chain of activin-A, a member of the transforming growth factor beta (TGF beta) superfamily, or the precursor of von Willebrand factor, all three proteins appeared capable of processing these substrates. Our studies indicate that the Dfur1 gene encodes structurally different subtilisin-like proprotein processing enzymes with distinct physiological functions in Drosophila. Images PMID:8491178

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

    PubMed

    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. 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

  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. 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

  14. Characterization of the six zebrafish clade B fibrillar procollagen genes, with evidence for evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide.

    PubMed

    Hoffman, Guy G; Branam, Amanda M; Huang, Guorui; Pelegri, Francisco; Cole, William G; Wenstrup, Richard M; Greenspan, Daniel S

    2010-05-01

    Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I-III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-alpha1(V), pro-alpha3(V), pro-alpha1(XI) and pro-alpha2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-alpha1(V), pro-alpha3(V)a and b, pro-alpha1(XI)a and b, and pro-alpha2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH(2)-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system. PMID:20102740

  15. Characterization of the Six Zebrafish Clade B Fibrillar Procollagen Genes, with Evidence for Evolutionarily Conserved Alternative Splicing within the pro-α1(V) C-propeptide

    PubMed Central

    Hoffman, Guy G.; Branam, Amanda M.; Huang, Guorui; Pelegri, Francisco; Cole, William G.; Wenstrup, Richard M.; Greenspan, Daniel S.

    2010-01-01

    Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I–III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-α1(V), pro-α3(V), pro-α1(XI) and pro-α2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-α1(V), proα3(V)a and b, pro-α1(XI)a and b, and pro-α2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH2-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-α1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system. PMID:20102740

  16. 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...

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. AVISPA: a web tool for the prediction and analysis of alternative splicing.

    PubMed

    Barash, Yoseph; Vaquero-Garcia, Jorge; González-Vallinas, Juan; Xiong, Hui Yuan; Gao, Weijun; Lee, Leo J; Frey, Brendan J

    2013-01-01

    Transcriptome complexity and its relation to numerous diseases underpins the need to predict in silico splice variants and the regulatory elements that affect them. Building upon our recently described splicing code, we developed AVISPA, a Galaxy-based web tool for splicing prediction and analysis. Given an exon and its proximal sequence, the tool predicts whether the exon is alternatively spliced, displays tissue-dependent splicing patterns, and whether it has associated regulatory elements. We assess AVISPA's accuracy on an independent dataset of tissue-dependent exons, and illustrate how the tool can be applied to analyze a gene of interest. AVISPA is available at http://avispa.biociphers.org. PMID:24156756

  2. 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

  3. 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

  4. Involvement of Alternative Splicing in Barley Seed Germination

    PubMed Central

    Zhang, Qisen; Zhang, Xiaoqi; Wang, Songbo; Tan, Cong; Zhou, Gaofeng; Li, Chengdao

    2016-01-01

    Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3’ splicing (34%-45%), intron retention (32%-34%) and alternative 5’ splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination. PMID:27031341

  5. Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA.

    PubMed Central

    Streuli, M; Saito, H

    1989-01-01

    Tissue-specific alternative splicing is an important mechanism for controlling gene expression. Exons 4, 5 and 6 of the human leukocyte common antigen (LCA) gene are included in B cell mRNA but excluded from thymocyte mRNA by differential splicing. In order to study this tissue-specific alternative splicing, we constructed mini-genes that contain only a few of the LCA exons and the SV40 promoter. Mouse B cells and thymocytes were transfected with these mini-gene constructs and the structures of mRNAs were determined by primer extension analysis. The results show that the same primary transcript is spliced alternatively in B cells and thymocytes. This finding suggests that there is a tissue-specific trans-acting factor that regulates the alternative splicing of LCA pre-mRNA. By making various deletion mutants, cis-elements necessary for tissue-specific splicing were confined within the alternatively spliced exons and their immediate flanking intron sequences. Furthermore, linker scanning analysis shows that there are at least three distinct cis-elements within the LCA exon 4 sequence that are required for tissue-specific alternative splicing. Possible mechanisms of LCA alternative splicing are discussed. Images PMID:2524382

  6. Molecular Characterization of the α-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.; André, 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

  7. 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

  8. 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

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

    PubMed Central

    Alamancos, Gael P.; Pagès, Amadís; Trincado, Juan L.; Bellora, Nicolás; 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

  10. Identification of cells deficient in signaling-induced alternative splicing by use of somatic cell genetics.

    PubMed Central

    Sheives, Paul; Lynch, Kristen W

    2002-01-01

    In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues. PMID:12515380

  11. 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, Tomaž; 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

  12. 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, Tomaž; 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

  13. 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

  14. Phytochrome controls alternative splicing to mediate light responses in Arabidopsis.

    PubMed

    Shikata, Hiromasa; Hanada, Kousuke; Ushijima, Tomokazu; Nakashima, Moeko; Suzuki, Yutaka; Matsushita, Tomonao

    2014-12-30

    Plants monitor the ambient light conditions using several informational photoreceptors, including red/far-red light absorbing phytochrome. Phytochrome is widely believed to regulate the transcription of light-responsive genes by modulating the activity of several transcription factors. Here we provide evidence that phytochrome significantly changes alternative splicing (AS) profiles at the genomic level in Arabidopsis, to approximately the same degree as it affects steady-state transcript levels. mRNA sequencing analysis revealed that 1,505 and 1,678 genes underwent changes in their AS and steady-state transcript level profiles, respectively, within 1 h of red light exposure in a phytochrome-dependent manner. Furthermore, we show that splicing factor genes were the main early targets of AS control by phytochrome, whereas transcription factor genes were the primary direct targets of phytochrome-mediated transcriptional regulation. We experimentally validated phytochrome-induced changes in the AS of genes that are involved in RNA splicing, phytochrome signaling, the circadian clock, and photosynthesis. Moreover, we show that phytochrome-induced AS changes of SPA1-RELATED 3, the negative regulator of light signaling, physiologically contributed to promoting photomorphogenesis. Finally, photophysiological experiments demonstrated that phytochrome transduces the signal from its photosensory domain to induce light-dependent AS alterations in the nucleus. Taking these data together, we show that phytochrome directly induces AS cascades in parallel with transcriptional cascades to mediate light responses in Arabidopsis. PMID:25512548

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

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

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

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  17. Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

    PubMed Central

    Bonomi, Serena; Gallo, Stefania; Catillo, Morena; Pignataro, Daniela; Biamonti, Giuseppe; Ghigna, Claudia

    2013-01-01

    Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments. PMID:24285959

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

    PubMed

    Selvanathan, Saravana P; Graham, Garrett T; 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; Üren, Aykut; Toretsky, Jeffrey A

    2015-03-17

    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 intron-exon 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 γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 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-4-279 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

  19. 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.; Üren, 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 intron–exon 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 γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 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-4–279 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. The CASR gene: alternative splicing and transcriptional control, and calcium-sensing receptor (CaSR) protein: structure and ligand binding sites.

    PubMed

    Hendy, Geoffrey N; Canaff, Lucie; Cole, David E C

    2013-06-01

    The calcium-sensing receptor (CaSR) is a G protein-coupled receptor encoded by a single copy gene. The human CASR gene spans ~103-kb and has eight exons. Promoters P1 and P2 drive transcription of exons 1A and 1B, respectively, encoding alternative 5'-UTRs that splice to exon 2 encoding the common part of the 5'-UTR. Exons 2-7 encode the CaSR protein of 1078 amino acids. Functional elements responsive to 1,25-dihydroxyvitamin D, proinflammatory cytokines, and glial cells missing-2 are present in the CASR promoters. Evolutionarily, the exon structure, first seen in aquatic vertebrates, is well-conserved with a single linkage disequilibrium haplotype block for protein coding exons 2-7. Structural features of the human CaSR protein are: an N-terminal signal peptide (19 amino acids (aa)); an extracellular domain (~600 aa) having a bi-lobed Venus Flytrap (VFT) domain with several Ca(2+)-binding sites; and a nine-cysteines domain that transduces the activation signal to the 7-transmembrane domain (250 aa) and the C-terminal tail (216 aa). PMID:23856260

  5. An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms.

    PubMed Central

    Tanaka, T; Tanaka, K; Ogawa, S; Kurokawa, M; Mitani, K; Nishida, J; Shibata, Y; Yazaki, Y; Hirai, H

    1995-01-01

    The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony-stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a. Images PMID:7530657

  6. Detection and quantification of alternative splice sites in Arabidopsis genes AtDCL2 and AtPTB2 with highly sensitive surface enhanced Raman spectroscopy (SERS) and gold nanoprobes.

