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1

Evolution of alternative splicing after gene duplication  

PubMed Central

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.

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

2006-01-01

2

Gene and alternative splicing annotation with AIR  

PubMed Central

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.

Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

2005-01-01

3

Gene and alternative splicing annotation with AIR.  

PubMed

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

2005-01-01

4

Prediction of Alternative Splice Sites in Human Genes  

Microsoft Academic Search

This thesis addresses the problem of predicting alternative splice sites in human genes. The most common way to identify alternative splice sites are the use of expressed sequence tags and microarray data. Since genes only produce alternative proteins under certain conditions, these methods are limited to detecting only alternative splice sites in genes whose alternative protein forms are expressed under

Douglas Simmons

2007-01-01

5

Integrating alternative splicing detection into gene prediction  

PubMed Central

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.

Foissac, Sylvain; Schiex, Thomas

2005-01-01

6

Gene Array Analyzer: alternative usage of gene arrays to study alternative splicing events  

PubMed Central

Exon arrays are regularly used to analyze differential splicing events. GeneChip Gene 1.0 ST Arrays (gene arrays) manufactured by Affymetrix, Inc. are primarily used to determine expression levels of transcripts, although their basic design is rather similar to GeneChip Exon 1.0 ST Arrays (exon arrays). Here, we show that the newly developed Gene Array Analyzer (GAA), which evolved from our previously published Exon Array Analyzer (EAA), enables economic and user-friendly analysis of alternative splicing events using gene arrays. To demonstrate the applicability of GAA, we profiled alternative splicing events during embryonic heart development. In addition, we found that numerous developmental splicing events are also activated under pathological conditions. We reason that the usage of GAA considerably expands the analysis of gene expression based on gene arrays and supplies an additional level of information without further costs and with only little effort.

Jenniches, Katharina; De Gaspari, Piera; John, David; grosse Kreymborg, Karsten; Braun, Thomas; Uchida, Shizuka

2012-01-01

7

The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing  

Microsoft Academic Search

RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding

Long Ma; Xiaoyang Gao; Jintao Luo; Liange Huang; Yanling Teng; H. Robert Horvitz

2012-01-01

8

Alternative splicing in ascomycetes.  

PubMed

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation. PMID:23515838

Kempken, Frank

2013-03-21

9

Identification of three mouse ?-opioid receptor (MOR) gene ( Oprm1) splice variants containing a newly identified alternatively spliced exon  

Microsoft Academic Search

The mouse ?-opioid receptor gene, Oprm1, is recognized currently to contain 17 alternatively spliced exons that generate 24 splice variants encoding at least 11 morphine-binding isoforms of the receptor. Here, we identify three new MOR splice variants that contain a previously undescribed exon, exon 18, and provide evidence that they are expressed in two mouse strains. The transcripts containing the

Glenn A. Doyle; X. Rebecca Sheng; Sharon S. J. Lin; Dorothy E. Grice; Russell J. Buono; Thomas N. Ferraro; Wade H. Berrettini

2007-01-01

10

The dystrophin gene is alternatively spliced throughout its coding sequence.  

PubMed

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability. PMID:12062429

Sironi, M; Cagliani, R; Pozzoli, U; Bardoni, A; Comi, G P; Giorda, R; Bresolin, N

2002-04-24

11

EuSplice: a unified resource for the analysis of splice signals and alternative splicing in eukaryotic genes  

Microsoft Academic Search

Motivation: Despite increased availability of genome annotation data, a comprehensive resource for in-depth analysis of splice signal distributions and alternative splicing (AS) patterns in eukaryote genomes is still lacking. To meet this need, we have developed EuSplice—a unique splice-centric database which provides reliable splice signal and AS information for 23 eukaryotes. Results: The EuSplice database contains 95822 AS events and

Ashwini Bhasi; Ram Vinay Pandey; Suriya Prabha Utharasamy; Periannan Senapathy

2007-01-01

12

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

SciTech Connect

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.