    PubMed

    Kadam, Ulhas S; Schulz, Burkhard; Irudayaraj, Joseph

    2014-05-01

    Alternative splicing (AS) increases the size of the transcriptome and proteome to enhance the physiological capacity of cells. We demonstrate surface enhanced Raman spectroscopy (SERS) in combination with a DNA hybridization analytical platform to identify and quantify AS genes in plants. AS in AtDCL2 and AtPTB2 were investigated using non-fluorescent Raman probes using a 'sandwich assay'. Utilizing Raman probes conjugated to gold nanoparticles we demonstrate the recognition of RNA sequences specific to AtDCL2 and AtPTB2 splice junction variants with detection sensitivity of up to 0.1 fM. PMID:24631541

  7. Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements.

    PubMed

    D'Souza, I; Poorkaj, P; Hong, M; Nochlin, D; Lee, V M; Bird, T D; Schellenberg, G D

    1999-05-11

    Frontotemporal dementia with parkinsonism, chromosome 17 type (FTDP-17) is caused by mutations in the tau gene, and the signature lesions of FTDP-17 are filamentous tau inclusions. Tau mutations may be pathogenic either by altering protein function or gene regulation. Here we show that missense, silent, and intronic tau mutations can increase or decrease splicing of tau exon 10 (E10) by acting on 3 different cis-acting regulatory elements. These elements include an exon splicing enhancer that can either be strengthened (mutation N279(K)) or destroyed (mutation Delta280(K)), resulting in either constitutive E10 inclusion or the exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an exon splicing silencer, the function of which is abolished by a silent FTDP-17 mutation (L284(L)), resulting in excess E10 inclusion. A third element inhibiting E10 splicing is contained in the intronic sequences directly flanking the 5' splice site of E10 and intronic FTDP-17 mutations in this element enhance E10 inclusion. Thus, tau mutations cause FTDP-17 by multiple pathological mechanisms, which may explain the phenotypic heterogeneity observed in FTDP-17, as exemplified by an unusual family described here with tau pathology as well as amyloid and neuritic plaques. PMID:10318930

  8. 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

  9. 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

  10. An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

    PubMed Central

    Buettner, R; Kannan, P; Imhof, A; Bauer, R; Yim, S O; Glockshuber, R; Van Dyke, M W; Tainsky, M A

    1993-01-01

    AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts. Images PMID:8321221

  11. 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...

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. A general definition and nomenclature for alternative splicing events.

    PubMed

    Sammeth, Michael; Foissac, Sylvain; Guigó, 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 part--in human more than a quarter-of 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

  19. A General Definition and Nomenclature for Alternative Splicing Events

    PubMed Central

    Guigó, 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 part—in human more than a quarter—of 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

  20. Psip1/Ledgf p52 Binds Methylated Histone H3K36 and Splicing Factors and Contributes to the Regulation of Alternative Splicing

    PubMed Central

    Pradeepa, Madapura M.; Sutherland, Heidi G.; Ule, Jernej; Grimes, Graeme R.; Bickmore, Wendy A.

    2012-01-01

    Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing. PMID:22615581

  1. 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

  2. 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

  3. Evolution of exon-intron structure and alternative splicing.

    PubMed

    Koralewski, Tomasz E; Krutovsky, Konstantin V

    2011-01-01

    Despite significant advances in high-throughput DNA sequencing, many important species remain understudied at the genome level. In this study we addressed a question of what can be predicted about the genome-wide characteristics of less studied species, based on the genomic data from completely sequenced species. Using NCBI databases we performed a comparative genome-wide analysis of such characteristics as alternative splicing, number of genes, gene products and exons in 36 completely sequenced model species. We created statistical regression models to fit these data and applied them to loblolly pine (Pinus taeda L.), an example of an important species whose genome has not been completely sequenced yet. Using these models, the genome-wide characteristics, such as total number of genes and exons, can be roughly predicted based on parameters estimated from available limited genomic data, e.g. exon length and exon/gene ratio. PMID:21464961

  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. 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

  6. RON alternative splicing regulation in primary ovarian cancer.

    PubMed

    Mayer, Sebastian; Hirschfeld, Marc; Jaeger, Markus; Pies, Susanne; Iborra, Severine; Erbes, Thalia; Stickeler, Elmar

    2015-07-01

    The proto-oncogene recepteur d'origine nantais (RON, MST1R) and its alternatively spliced variants are involved in various tumor biological processes, such as cell motility, adhesion, proliferation, apoptosis and epithelial-to-mesenchymal transition (EMT). RON overexpression and the occurrence of specific alternatively spliced RON isoforms have been detected in ovarian cancer. In the present study, we evaluated the role and regulation of cancer-related RON splicing isoforms in primary ovarian cancer. Expression of RON variants (RONΔ165, RONΔ160) was determined in 45 primary ovarian cancer and 4 physiological ovarian tissue specimens by RT-PCR and western blot analysis. The results were correlated to clinicopathological parameters. Additionally, expression of splicing factors with known involvement in RON alternative splicing regulation was examined. Increased RON levels were detected in all tumor samples (p=0.001) without differences between the primary tumors and metastases. Alternative RON variants were present in the majority of tumor samples (39 of 45; 86.67%). Potential RONΔ165 occurred more often (82.22%) than potential RONΔ160 or RONΔ155 (24.40%). Several significant correlations of RON and splicing factor expression [e.g. ASF/SFRS1 (p=0.035)] were detected. Correlations of RON expression to clinicopathological parameters were not observed. Significant splicing factor interactions (e.g. SRp55/SRp75: p<0.001) were observed in tumor samples with alternative RON splicing. Our data demonstrated upregulated RON isoform expression and significant changes in splicing factor expression in primary ovarian cancer. These findings account for an essential regulatory interplay of splicing factor-driven alterations in the RON alternative splicing pattern with subsequent tumor biological consequences in ovarian cancer. PMID:25997828

  7. 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

  8. 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

  9. 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

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

    PubMed

    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

  11. 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

  12. 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

  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. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome.

    PubMed

    Finley, Jahahreeh

    2015-09-01

    Although the use of antiretroviral therapy (ART) has proven highly effective in controlling and suppressing HIV-1 replication, the persistence of latent but replication-competent proviruses in a small subset of CD4(+) memory T cells presents significant challenges to viral eradication from infected individuals. Attempts to eliminate latent reservoirs are epitomized by the 'shock and kill' approach, a strategy involving the combinatorial usage of compounds that influence epigenetic modulation and initiation of proviral transcription. However, efficient regulation of viral pre-mRNA splicing through manipulation of host cell splicing machinery is also indispensible for HIV-1 replication. Interestingly, aberrant alternative splicing of the LMNA gene via the usage of a cryptic splice site has been shown to be the cause of most cases of Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic condition characterized by an accelerated aging phenotype due to the accumulation of a truncated form of lamin A known as progerin. Recent evidence has shown that inhibition of the splicing factors ASF/SF2 (or SRSF1) and SRp55 (or SRSF6) leads to a reduction or an increase in progerin at both the mRNA and protein levels, respectively, thus altering the LMNA pre-mRNA splicing ratio. It is also well-established that during the latter stages of HIV-1 infection, an increase in the production and nuclear export of unspliced viral mRNA is indispensible for efficient HIV-1 replication and that the presence of ASF/SF2 leads to excessive viral pre-mRNA splicing and a reduction of unspliced mRNA, while the presence of SRp55 inhibits viral pre-mRNA splicing and aids in the generation and translation of unspliced HIV-1 mRNAs. The splicing-factor associated protein and putative mitochondrial chaperone p32 has also been shown to inhibit ASF/SF2, increase unspliced HIV-1 viral mRNA, and enhance mitochondrial DNA replication and oxidative phosphorylation. It is our hypothesis that activation of AMPK, a master regulator of cellular metabolism which has been shown to activate PKC-theta (θ) and is essential for T cell activation, may modulate the splicing activities of SRp55 as well as enhance a p32-mediated inhibition of ASF/SF2-induced alternative splicing, potentially correcting aberrant alternative splicing in the LMNA gene and reactivating latent viral HIV-1 reservoirs. Moreover, similar epigenetic modifications and cell cycle regulators also characterize the analogous stages of premature senescence in progeroid cells and latency in HIV-1 infected T cells. AMPK-activating compounds including metformin and resveratrol may thus embody a novel treatment paradigm linking the pathophysiology of HGPS with that of HIV-1 latency. PMID:26115946

  15. 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.

  16. 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

  17. 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

  18. Female-specific insect lethality engineered using alternative splicing.

    PubMed

    Fu, Guoliang; Condon, Kirsty C; Epton, Matthew J; Gong, Peng; Jin, Li; Condon, George C; Morrison, Neil I; Dafa'alla, Tarig H; Alphey, Luke

    2007-03-01

    The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters. PMID:17322873

  19. Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

    PubMed Central

    Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M.

    2010-01-01

    CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. PMID:20558599

  20. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-01-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled. PMID:18508565

  1. Validation of alternative transcript splicing in chicken lines that differ in genetic resistance to Marek’s disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Utilizing RNA-seq data, 1,574 candidate genes with alternative splicing were previously identified between two chicken lines that differ in Marek’s disease (MD) genetic resistance under control and Marek’s disease virus infection conditions. After filtering out 1,530 genes with splice variants in th...

  2. Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

    PubMed

    Wang, Xiu-Ye; Zheng, Zeng-Zhang; Song, Hong-Sheng; Xu, Yong-Zhen

    2014-01-01

    Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5' splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5' splice site at intron 3 of Lepidopteran dsx. We propose that the 5' splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5' splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx. PMID:24239545

  3. 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

  4. 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

  5. Genome-wide association between DNA methylation and alternative splicing in an invertebrate

    PubMed Central

    2012-01-01

    Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution. PMID:22978521

  6. 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

  7. 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

  8. 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.

  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. 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 PPARγ 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 ZNF638’s 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

  11. 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

  12. A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the μ Opioid Receptor Gene OPRM1 via hnRNPH Interactions

    PubMed Central

    Xu, Jin; Lu, Zhigang; Xu, Mingming; Pan, Ling; Deng, Yi; Xie, Xiaohu; Liu, Huifen; Ding, Shixiong; Hurd, Yasmin L.; Pasternak, Gavril W.; Klein, Robert J.; Cartegni, Luca

    2014-01-01

    Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction. PMID:25122903

  13. A heroin addiction severity-associated intronic single nucleotide polymorphism modulates alternative pre-mRNA splicing of the μ opioid receptor gene OPRM1 via hnRNPH interactions.

    PubMed

    Xu, Jin; Lu, Zhigang; Xu, Mingming; Pan, Ling; Deng, Yi; Xie, Xiaohu; Liu, Huifen; Ding, Shixiong; Hurd, Yasmin L; Pasternak, Gavril W; Klein, Robert J; Cartegni, Luca; Zhou, Wenhua; Pan, Ying-Xian

    2014-08-13

    Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction. PMID:25122903

  14. 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

  15. Modulation of alternative splicing with chemical compounds in new therapeutics for human diseases.

    PubMed

    Ohe, Kenji; Hagiwara, Masatoshi

    2015-04-17

    Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the "genome code," have been found to result in an aberrant mis-spliced "transcript code" with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced "transcript code". We will briefly mention about how to obtain splicing-modifying-compounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acid-based strategies. The information on their involvement in mis-splicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy. PMID:25560473

  16. 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

  17. 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

  18. 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

  19. 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

  20. Alternative splicing modulated by genetic variants demonstrates accelerated evolution regulated by highly conserved proteins.

    PubMed

    Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Lin, Xianzhi; Chan, Tak-Ming; Wang, Rena; Xiao, Xinshu

    2016-04-01

    Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting thatcis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon. PMID:26888265

  1. 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

  2. Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins.

    PubMed

    Hao, Yanqi; Colak, Recep; Teyra, Joan; Corbi-Verge, Carles; Ignatchenko, Alexander; Hahne, Hannes; Wilhelm, Mathias; Kuster, Bernhard; Braun, Pascal; Kaida, Daisuke; Kislinger, Thomas; Kim, Philip M

    2015-07-14

    Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse), which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS) spectra for an overall AU-ROC of 0.85. We predict that around 32% of "exon skipping" alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing. PMID:26146086

  3. Expression of Two Novel Alternatively Spliced COL2A1 Isoforms During Chondrocyte Differentiation

    PubMed Central

    McAlinden, Audrey; Johnstone, Brian; Kollar, John; Kazmi, Najam; Hering, Thomas M.

    2008-01-01

    Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by use of an alternative 5’ splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by use of another 5’ alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5’ splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites. PMID:18023161

  4. Role of Acinus in regulating retinoic acid-responsive gene pre-mRNA splicing.

    PubMed

    Wang, Fang; Soprano, Kenneth J; Soprano, Dianne Robert

    2015-04-01

    Acinus-S' is a corepressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study, the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters, while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARβ and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription. PMID:25205379

  5. Role of Acinus in Regulating Retinoic Acid-Responsive Gene Pre-mRNA Splicing

    PubMed Central

    Wang, Fang; Soprano, Kenneth J.; Soprano, Dianne Robert

    2014-01-01

    Acinus-S’ is a co-repressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S’, with the activity of Acinus-L higher than that of Acinus-S’, increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5′ splice site but not a RA-responsive minigene containing a strong 5′ splice site. RA treatment further enhances the splicing of the weak 5′ splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5′ splice site of these two genes in a RA-dependent manner for RARβ and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription. PMID:25205379

  6. 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

  7. 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

  8. DARPP-32 binds to tra2-beta1 and influences alternative splicing

    PubMed Central

    Benderska, Natalya; Becker, Kristina; Girault, Jean-Antoine; Becker, Cord-Michael; Andreadis, Athena; Stamm, Stefan

    2010-01-01

    The majority of human genes undergo alternative splicing, which is frequently altered in response to physiological stimuli. DARPP-32 (Dopamine and cAMP regulated phosphoprotein, 32 kD) is a component of PKA-dependent signaling pathways. Here we show that DARPP-32 binds directly to the splicing factor tra2-beta1 (transformer 2). DARPP-32 changes the usage of tra2-beta1 dependent alternative exons in a concentration dependent manner, suggesting that the DARPP-32:tra2-beta1 interaction is a molecular link between signaling pathways and pre-mRNA processing. PMID:20074680

  9. Mapping of the human BAX gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX{delta}

    SciTech Connect

    Apte, S.S.; Olsen, B.R.; Mattei, M.G.

    1995-04-10

    The BAX gene is a member of the Bcl-2 gene family; it encodes a 21-kDa protein whose association with Bcl-2 is believed to play a critical role in regulating apoptosis. Through analysis of human-hamster somatic cell hybrid DNA and by in situ hybridization to metaphase chromosomes, we have determined that the human BAX gene is located in the q13.3-q13.4 region of human chromosome 19. We have also isolated a BAX cDNA clone in which that part of the mRNA encoded by exon 3 is absent. The skipping of exon 3 and the resultant splicing of exons 2 and 4 maintains the original reading frame and predicts the existence of an interstitially truncated form of the major Bax protein (Bax{alpha}), termed Bax{delta}. Unlike two previously described variant forms of Bax{alpha} (Bax{beta} and Bax{tau}), Bax{delta} retains the functionally critical C-terminal membrane anchor region as well as the Bcl-2 homology 1 and 2 (BH1 and BH2) domains. 10 refs., 1 fig., 1 tab.

  10. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    PubMed

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein. PMID:26609938

  11. 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...

  12. New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process.

    PubMed

    Dehghanian, Fariba; Hojati, Zohreh; Kay, Maryam

    2014-10-01

    Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angio-genic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases. PMID:25414781

  13. 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.

  14. 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.

  15. Robust detection of alternative splicing in a population of single cells.

    PubMed

    Welch, Joshua D; Hu, Yin; Prins, Jan F

    2016-05-01

    Single cell RNA-seq experiments provide valuable insight into cellular heterogeneity but suffer from low coverage, 3' bias and technical noise. These unique properties of single cell RNA-seq data make study of alternative splicing difficult, and thus most single cell studies have restricted analysis of transcriptome variation to the gene level. To address these limitations, we developed SingleSplice, which uses a statistical model to detect genes whose isoform usage shows biological variation significantly exceeding technical noise in a population of single cells. Importantly, SingleSplice is tailored to the unique demands of single cell analysis, detecting isoform usage differences without attempting to infer expression levels for full-length transcripts. Using data from spike-in transcripts, we found that our approach detects variation in isoform usage among single cells with high sensitivity and specificity. We also applied SingleSplice to data from mouse embryonic stem cells and discovered a set of genes that show significant biological variation in isoform usage across the set of cells. A subset of these isoform differences are linked to cell cycle stage, suggesting a novel connection between alternative splicing and the cell cycle. PMID:26740580

  16. Robust detection of alternative splicing in a population of single cells

    PubMed Central

    Welch, Joshua D.; Hu, Yin; Prins, Jan F.