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

2008-11-07

13

Identification of five mouse ?-opioid receptor (MOR) gene ( Oprm1) splice variants containing a newly identified alternatively spliced exon  

Microsoft Academic Search

The mouse ?-opioid receptor gene, Oprm1, currently contains 18 recognized alternatively spliced exons [Doyle, G.A., Sheng, X.R., Lin, S.S.J., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007. Identification of three mouse ?-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388 (1–2) 135–147, in press (doi:10.1016\\/j.gene.2006.10.017). Electronic publication 2006 November 1

Glenn A. Doyle; X. Rebecca Sheng; Sharon S. J. Lin; Dorothy E. Grice; Russell J. Buono; Thomas N. Ferraro; Wade H. Berrettini

2007-01-01

14

Discovery of Gene Families and Alternatively Spliced Variants by RecA-Mediated Cloning  

Microsoft Academic Search

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene

Hong Zeng; Elizabeth Allen; Chris W. Lehman; R. Geoffrey Sargent; Sushma Pati; David A. Zarling

2002-01-01

15

Alternative splice variants of the human PD1 gene  

Microsoft Academic Search

PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1?ex2, PD-1?ex3, PD-1?ex2,3, and PD-1?ex2,3,4) in addition to the full length isoform. PD-1?ex2 and PD-1?ex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out.

Christian Nielsen; Line Ohm-Laursen; Torben Barington; Steffen Husby; Søren T. Lillevang

2005-01-01

16

Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript  

PubMed Central

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Standiford, D. M.; Davis, M. B.; Sun, W.; Emerson-Jr., C. P.

1997-01-01

17

Copy Number Variations in Alternative Splicing Gene Networks Impact Lifespan  

PubMed Central

Longevity has a strong genetic component evidenced by family-based studies. Lipoprotein metabolism, FOXO proteins, and insulin/IGF-1 signaling pathways in model systems have shown polygenic variations predisposing to shorter lifespan. To test the hypothesis that rare variants could influence lifespan, we compared the rates of CNVs in healthy children (0–18 years of age) with individuals 67 years or older. CNVs at a significantly higher frequency in the pediatric cohort were considered risk variants impacting lifespan, while those enriched in the geriatric cohort were considered longevity protective variants. We performed a whole-genome CNV analysis on 7,313 children and 2,701 adults of European ancestry genotyped with 302,108 SNP probes. Positive findings were evaluated in an independent cohort of 2,079 pediatric and 4,692 geriatric subjects. We detected 8 deletions and 10 duplications that were enriched in the pediatric group (P?=?3.33×10?8–1.6×10?2 unadjusted), while only one duplication was enriched in the geriatric cohort (P?=?6.3×10?4). Population stratification correction resulted in 5 deletions and 3 duplications remaining significant (P?=?5.16×10?5–4.26×10?2) in the replication cohort. Three deletions and four duplications were significant combined (combined P?=?3.7×10?4?3.9×10?2). All associated loci were experimentally validated using qPCR. Evaluation of these genes for pathway enrichment demonstrated ?50% are involved in alternative splicing (P?=?0.0077 Benjamini and Hochberg corrected). We conclude that genetic variations disrupting RNA splicing could have long-term biological effects impacting lifespan.

Glessner, Joseph T.; Smith, Albert Vernon; Panossian, Saarene; Kim, Cecilia E.; Takahashi, Nagahide; Thomas, Kelly A.; Wang, Fengxiang; Seidler, Kallyn; Harris, Tamara B.; Launer, Lenore J.; Keating, Brendan; Connolly, John; Sleiman, Patrick M. A.; Buxbaum, Joseph D.; Grant, Struan F. A.; Gudnason, Vilmundur; Hakonarson, Hakon

2013-01-01

18

Alternative splicing of the FMR1 gene in human fetal brain neurons  

SciTech Connect

The alternative splicing expression of the FMR1 gene was reported in several human and mouse tissues. Five regions of FMR1 gene can be alternatively spliced, but the combination of them has not been investigated fully. We reported here the analysis of alternative splicing pattern of the FMR1 gene in cultured fetal human neurons, using a RT-PCR and cloning strategy. Eleven splicing types were cloned and different isoforms were not equally represented. The dominant isoform represents nearly 40%, and the other isoforms were relatively rare. One isoform has a different carboxyl-terminus. Most of the alternative spliced regions appear hydrophilic; thus, they may locate on the surface of the FMR1 protein. 16 refs., 2 figs.