    2016-01-01

    Single cell RNA-seq experiments provide valuable insight into cellular heterogeneity but suffer from low coverage, 3′ bias and technical noise. These unique properties of single cell RNA-seq data make study of alternative splicing difficult, and thus most single cell studies have restricted analysis of transcriptome variation to the gene level. To address these limitations, we developed SingleSplice, which uses a statistical model to detect genes whose isoform usage shows biological variation significantly exceeding technical noise in a population of single cells. Importantly, SingleSplice is tailored to the unique demands of single cell analysis, detecting isoform usage differences without attempting to infer expression levels for full-length transcripts. Using data from spike-in transcripts, we found that our approach detects variation in isoform usage among single cells with high sensitivity and specificity. We also applied SingleSplice to data from mouse embryonic stem cells and discovered a set of genes that show significant biological variation in isoform usage across the set of cells. A subset of these isoform differences are linked to cell cycle stage, suggesting a novel connection between alternative splicing and the cell cycle. PMID:26740580

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Identification of genetic variants associated with alternative splicing using sQTLseekeR

    PubMed Central

    Monlong, Jean; Calvo, Miquel; Ferreira, Pedro G.; Guigó, Roderic

    2014-01-01

    Identification of genetic variants affecting splicing in RNA sequencing population studies is still in its infancy. Splicing phenotype is more complex than gene expression and ought to be treated as a multivariate phenotype to be recapitulated completely. Here we represent the splicing pattern of a gene as the distribution of the relative abundances of a gene’s alternative transcript isoforms. We develop a statistical framework that uses a distance-based approach to compute the variability of splicing ratios across observations, and a non-parametric analogue to multivariate analysis of variance. We implement this approach in the R package sQTLseekeR and use it to analyze RNA-Seq data from the Geuvadis project in 465 individuals. We identify hundreds of single nucleotide polymorphisms (SNPs) as splicing QTLs (sQTLs), including some falling in genome-wide association study SNPs. By developing the appropriate metrics, we show that sQTLseekeR compares favorably with existing methods that rely on univariate approaches, predicting variants that behave as expected from mutations affecting splicing. PMID:25140736

  4. 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

  5. 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

  6. 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

  7. 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

  8. TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing.

    PubMed

    Marina, Ryan J; Sturgill, David; Bailly, Marc A; Thenoz, Morgan; Varma, Garima; Prigge, Maria F; Nanan, Kyster K; Shukla, Sanjeev; Haque, Nazmul; Oberdoerffer, Shalini

    2016-01-01

    Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins. PMID:26711177

  9. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

    PubMed Central

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2014-01-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins. PMID:24213538

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Genome-Wide Analysis of Alternative Splicing in Zea mays: Landscape and Genetic Regulation[C][W

    PubMed Central

    Thatcher, Shawn R.; Zhou, Wengang; Leonard, April; Wang, Bing-Bing; Beatty, Mary; Zastrow-Hayes, Gina; Zhao, Xiangyu; Baumgarten, Andy; Li, Bailin

    2014-01-01

    Alternative splicing enhances transcriptome diversity in all eukaryotes and plays a role in plant tissue identity and stress adaptation. To catalog new maize (Zea mays) transcripts and identify genomic loci that regulate alternative splicing, we analyzed over 90 RNA-seq libraries from maize inbred lines B73 and Mo17, as well as Syn10 doubled haploid lines (progenies from B73 × Mo17). Transcript discovery was augmented with publicly available data from 14 maize tissues, expanding the maize transcriptome by more than 30,000 and increasing the percentage of intron-containing genes that undergo alternative splicing to 40%. These newly identified transcripts greatly increase the diversity of the maize proteome, sometimes coding for entirely different proteins compared with their most similar annotated isoform. In addition to increasing proteome diversity, many genes encoding novel transcripts gained an additional layer of regulation by microRNAs, often in a tissue-specific manner. We also demonstrate that the majority of genotype-specific alternative splicing can be genetically mapped, with cis-acting quantitative trait loci (QTLs) predominating. A large number of trans-acting QTLs were also apparent, with nearly half located in regions not shown to contain genes associated with splicing. Taken together, these results highlight the currently underappreciated role that alternative splicing plays in tissue identity and genotypic variation in maize. PMID:25248552

  20. 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

  1. 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

  2. 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 Central

    Kabat, Jennifer L.; Barberan-Soler, Sergio; Zahler, Alan M.

    2009-01-01

    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

  3. 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

  4. Analyzing alternative splicing data of splice junction arrays from Parkinson patients' leukocytes before and after deep brain stimulation as compared with control donors

    PubMed Central

    Soreq, Lilach; Salomonis, Nathan; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2015-01-01

    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

  5. 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 Krüppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krüppel-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

  6. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    SciTech Connect

    Giblin, P.; Kavathas, P. ); Ledbetter, J.A. )

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  7. 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

  8. Alternative Splicing QTLs in European and African Populations

    PubMed Central

    Ongen, Halit; Dermitzakis, Emmanouil T.

    2015-01-01

    With the advent of RNA-sequencing technology, we can detect different types of alternative splicing and determine how DNA variation regulates splicing. However, given the short read lengths used in most population-based RNA-sequencing experiments, quantifying transcripts accurately remains a challenge. Here we present a method, Altrans, for discovery of alternative splicing quantitative trait loci (asQTLs). To assess the performance of Altrans, we compared it to Cufflinks and MISO in simulations and Cufflinks for asQTL discovery. Simulations show that in the presence of unannotated transcripts, Altrans performs better in quantifications than Cufflinks and MISO. We have applied Altrans and Cufflinks to the Geuvadis dataset, which comprises samples from European and African populations, and discovered (FDR = 1%) 1,427 and 166 asQTLs with Altrans and 1,737 and 304 asQTLs with Cufflinks for Europeans and Africans, respectively. We show that, by discovering a set of asQTLs in a smaller subset of European samples and replicating these in the remaining larger subset of Europeans, both methods achieve similar replication levels (95% for both methods). We find many Altrans-specific asQTLs, which replicate to a high degree (93%). This is mainly due to junctions absent from the annotations and hence not tested with Cufflinks. The asQTLs are significantly enriched for biochemically active regions of the genome, functional marks, and variants in splicing regions, highlighting their biological relevance. We present an approach for discovering asQTLs that is a more direct assessment of splicing compared to other methods and is complementary to other transcript quantification methods. PMID:26430802

  9. The role of HMGCR alternative splicing in statin efficacy

    PubMed Central

    Medina, Marisa Wong; Krauss, Ronald M.

    2009-01-01

    Statins, or 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors, are widely prescribed to lower plasma cholesterol levels and reduce cardiovascular disease (CVD) risk. Despite the well documented efficacy of statins, there is large inter-individual variation in response. Using a panel of immortalized lymphocyte cell lines incubated with simvastatin, we recently found that the magnitude of expression of an alternatively spliced HMGCR transcript lacking exon 13 was inversely correlated with in vivo reductions of total cholesterol, LDL cholesterol, apoB, and triglycerides following statin treatment of the individuals from whom the cells were derived. This review will discuss the potential significance of alternative splicing as a mechanism contributing to variation in statin efficacy as well as the utility of immortalized lymphocyte cell lines for identifying pharmacogenetically relevant polymorphisms and molecular mechanisms. PMID:20005478

  10. [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

  11. SRSF1-Regulated Alternative Splicing in Breast Cancer.

    PubMed

    Anczuków, Olga; Akerman, Martin; Cléry, Antoine; Wu, Jie; Shen, Chen; Shirole, Nitin H; Raimer, Amanda; Sun, Shuying; Jensen, Mads A; Hua, Yimin; Allain, Frédéric 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

  12. 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

  13. Exonal elements and factors involved in the depolarization-induced alternative splicing of neurexin 2.

    PubMed

    Rozic, G; Lupowitz, Z; Zisapel, N

    2013-05-01

    The neurexin genes (NRXN1, NRXN2, and NRXN3) encode polymorphic presynaptic proteins that are implicated in synaptic plasticity and memory processing. In rat brain neurons grown in culture, depolarization induces reversible, calcium-dependent, repression of NRXN2α exon 11 (E11) splicing. Using Neuro2a cells as a model, we explored E11 cis elements and trans-acting factors involved in alternative splicing of NRXN2α E11 pre-mRNA under basal and depolarization conditions. E11 mutation studies revealed two motifs, CTGCCTG (enhancer) and GCACCCA (suppressor) regulating NRXN2α E11 alternative splicing. Subsequent E11 RNA affinity pull-down experiments demonstrated heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP L binding to this exon. Under depolarization, the amount of E11-bound hnRNP L (but not of hnRNP K) increased, in parallel to NRXN2α E11 splicing repression. Depletion of hnRNP K or hnRNP L in the Neuro2a cells by specific siRNAs enhanced NRXN2α E11 splicing and ablated the depolarization-induced repression of this exon. In addition, depolarization suppressed whereas hnRNP K depletion enhanced NRXN2α expression. These results indicate a role for hnRNP K in regulation of NRXN2α expression and of hnRNP L in the activity-dependent alternative splicing of neurexins which may potentially govern trans-synaptic signaling required for memory processing. PMID:23180095

  14. A mutation in the AtPRP4 splicing factor gene suppresses seed development in Arabidopsis.

    PubMed

    Raab, S; Hoth, S

    2007-05-01

    The spliceosome catalyzes alternative splicing of many genes in eucaryotic cells. This leads to the expression of distinct proteins. Components of the spliceosome are conserved in mammals and plants. Because splicing can be affected by environmental stress, we analyzed the regulation of splicing-related genes that encode small nuclear ribonucleoprotein particle (snRNP) proteins by the stress hormone abscisic acid (ABA). The transcript abundance of about 25 % of those genes was changed by at least 1.5-fold after addition of ABA. The U4/U6-specific snRNP gene AtPRP4 was strongly repressed by ABA. The homozygous knock-out of AtPRP4 resulted in the suppression of seed development suggesting that the gene product of this stress hormone-regulated gene is crucial for normal seed development. PMID:17236097

  15. 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

  16. 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

  17. 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

  18. 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

  19. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

    PubMed

    Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

    2010-10-01

    Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably 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 Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-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 but the basal response to noxious thermal stimuli is not altered. 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

  20. Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

    PubMed

    Lind, Ulrika; Alm Rosenblad, Magnus; Wrange, Anna-Lisa; Sundell, Kristina S; Jonsson, Per R; André, 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

  1. The haplo-spliceo-transcriptome: common variations in alternative splicing in the human population

    PubMed Central

    Graveley, Brenton R.

    2008-01-01

    Numerous inherited human genetic disorders are caused by defects in pre-mRNA splicing. Two recent studies have added a new twist to the link between genetic variation and pre-mRNA splicing by identifying SNPs that correlate with heritable changes in alternative splicing but do not cause disease. This suggests that allele-specific alternative splicing is a mechanism that accounts for individual variation in the human population. PMID:18054116

  2. Alternative splicing of SV40 early pre-mRNA in vitro.

    PubMed Central

    van Santen, V L; Spritz, R A

    1986-01-01

    Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vivo, cleavage at the T antigen 5' splice site is more efficient than cleavage at the t antigen 5' splice site in vitro, although both of these 5' splice sites are utilized relatively inefficiently in vitro. The ratio of cleavage at the T and t antigen 5' splice sites is not changed significantly by a number of alterations in the conditions under which the in vitro splicing reactions are carried out. Images PMID:3027668

  3. 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

  4. Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Zimmerly, Steven

    2014-06-01

    Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. PMID:24751650

  5. 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

  6. 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.

  7. 20-hydroxyecdysone mediates non-canonical regulation of mosquito vitellogenins through alternative splicing

    PubMed Central

    Provost-Javier, K. N.; Rasgon, J. L.

    2015-01-01

    Vitellogenesis is one of the most well-studied physiological processes in mosquitoes. Expression of mosquito vitellogenin genes is classically described as being restricted to female adult reproduction. We report premature vitellogenin transcript expression in three vector mosquitoes: Culex tarsalis, Aedes aegypti and Anopheles gambiae. Vitellogenins expressed during non-reproductive stages are alternatively spliced to retain their first intron and encode premature termination codons. We show that intron retention results in transcript degradation by translation-dependent nonsense-mediated mRNA decay. This is probably an example of regulated unproductive splicing and translation (RUST), a mechanism known to regulate gene expression in numerous organisms but which has never been described in mosquitoes. We demonstrate that the hormone 20-hydroxyecdysone (20E) is responsible for regulating post-transcriptional splicing of vitellogenin. After exposure of previtellogenic fat bodies to 20E, vitellogenin expression switches from a non-productive intron-retaining transcript to a spliced protein-coding transcript. This effect is independent of factors classically known to influence transcription, such as juvenile hormone-mediated competence and amino acid signalling through the target of rapamycin pathway. Non-canonical regulation of vitellogenesis through RUST is a novel role for the multifunctional hormone 20E, and may have important implications for general patterns of gene regulation in mosquitoes. PMID:24720618

  8. 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

  9. 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

  10. Coordinated tissue-specific regulation of adjacent alternative 3' splice sites in C. elegans.

    PubMed

    Ragle, James Matthew; Katzman, Sol; Akers, Taylor F; Barberan-Soler, Sergio; Zahler, Alan M

    2015-07-01

    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

  11. Regulation of alternative splicing through coupling with transcription and chromatin structure.

    PubMed

    Naftelberg, Shiran; Schor, Ignacio E; Ast, Gil; Kornblihtt, Alberto R

    2015-01-01

    Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing. PMID:26034889

  12. The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels.

    PubMed

    Allen, Summer E; Darnell, Robert B; Lipscombe, Diane

    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 theCaV2 .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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Control of myosin-I force sensing by alternative splicing.

    PubMed

    Laakso, Joseph M; Lewis, John H; Shuman, Henry; Ostap, E Michael

    2010-01-12

    Myosin-Is are molecular motors that link cellular membranes to the actin cytoskeleton, where they play roles in mechano-signal transduction and membrane trafficking. Some myosin-Is are proposed to act as force sensors, dynamically modulating their motile properties in response to changes in tension. In this study, we examined force sensing by the widely expressed myosin-I isoform, myo1b, which is alternatively spliced in its light chain binding domain (LCBD), yielding proteins with lever arms of different lengths. We found the actin-detachment kinetics of the splice isoforms to be extraordinarily tension-sensitive, with the magnitude of tension sensitivity to be related to LCBD splicing. Thus, in addition to regulating step-size, motility rates, and myosin activation, the LCBD is a key regulator of force sensing. We also found that myo1b is substantially more tension-sensitive than other myosins with similar length lever arms, indicating that different myosins have different tension-sensitive transitions. PMID:20080738

  18. 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

  19. Genome-Wide Detection of Condition-Sensitive Alternative Splicing in Arabidopsis Roots1[C][W

    PubMed Central

    Li, Wenfeng; Lin, Wen-Dar; Ray, Prasun; Lan, Ping; Schmidt, Wolfgang

    2013-01-01

    Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms. PMID:23735510

  20. Purifying Selection on Exonic Splice Enhancers in Intronless Genes.

    PubMed

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  1. Purifying Selection on Exonic Splice Enhancers in Intronless Genes

    PubMed Central

    Savisaar, Rosina; Hurst, Laurence D.

    2016-01-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  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. Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

    PubMed Central

    Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

    2012-01-01

    Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

  6. Mnk2 alternative splicing modulates the p38-MAPK pathway and impacts Ras-induced transformation.

    PubMed

    Maimon, Avraham; Mogilevsky, Maxim; Shilo, Asaf; Golan-Gerstl, Regina; Obiedat, Akram; Ben-Hur, Vered; Lebenthal-Loinger, Ilana; Stein, Ilan; Reich, Reuven; Beenstock, Jonah; Zehorai, Eldar; Andersen, Claus L; Thorsen, Kasper; Orntoft, Torben F; Davis, Roger J; Davidson, Ben; Mu, David; Karni, Rotem

    2014-04-24

    The kinase Mnk2 is a substrate of the MAPK pathway and phosphorylates the translation initiation factor eIF4E. In humans, MKNK2, the gene encoding for Mnk2, is alternatively spliced yielding two splicing isoforms with differing last exons: Mnk2a, which contains a MAPK-binding domain, and Mnk2b, which lacks it. We found that the Mnk2a isoform is downregulated in breast, lung, and colon tumors and is tumor suppressive. Mnk2a directly interacts with, phosphorylates, activates, and translocates p38α-MAPK into the nucleus, leading to activation of its target genes, increasing cell death and suppression of Ras-induced transformation. Alternatively, Mnk2b is pro-oncogenic and does not activate p38-MAPK, while still enhancing eIF4E phosphorylation. We further show that Mnk2a colocalization with p38α-MAPK in the nucleus is both required and sufficient for its tumor-suppressive activity. Thus, Mnk2a downregulation by alternative splicing is a tumor suppressor mechanism that is lost in some breast, lung, and colon tumors. PMID:24726367

  7. Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis.

    PubMed

    Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

    2015-01-01

    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 SGR5α transcription factor and the truncated SGR5β form lacking functional ZF motifs. The SGR5β form inhibits SGR5α 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 SGR5β proteins. Accordingly, transgenic plants overexpressing SGR5β 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

  8. 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

  9. 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

  10. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins.