Tao Huang; Yan Shen; Xue-bin Qin; Guan-Yun Wu [Chinese Academy of Medical Sciences, Beijing (China)] [and others

1996-08-09

19

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency  

Microsoft Academic Search

Alternative splicing of the Drosophila melanogaster Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDLac and RDLbd (DRC17-1-2), differ in their apparent agonist affinity. Here, we report the cloning of a third splice variant of Rdl, RDLad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following

A. M Hosie; S. D Buckingham; J. K Presnail; D. B Sattelle

2001-01-01

20

Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes  

PubMed Central

Background Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla [2] indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism. Reviewers This article was reviewed by Dr. Eugene Koonin, Dr. Chungoo Park (nominated by Dr. Kateryna Makova), and Dr. Marcelo Yanovsky (nominated by Dr. Valerian Dolja).

2012-01-01

21

Regulation of Alternative Splicing by Histone Modifications  

Microsoft Academic Search

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

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

2010-01-01

22

Understanding alternative splicing: towards a cellular code  

Microsoft Academic Search

In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms — thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global

Arianne J. Matlin; Francis Clark; Christopher W. J. Smith

2005-01-01

23

Calculation of Splicing Potential from the Alternative Splicing Mutation Database  

Microsoft Academic Search

BACKGROUND: The Alternative Splicing Mutation Database (ASMD) presents a collection of all known mutations inside human exons which affect splicing enhancers and silencers and cause changes in the alternative splicing pattern of the corresponding genes. FINDINGS: An algorithm was developed to derive a Splicing Potential (SP) table from the ASMD information. This table characterizes the influence of each oligonucleotide on

Jason M Bechtel; Preeti Rajesh; Irina Ilikchyan; Ying Deng; Pankaj K Mishra; Qi Wang; Xiaochun Wu; Kirill A Afonin; William E Grose; Ye Wang; Sadik Khuder; Alexei Fedorov

2008-01-01

24

Alternative Splicing of the Amelogenin Gene in a Caudate Amphibian, Plethodoncinereus  

PubMed Central

As the major enamel matrix protein contributing to tooth development, amelogenin has been demonstrated to play a crucial role in tooth enamel formation. Previous studies have revealed amelogenin alternative splicing as a mechanism for amelogenin heterogeneous expression in mammals. While amelogenin and its splicing forms in mammalian vertebrates have been characterized, splicing variants of amelogenin gene still remains largely unknown in non-mammalian species. Here, using PCR and sequence analysis we discovered two novel amelogenin transcript variants in tooth organ extracts from a caudate amphibian, the salamander Plethodoncinereus. The one was shorter -S- (416 nucleotides including untranslated regions, 5 exons) and the other larger -L- (851 nt, 7 exons) than the previously published “normal” gene in this species -M- (812 nucleotides, 6 exons). This is the first report demonstrating the amelogenin alternative splicing in amphibian, revealing a unique exon 2b and two novel amelogenin gene transcripts in Plethodoncinereus.

Wang, Xinping; Xing, Zeli; Zhang, Xichen; Zhu, Lisai; Diekwisch, Thomas G. H.

2013-01-01

25

Alternative splicing and muscular dystrophy  

PubMed Central

Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle.

Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

2013-01-01

26

Alternative splicing and gene duplication in the evolution of the FoxP gene subfamily.  

PubMed

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1-P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system. PMID:20651048

Santos, M Emília; Athanasiadis, Alekos; Leitão, Alexandre B; DuPasquier, Louis; Sucena, Elio

2010-07-22

27

Alternative Splicing and Gene Duplication in the Evolution of the FoxP Gene Subfamily  

PubMed Central

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1–P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system.

Santos, M. Emilia; Athanasiadis, Alekos; Leitao, Alexandre B.; DuPasquier, Louis; Sucena, Elio

2011-01-01

28

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

SciTech Connect

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.