    PubMed

    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

    2015-11-01

    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. 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

  12. Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells

    PubMed Central

    Calabretta, Sara; Bielli, Pamela; Passacantilli, Ilaria; Pilozzi, Emanuela; Fendrich, Volker; Capurso, Gabriele; Delle Fave, Gianfranco; Sette, Claudio

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favour the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, up-regulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to up-regulation of PTBP1 and modulation of PKM alternative splicing in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy. PMID:26234680

  13. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain

    PubMed Central

    Rogelj, Boris; Easton, Laura E.; Bogu, Gireesh K.; Stanton, Lawrence W.; Rot, Gregor; Curk, Tomaž; Zupan, Blaž; Sugimoto, Yoichiro; Modic, Miha; Haberman, Nejc; Tollervey, James; Fujii, Ritsuko; Takumi, Toru; Shaw, Christopher E.; Ule, Jernej

    2012-01-01

    Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS−/− brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development. PMID:22934129

  14. ASPicDB: a database web tool for alternative splicing analysis.

    PubMed

    D'Antonio, Mattia; Castrgnanò, Tiziana; Pallocca, Matteo; D'Erchia, Anna Maria; Picardi, Ernesto; Pesole, Graziano

    2015-01-01

    Alternative splicing (AS) is a basic molecular phenomenon that increases the functional complexity of higher eukaryotic transcriptomes. Indeed, through AS individual gene loci can generate multiple RNAs from the same pre-mRNA. AS has been investigated in a variety of clinical and pathological studies, such as the transcriptome regulation in cancer. In human, recent works based on massive RNA sequencing indicate that >95 % of pre-mRNAs are processed to yield multiple transcripts. Given the biological relevance of AS, several computational efforts have been done leading to the implementation of novel algorithms and specific specialized databases. Here we describe the web application ASPicDB that allows the recovery of detailed biological information about the splicing mechanism. ASPicDB provides powerful querying systems to interrogate AS events at gene, transcript, and protein levels. Finally, ASPicDB includes web visualization instruments to browse and export results for further off-line analyses. PMID:25577391

  15. Structural and functional analyses of Barth syndrome-causing mutations and alternative splicing in the tafazzin acyltransferase domain.

    PubMed

    Hijikata, Atsushi; Yura, Kei; Ohara, Osamu; Go, Mitiko

    2015-06-01

    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

  16. Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A

    PubMed Central

    Corrionero, Anna; Miñana, Belén; Valcárcel, Juan

    2011-01-01

    Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5′ of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3′ splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition. PMID:21363963

  17. Multiple non-coding exons and alternative splicing in the mouse Mas protooncogene.

    PubMed

    Alenina, Natalia; Böhme, Ilka; Bader, Michael; Walther, Thomas

    2015-09-01

    The Mas protooncogene encodes a G protein-coupled receptor with the common seven transmembrane domains, expressed mainly in the testis and brain. We provided evidence that Mas is a functional angiotensin-(1-7) receptor and can interact with the angiotensin II type 1 (AT1) receptor. The gene is transcriptionally regulated during development in the brain and testis, but its structure was unresolved. In this study we used 5'- and 3'-RACE, RT-PCR, and RNase-protection assays to elucidate the complete Mas gene structure and organization. We identified 12 exons in the mouse Mas gene with 11 in the 5' untranslated mRNA, which can be alternatively spliced. We also showed that Mas transcription can start from 4 tissue-specific promoters, whereby testis-specific Mas mRNA is transcribed from two upstream promoters, and the expression of Mas in the brain starts from two downstream promoters. Alternative splicing and multiple promoter usage result in at least 12 Mas transcripts in which different 5' untranslated regions are fused to a common coding sequence. Moreover, termination of Mas mRNA is regulated by two different polyadenylation signals. The gene spans approximately 27 kb, and the longest detected mRNA contains 2,451 bp. Thus, our results characterize the Mas protooncogene as the gene with the most complex gene structure of all described members of the gene family coding for G protein-coupled receptors. PMID:26003294

  18. 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

  19. Novel female-specific trans-spliced and alternative splice forms of dsx in the silkworm Bombyx mori.

    PubMed

    Duan, Jianping; Xu, Hanfu; Wang, Feng; Ma, Sanyuan; Zha, Xingfu; Guo, Huizhen; Zhao, Ping; Xia, Qingyou

    2013-02-15

    The Bombyx mori doublesex gene (Bmdsx) plays an important role in somatic sexual development. Its pre-mRNA splices in a sex-specific manner to generate two female-specific and one male-specific splice forms. The present study investigated six novel dsx variants generated by trans-splicing between female dsx transcripts and two additional novel genes, dsr1 and dsr2. Expression analysis indicated that Bmdsx-dsr1 represented splicing noise, whereas dsr2, which trans-spliced with dsx to generate five variants, regulated the expression of the female-specific B. mori dsx transcript Bmdsx(F)s. We unexpectedly found a novel exon 2n insertion during Bmdsx transcription, which did not influence the validity of the novel protein, BmDSX(F3). Ectopic expression of BmDSX(F3) repressed the pheromone-binding protein gene and the testis-specific gene A2 in males, and activated of the storage protein 1 gene. Our findings suggest that trans-splicing is a novel regulatory function of Bmdsx, which participates in female sexual development by regulating the expression of three BmDSX(F) proteins. PMID:23261436

  20. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

    PubMed Central

    Lou, H; Yang, Y; Cote, G J; Berget, S M; Gagel, R F

    1995-01-01

    Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing. PMID:8524281

  1. Structural determinants for alternative splicing regulation of the MAPT pre-mRNA

    PubMed Central

    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

  2. HNRNPA1 regulates HMGCR alternative splicing and modulates cellular cholesterol metabolism

    PubMed Central

    Yu, Chi-Yi; Theusch, Elizabeth; Lo, Kathleen; Mangravite, Lara M.; Naidoo, Devesh; Kutilova, Mariya; Medina, Marisa W.

    2014-01-01

    3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) encodes the rate-limiting enzyme in the cholesterol biosynthesis pathway and is inhibited by statins, a class of cholesterol-lowering drugs. Expression of an alternatively spliced HMGCR transcript lacking exon 13, HMGCR13(−), has been implicated in the variation of plasma LDL-cholesterol (LDL-C) and is the single most informative molecular marker of LDL-C response to statins. Given the physiological importance of this transcript, our goal was to identify molecules that regulate HMGCR alternative splicing. We recently reported gene expression changes in 480 lymphoblastoid cell lines (LCLs) after in vitro simvastatin treatment, and identified a number of statin-responsive genes involved in mRNA splicing. Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) was chosen for follow-up since rs3846662, an HMGCR SNP that regulates exon 13 skipping, was predicted to alter an HNRNPA1 binding motif. Here, we not only demonstrate that rs3846662 modulates HNRNPA1 binding, but also that sterol depletion of human hepatoma cell lines reduced HNRNPA1 mRNA levels, an effect that was reversed with sterol add-back. Overexpression of HNRNPA1 increased the ratio of HMGCR13(−) to total HMGCR transcripts by both directly increasing exon 13 skipping in an allele-related manner and specifically stabilizing the HMGCR13(−) transcript. Importantly, HNRNPA1 overexpression also diminished HMGCR enzyme activity, enhanced LDL-C uptake and increased cellular apolipoprotein B (APOB). rs1920045, an SNP associated with HNRNPA1 exon 8 alternative splicing, was also associated with smaller statin-induced reduction in total cholesterol from two independent clinical trials. These results suggest that HNRNPA1 plays a role in the variation of cardiovascular disease risk and statin response. PMID:24001602

  3. 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 4–6 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 24 hpf, 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 4–6), 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 4–5–6) 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

  4. Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2.

    PubMed

    Grammatikakis, Ioannis; Zhang, Peisu; Panda, Amaresh C; Kim, Jiyoung; Maudsley, Stuart; Abdelmohsen, Kotb; Yang, Xiaoling; Martindale, Jennifer L; Motiño, Omar; Hutchison, Emmette R; Mattson, Mark P; Gorospe, Myriam

    2016-05-01

    During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH) as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation. PMID:27117401

  5. 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

  6. The role played by alternative splicing in antigenic variability in human endo-parasites.

    PubMed

    Hull, Rodney; Dlamini, Zodwa

    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 host's 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

  7. 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

  8. 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 host’s 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

  9. Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

    PubMed

    Yap, Karen; Xiao, Yixin; Friedman, Brad A; Je, H Shawn; Makeyev, Eugene V

    2016-05-10

    Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons. PMID:27134173

  10. 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

  11. 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

  12. Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.

    PubMed

    Zhang, Xiao-Ning; Mount, Stephen M

    2009-07-01

    The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3' splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions. PMID:19403727

  13. My road to alternative splicing control: from simple paths to loops and interconnections.

    PubMed

    Chabot, Benoit

    2015-06-01

    With the functional importance of alternative splicing being validated in nearly every mammalian biological system and implicated in many human diseases, it is now crucial to identify the molecular programs that control the production of splice variants. In this article, I will survey how our knowledge of the basic principles of alternative splicing control evolved over the last 25 years. I will also describe how investigation of the splicing control of an apoptotic regulator led us to identify novel effectors and revealed the existence of converging pathways linking splicing decisions to DNA damage. Finally, I will review how our efforts at developing tools designed to monitor and redirect splicing helped assess the impact of misregulated splicing in cancer. PMID:25759250

  14. 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

  15. 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

  16. 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

  17. Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis

    PubMed Central

    Cheng, Albert W.; Shi, Jiahai; Wong, Piu; Luo, Katherine L.; Trepman, Paula; Wang, Eric T.; Choi, Heejo; Lodish, Harvey F.

    2014-01-01

    The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation. PMID:24869935

  18. 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

  19. Transcriptome-wide targets of alternative splicing by RBM4 and possible role in cancer.

    PubMed

    Markus, M Andrea; Yang, Yee Hwa J; Morris, Brian J

    2016-04-01

    This study determined transcriptome-wide targets of the splicing factor RBM4 using Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays and HeLa cells treated with RBM4-specific siRNA. This revealed 238 transcripts that were targeted for alternative splicing. Cross-linking and immunoprecipitation experiments identified 945 RBM4 targets in mouse HEK293 cells, 39% of which were ascribed to "alternative splicing" by in silico pathway analysis. Mouse embryonic stem cells transfected with Rbm4 siRNA hairpins exhibited reduced colony numbers and size consistent with involvement of RBM4 in cell proliferation. RBM4 cDNA probing of a cancer cDNA array involving 18 different tumor types from 13 different tissues and matching normal tissue found overexpression of RBM4 mRNA (p<0.01) in cervical, breast, lung, colon, ovarian and rectal cancers. Many RBM4 targets we identified have been implicated in these cancers. In conclusion, our findings reveal transcriptome-wide targets of RBM4 and point to potential cancer-related targets and mechanisms that may involve RBM4. PMID:26898347

  20. 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

  1. 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

  2. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  3. 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

  4. Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

    PubMed Central

    Hong, Yoonki; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

    2016-01-01

    Background Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required. PMID:27066085

  5. 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

  6. Innovations in Proteomic Profiling of Cancers: Alternative Splice Variants as a New Class of Cancer Biomarker Candidates and Bridging of Proteomics with Structural Biology

    PubMed Central

    Omenn, Gilbert S.; Menon, Rajasree; Zhang, Yang

    2013-01-01

    Alternative splicing allows a single gene to generate multiple RNA transcripts which can be translated into functionally diverse protein isoforms. Current knowledge of splicing is derived mainly from RNA transcripts, with very little known about the expression level, 3D structures, and functional differences of the proteins. Splicing is a remarkable phenomenon of molecular and biological evolution. Studies which simply report up-regulation or down-regulation of protein or mRNA expression are confounded by the effects of mixtures of these isoforms. Besides understanding the net biological effects of the mixtures, we may be able to develop biomarker tests based on the observable differential expression of particular splice variants or combinations of splice variants in specific disease states. Here we review our work on differential expression of splice variant proteins in cancers and the feasibility of integrating proteomic analysis with structure-based conformational predictions of the differences between such isoforms. PMID:23603631

  7. 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

  8. Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.

    PubMed

    Calabretta, S; Bielli, P; Passacantilli, I; Pilozzi, E; Fendrich, V; Capurso, G; Fave, G Delle; Sette, C

    2016-04-21

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy. PMID:26234680

  9. 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

  10. 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

  11. Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

    PubMed

    Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

    2016-06-01

    The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates. PMID:26792793

  12. 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.

  13. 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

  14. PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing

    PubMed Central

    Taube, Jennifer R.; Sperle, Karen; Banser, Linda; Seeman, Pavel; Cavan, Barbra Charina V.; Garbern, James Y.; Hobson, Grace M.

    2014-01-01

    Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5′ splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD. PMID:24890387

  15. 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

  16. 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

  17. 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

  18. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    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

  19. Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

    PubMed Central

    Scholthof, Karen-Beth G.

    2015-01-01

    In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes. PMID:25634987

  20. ABERRANT SPLICING OF A BRAIN-ENRICHED ALTERNATIVE EXON ELIMINATES TUMOR SUPPRESSOR FUNCTION AND PROMOTES ONCOGENE FUNCTION DURING BRAIN TUMORIGENESIS

    PubMed Central

    Bredel, Markus; Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; Elverfeldt, Dominik v.; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.

    2014-01-01

    BACKGROUND: Tissue-specific alternative splicing is known to be critical to emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionary-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence activity in signaling pathways to profound biological effect. Given that tissue-specific splicing has a determinative role in brain development and the enrichment of genes containing tissue-specific exons for proteins with roles in signaling and development, it is thus plausible that changes in such exons could rewire normal neurogenesis towards malignant transformation. METHODS: We used integrated molecular genetic and cell biology analyses, computational biology, animal modeling, and clinical patient profiles to characterize the effect of aberrant splicing of a brain-enriched alternative exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7) on oncogene regulation and brain tumorigenesis. RESULTS: We show that aberrant splicing of a tissue-specific cassette exon in ANXA7 diminishes endosomal targeting and consequent termination of the signal of the EGFR oncoprotein during brain tumorigenesis. Splicing of this exon is mediated by the ribonucleoprotein Polypyrimidine Tract-Binding Protein 1 (PTBP1), which is normally repressed during brain development but, we find, is excessively expressed in glioblastomas through either gene amplification or loss of a neuron-specific microRNA, miR-124. Silencing of PTBP1 attenuates both malignancy and angiogenesis in a stem cell-derived glioblastoma animal model characterized by a high native propensity to generate tumor endothelium or vascular pericytes to support tumor growth. We show that EGFR amplification and PTBP1 overexpression portend a similarly poor clinical outcome, further highlighting the importance of PTBP1-mediated activation of EGFR. CONCLUSIONS: Our data illustrate how anomalous splicing of a tissue-regulated exon in a constituent of an oncogenic signaling pathway eliminates its tumor suppressor function and promotes tumorigenesis. This paradigm of malignant glial transformation as a consequence of tissue-specific alternative exon splicing in a tumor suppressor, may have widespread applicability in explaining how changes in critical tissue-specific regulatory mechanisms reprogram normal development to oncogenesis. SECONDARY CATEGORY: n/a.

  1. Evidence for differential alternative splicing in blood of young boys with autism spectrum disorders

    PubMed Central

    2013-01-01

    Background Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. Methods RNA from blood was processed on whole genome exon arrays for 2-4–year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). Results A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT). Conclusions These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods. PMID:24007566

  2. MiasDB: A Database of Molecular Interactions Associated with Alternative Splicing of Human Pre-mRNAs

    PubMed Central

    Xing, Yongqiang; Zhao, Xiujuan; Yu, Tao; Liang, Dong; Li, Jun; Wei, Guanyun; Liu, Guoqing; Cui, Xiangjun; Zhao, Hongyu; Cai, Lu

    2016-01-01

    Alternative splicing (AS) is pervasive in human multi-exon genes and is a major contributor to expansion of the transcriptome and proteome diversity. The accurate recognition of alternative splice sites is regulated by information contained in networks of protein-protein and protein-RNA interactions. However, the mechanisms leading to splice site selection are not fully understood. Although numerous databases have been built to describe AS, molecular interaction databases associated with AS have only recently emerged. In this study, we present a new database, MiasDB, that provides a description of molecular interactions associated with human AS events. This database covers 938 interactions between human splicing factors, RNA elements, transcription factors, kinases and modified histones for 173 human AS events. Every entry includes the interaction partners, interaction type, experimental methods, AS type, tissue specificity or disease-relevant information, a simple description of the functionally tested interaction in the AS event and references. The database can be queried easily using a web server (http://47.88.84.236/Miasdb). We display some interaction figures for several genes. With this database, users can view the regulation network describing AS events for 12 given genes. PMID:27167218

  3. New insights into the genomic organization and splicing of the doublesex gene, a terminal regulator of sexual differentiation in the silkworm Bombyx mori.