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

2009-02-03

29

Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F.  

PubMed

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5' splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes. PMID:23284676

Wang, Erming; Aslanzadeh, Vahid; Papa, Filomena; Zhu, Haiyan; de la Grange, Pierre; Cambi, Franca

2012-12-17

30

CLONING, SEQUENCING, AND CHARACTERIZATION OF ALTERNATIVELY SPLICED GLUTAREDOXIN CDNA AND ITS GENOMIC GENE  

Technology Transfer Automated Retrieval System (TEKTRAN)

Alternatively spliced human glutaredoxin (GRXas) cDNA was isolated from a neutrophil cDNA library using the 32P-labeled glutaredoxin cDNA probe in non-stringent conditions. The sequence of this GRXas cDNA indicates that the open reading frame (ORF) of the gene is identical to the ORF of the previou...

31

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene  

PubMed Central

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3? splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis-acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3? splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.

Laverdiere, M.; Beaudoin, J.; Lavigueur, Alain

2000-01-01

32

Structure and Expression of Phosphoenolpyruvate Carboxylase Kinase Genes in Solanaceae. A Novel Gene Exhibits Alternative Splicing1  

PubMed Central

Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism in higher plants. Two PPCK (PEPc kinase) genes have been identified in tomato (Lycopersicon esculentum cv Alicante), hereafter termed LePPCK1 and LePPCK2. The function of the gene products has been confirmed by transcription of full-length cDNAs, translation, and in vitro assay of kinase activity. Previously studied PPCK genes contain a single intron. LePPCK2 also contains a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts depends on tissue and conditions. Expression of LePPCK2 was markedly increased during fruit ripening. In ripe Alicante fruit, the locule and seeds contained only the correctly spliced LePPCK2 transcripts, whereas in ripe fruit of the tomato greenflesh mutant, they contained correctly and incorrectly spliced transcripts. Potato (Solanum tuberosum) contains genes that are very similar to LePPCK1, and LePPCK2; StPPCK2 exhibits alternative splicing. Aubergine (Solanum melongena) and tobacco (Nicotiana tabacum) also contain a PPCK2 gene; the sequence of the alternatively spliced intron is highly conserved between all four species. The data suggest that the two PPCK genes have different roles in tissue-specific regulation of PEPc and that the alternative splicing of PPCK2 transcripts is functionally significant.

Marsh, Justin T.; Sullivan, Stuart; Hartwell, James; Nimmo, Hugh G.

2003-01-01

33

Regulation of Alternative Splicing by Histone Modifications  

PubMed Central

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.

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

2010-01-01

34

Regulation of alternative splicing by histone modifications.  

PubMed

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

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

2010-02-04

35

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency.  

PubMed

Alternative splicing of the Drosophila melanogaster Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDL(ac) and RDL(bd) (DRC17-1-2), differ in their apparent agonist affinity. Here, we report the cloning of a third splice variant of Rdl, RDL(ad). Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The EC(so) values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDL(ad) homomeric receptor differed from those previously described for RDL(ac) and DRC17-1-2 receptors. In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors. Thus, using expression in Xenopus oocytes, we have demonstrated differences in agonist potency for the neurotransmitter GABA (and four analogues) between splice variant products of the Drosophila melanogaster Rdl gene encoding homomer-forming GABA receptor subunits. PMID:11226707

Hosie, A M; Buckingham, S D; Presnail, J K; Sattelle, D B

2001-01-01

36

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

PubMed Central

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.

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

2013-01-01

37

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

PubMed

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

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

2013-05-01

38

The Application of Alternative Splicing Graphs in Quantitative Analysis of Alternative Splicing Form from EST Database  

Microsoft Academic Search

Alternative splicing of a single pre-mRNA can give rise to different mRNA transcripts. Alternative splicing of pre-messenger RNA is an important layer of gene expression regulation in eukaryotic cell. Consequently, alternative splicing is an important mechanism for generating protein diversity from a single gene. Although alternative splicing is an important biological process, standard molecular biology techniques have only identified several

Hsun-chang Chang; Po-shun Yu; Tze-wei Huang; Yaw-ling Lin; Fang-rong Hsu

2004-01-01

39

Alternative splicing in bone following mechanical loading  

Microsoft Academic Search

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

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

2011-01-01

40

Aberrant Alternative Splicing Is Another Hallmark of Cancer  

PubMed Central

The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

Ladomery, Michael

2013-01-01

41

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

PubMed Central

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

Foissac, Sylvain; Sammeth, Michael

2007-01-01

42

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

PubMed Central

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

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

2011-01-01

43

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression  

PubMed Central

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 21 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3? splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element’s identity and changes in cellular splicing factors under physiological or pathological conditions.

Zheng, Zhi-Ming

2008-01-01

44

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product  

Microsoft Academic Search

BACKGROUND: GFG\\/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species

Shuo Cheng Zhang; Kimberley A MacDonald; Mark Baguma-Nibasheka; Laurette Geldenhuys; Alan G Casson; Paul R Murphy

2008-01-01

45

Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing.  

PubMed

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents. PMID:21344922

Fan, Liying; Lagisetti, Chandraiah; Edwards, Carol C; Webb, Thomas R; Potter, Philip M

2011-03-07

46

Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing  

PubMed Central

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant anti-tumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, and allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity towards human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumor, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Fan, Liying; Lagisetti, Chandraiah; Edwards, Carol C.; Webb, Thomas R.; Potter, Philip M.

2011-01-01

47

Alternative transcription and splicing of the human porphobilinogen deaminase gene  

SciTech Connect

Porphobilinogen deaminase is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. The authors have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. They demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the ..beta..-globin gene promoters.

Chretien, S.; Dubart, A.; Beaupain, D.; Raich, N.; Grandchamp, B.; Rosa, J.; Goossens, M.; Romeo, P.H.

1988-01-01

48

Characterization, phylogeny, alternative splicing and expression of Sox30 gene  

Microsoft Academic Search

BACKGROUND: Members of the Sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. Among these members, Sox30 had been considered to exist only in mammals since its discovery, and its exact function remains unclear. RESULTS: Sox30 cDNA was cloned from the Nile tilapia

Fei Han; Zhijian Wang; Fengrui Wu; Zhihao Liu; Baofeng Huang; Deshou Wang

2010-01-01

49

Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets  

Microsoft Academic Search

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62\\/352 (?18%) of

Sergio Barberan-Soler; Alan M. Zahler

2008-01-01

50

Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation*  

PubMed Central

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (?II, ?I, ?III, and ?I), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1? or S2? binding pocket and results in the complete loss of the “47 loop,” reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Jackson, Nicole E.; Wang, Hong-Wei; Bryant, Katherine J.; McNeil, H. Patrick; Husain, Ahsan; Liu, Ke; Tedla, Nicodemus; Thomas, Paul S.; King, Garry C.; Hettiaratchi, Anusha; Cairns, Jennifer; Hunt, John E.

2008-01-01

51

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation.  

PubMed

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates. PMID:18854315

Jackson, Nicole E; Wang, Hong-Wei; Bryant, Katherine J; McNeil, H Patrick; Husain, Ahsan; Liu, Ke; Tedla, Nicodemus; Thomas, Paul S; King, Garry C; Hettiaratchi, Anusha; Cairns, Jennifer; Hunt, John E

2008-10-14

52

Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3? Splice Site Choice in Vertebrate 4.1 Genes  

PubMed Central

Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3? splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ?120 kb upstream of E2 an ultradistal intraexon, iEB, that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iEB in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iEB function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iER-mediated intrasplicing at the first available exons downstream of iER, namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3? splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3? splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity.