    PubMed

    Duan, Jianping; Xu, Hanfu; Guo, Huizhen; O'Brochta, David A; Wang, Feng; Ma, Sanyuan; Zhang, Liying; Zha, Xingfu; Zhao, Ping; Xia, Qingyou

    2013-01-01

    Sex-determination mechanisms differ among organisms. The primary mechanism is diverse, whereas the terminal regulator is relatively-conserved. We analyzed the transcripts of the Bombyx mori doublesex gene (Bmdsx), and reported novel results concerning the genomic organization and expression of Bmdsx. Bmdsx consists of nine exons and eight introns, of which two exons are novel and have not been reported previously. Bmdsx transcripts are spliced to generate seventeen alternatively-spliced forms and eleven putative trans-spliced variants. Thirteen of the alternatively-spliced forms and five of the putative trans-spliced forms are reported here for the first time. Sequence analysis predicts that ten female-specific, six male-specific splice forms and one splice form found in males and females will result in four female-specific, two male-specific Dsx proteins and one Dsx protein common to males and females. The Dsx proteins are expected to be functional and regulate downstream target genes. Some of the predicted Dsx proteins are described here for the first time. Therefore the expression of the dsx gene in B. mori results in a variety of cis- and trans-spliced transcripts and multiple Dsx proteins. These findings show that in B. mori there is a complicated pattern of dsx splicing, and that the regulation of splicing and sex-specific functions of lepidopteran dsx have evolved complexity. PMID:24244545

  4. Identification of recurrent regulated alternative splicing events across human solid tumors

    PubMed Central

    Danan-Gotthold, Miri; Golan-Gerstl, Regina; Eisenberg, Eli; Meir, Keren; Karni, Rotem; Levanon, Erez Y.

    2015-01-01

    Cancer is a complex disease that involves aberrant gene expression regulation. Discriminating the modified expression patterns driving tumor biology from the many that have no or little contribution is important for understanding cancer molecular basis. Recurrent deregulation patterns observed in multiple cancer types are enriched for such driver events. Here, we studied splicing alterations in hundreds of matched tumor and normal RNA-seq samples of eight solid cancer types. We found hundreds of cassette exons for which splicing was altered in multiple cancer types and identified a set of highly frequent altered splicing events. Specific splicing regulators, including RBFOX2, MBNL1/2 and QKI, appear to account for many splicing alteration events in multiple cancer types. Together, our results provide a first global analysis of regulated splicing alterations in cancer and identify common events with a potential causative role in solid tumor development. PMID:25908786

  5. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer

    PubMed Central

    Inoue, Kazushi; Fry, Elizabeth A.

    2015-01-01

    Breast cancer (BC) is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor) and human epidermal growth factor receptor 2 (HER2), play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER, HER2, and CD44 genes from the viewpoint of BC development. ERα36, a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2) has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms (CD44s, CD44v) play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci will provide keys to understand the pathogenesis, predict the prognosis, and choose specific therapies for BC. PMID:26692764

  6. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer.

    PubMed

    Inoue, Kazushi; Fry, Elizabeth A

    2015-01-01

    Breast cancer (BC) is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor) and human epidermal growth factor receptor 2 (HER2), play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER, HER2, and CD44 genes from the viewpoint of BC development. ERα36, a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2) has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms (CD44s, CD44v) play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci will provide keys to understand the pathogenesis, predict the prognosis, and choose specific therapies for BC. PMID:26692764

  7. RBM24 is a major regulator of muscle-specific alternative splicing.

    PubMed

    Yang, Jiwen; Hung, Lee-Hsueh; Licht, Thomas; Kostin, Sawa; Looso, Mario; Khrameeva, Ekaterina; Bindereif, Albrecht; Schneider, Andre; Braun, Thomas

    2014-10-13

    Cell-type-specific splicing generates numerous alternatively spliced transcripts playing important roles for organ development and homeostasis, but only a few tissue-specific splicing factors have been identified. We found that RBM24 governs a large number of muscle-specific splicing events that are critically involved in cardiac and skeletal muscle development and disease. Targeted inactivation of RBM24 in mice disrupted cardiac development and impaired sarcomerogenesis in striated muscles. In vitro splicing assays revealed that recombinant RBM24 is sufficient to promote muscle-specific exon inclusion in nuclear extracts of nonmuscle cells. Furthermore, we demonstrate that binding of RBM24 to an intronic splicing enhancer (ISE) is essential and sufficient to overcome repression of exon inclusion by an exonic splicing silencer (ESS) containing PTB and hnRNP A1/A2 binding sites. Introduction of ESS and ISE converted a constitutive exon into an RMB24-dependent alternative exon. We reason that RBM24 is a major regulator of alternative splicing in striated muscles. PMID:25313962

  8. Co-transcriptional regulation of alternative pre-mRNA splicing

    PubMed Central

    Shukla, Sanjeev; Oberdoerffer, Shalini

    2012-01-01

    While studies of alternative pre-mRNA splicing regulation have typically focused on RNA-binding proteins and their target sequences within nascent message, it is becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II) elongation and chromatin structure are intricately intertwined. The majority of introns in higher eukaryotes are excised prior to transcript release in a manner that is dependent on transcription through pol II. As a result of co-transcriptional splicing, variations in pol II elongation influence alternative splicing patterns, wherein a slower elongation rate is associated with increased inclusion of alternative exons within mature mRNA. Physiological barriers to pol II elongation, such as repressive chromatin structure, can thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can reciprocally influence pol II elongation and chromatin structure. Here, we highlight recent advances in co-transcriptional splicing that reveal an extensive network of coupling between splicing, transcription and chromatin remodeling complexes. PMID:22326677

  9. Integrative visual analysis of the effects of alternative splicing on protein domain interaction networks.

    PubMed

    Emig, Dorothea; Cline, Melissa S; Klein, Karsten; Kunert, Anne; Mutzel, Petra; Lengauer, Thomas; Albrecht, Mario

    2008-01-01

    Proteins and their interactions are essential for the functioning of all organisms and for understanding biological processes. Alternative splicing is an important molecular mechanism for increasing the protein diversity in eukaryotic cells. Splicing events that alter the protein structure and the domain composition can be responsible for the regulation of protein interactions and the functional diversity of different tissues. Discovering the occurrence of splicing events and studying protein isoforms have become feasible using Affymetrix Exon Arrays. Therefore, we have developed the versatile Cytoscape plugin DomainGraph that allows for the visual analysis of protein domain interaction networks and their integration with exon expression data. Protein domains affected by alternative splicing are highlighted and splicing patterns can be compared. PMID:20134061

  10. RNA sequencing reveals the role of splicing polymorphisms in regulating human gene expression

    PubMed Central

    Lalonde, Emilie; Ha, Kevin C.H.; Wang, Zibo; Bemmo, Amandine; Kleinman, Claudia L.; Kwan, Tony; Pastinen, Tomi; Majewski, Jacek

    2011-01-01

    Expression levels of many human genes are under the genetic control of expression quantitative trait loci (eQTLs). Despite technological advances, the precise molecular mechanisms underlying most eQTLs remain elusive. Here, we use deep mRNA sequencing of two CEU individuals to investigate those mechanisms, with particular focus on the role of splicing control loci (sQTLs). We identify a large number of genes that are differentially spliced between the two samples and associate many of those differences with nearby single nucleotide polymorphisms (SNPs). Subsequently, we investigate the potential effect of splicing SNPs on eQTL control in general. We find a significant enrichment of alternative splicing (AS) events within a set of highly confident eQTL targets discovered in previous studies, suggesting a role of AS in regulating overall gene expression levels. Next, we demonstrate high correlation between the levels of mature (exonic) and unprocessed (intronic) RNA, implying that ∼75% of eQTL target variance can be explained by control at the level of transcription, but that the remaining 25% may be regulated co- or post-transcriptionally. We focus on eQTL targets with discordant mRNA and pre-mRNA expression patterns and use four examples: USMG5, MMAB, MRPL43, and OAS1, to dissect the exact downstream effects of the associated genetic variants. PMID:21173033

  11. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    SciTech Connect

    Chiu Yali; Ouyang Pin . E-mail: ouyang@mail.cgu.edu.tw

    2006-03-10

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.

  12. MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma.

    PubMed

    Zhang, Shile; Wei, Jun S; Li, Samuel Q; Badgett, Tom C; Song, Young K; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C; Maris, John M; Guidry Auvil, Jamie M; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

    2016-02-28

    The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. PMID:26683771

  13. Identification of differential splicing genes in gliomas using exon expression profiling.

    PubMed

    Yu, Feng; Fu, Wei-Ming

    2015-02-01

    Diffuse gliomas are the most common type of malignant primary brain tumor, and their initiation and/or progression are often associated with alternative splicing. They produce an enormous economic burden on society and greatly impair the quality of life of those affected. The aim of the current study was to explore the differentially expressed genes (DEGs) observed in glioblastoma (GBM) and oligodendroglioma (OD) at the splicing level, and to analyze their functions in order to identify the underlying molecular mechanisms of gliomas. The exon‑level expression profile data GSE9385 was downloaded from the Gene Expression Omnibus database, and included 26 GBM samples, 22 OD samples and 6 control brain samples. The differentially expressed exon‑level probes were analyzed using the microarray detection of alternative splicing algorithm combined with the splicing index method, and the corresponding DEGs were identified. Next, a Gene Ontology enrichment analysis of the DEGs was performed. Additionally, the protein‑protein interaction (PPI) networks were constructed based on the depth‑first search algorithm. A total of 300 DEGs were identified to be shared