Parra, Marilyn K.; Gallagher, Thomas L.; Amacher, Sharon L.; Mohandas, Narla

2012-01-01

53

Regulated functional alternative splicing in Drosophila  

PubMed Central

Alternative splicing expands the coding capacity of metazoan genes, and it was largely genetic studies in the fruit-fly Drosophila melanogaster that established the principle that regulated alternative splicing results in tissue- and stage-specific protein isoforms with different functions in development. Alternative splicing is particularly prominent in germ cells, muscle and the central nervous system where it modulates the expression of various proteins including cell-surface molecules and transcription factors. Studies in flies have given us numerous insights into alternative splicing in terms of upstream regulation, the exquisite diversity of their forms and the key differential cellular functions of alternatively spliced gene products. The current inundation of transcriptome sequencing data from Drosophila provides an unprecedented opportunity to gain a comprehensive view of alternative splicing.

Venables, Julian P.; Tazi, Jamal; Juge, Francois

2012-01-01

54

Selecting for Functional Alternative Splices in ESTs  

Microsoft Academic Search

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 computatio nal 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 ne arly all

Zhengyan Kan; David States; Warren Gish

2002-01-01

55

Stress-induced alternative gene splicing in mind-body medicine.  

PubMed

Recent research documents how psychosocial stress can alter the expression of the acetylcholinesterase gene to generate at least 3 alternative proteins that are implicated in a wide variety of normal mind-body functions, as well as pathologies. These range from early embryological development, plasticity of the brain in adulthood, post-traumatic stress disorder (PTSD), and stress-associated dysfunctions of the central nervous, endocrine, and immune systems, to age-related neuropathologies. Such stress-induced alternative gene splicing is proposed here as a major mind-body pathway of psychosocial genomics-the modulation of gene expression by creative psychological, social, and cultural processes. We explore the types of research that are now needed to investigate how stress-induced alternative splicing of the acetylcholinesterase gene may play a pivotal role in the deep psychobiology of psychotherapy, meditation, spiritual rituals, and the experiencing of positive humanistic values that have been associated with mind-body medicine, such as compassion, beneficence, serenity, forgiveness, and gratitude. PMID:15356952

Rossi, Ernest Lawrence

2004-01-01

56

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

PubMed

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

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

2012-05-29

57

Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer  

Microsoft Academic Search

Alternative processing of pre-mRNA transcripts is a major source of protein diversity in eukaryotes and has been implicated in several disease processes including cancer. In this study we have performed a genome wide analysis of alternative splicing events in lung adenocarcinoma. We found that 2369 of the 17800 core Refseq genes appear to have alternative transcripts that are differentially expressed

Liqiang Xi; Andrew Feber; Vanita Gupta; Maoxin Wu; Andrew D. Bergemann; Rodney J. Landreneau; Virginia R. Litle; Arjun Pennathur; James D. Luketich; Tony E. Godfrey

2008-01-01

58

Identification of alternative 50\\/30 splice sites based on the mechanism of splice site competition  

Microsoft Academic Search

Alternative splicing plays an important role in regu- lating gene expression. Currently, most efficient methods use expressed sequence tags or microar- ray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alterna- tive splice events with them because of their in- herent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds

Huiyu Xia; Jianning Bi; Yanda Li

2006-01-01

59

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene.  

PubMed Central

The cystatin superfamily of cysteine protease inhibitors consists of three major families, including the stefins, cystatins and kininogens. However, the recent identification of several genes that possess sequence similarity with the cystatins but have different gene or protein structures indicates that several new cystatin families or subgroups of families might exist. We previously identified the cystatin-related epididymal spermatogenic (Cres) gene, which is related to the family 2 cystatins but exhibits highly tissue-specific expression in the reproductive tract. In the studies presented here, an analysis of gene structure as well as chromosomal mapping studies suggest that the Cres gene might represent a new subgroup within the family 2 cystatins. Although the Cres gene possesses an additional exon encoding 5' untranslated sequences, its coding exons are similar in size to the three coding exons of the cystatin family 2 genes, and the Cres exon/intron splice junctions occur in identical locations as in the cystatin C gene. Furthermore, chromosomal mapping studies show that the Cres gene co-segregates with the cystatin C gene on mouse chromosome 2. Similar to the cystatin family 2 proteins, the Cres protein possesses the type A and B disulphide loops that are necessary for cystatin folding. Interestingly, Cres protein also possesses half of a type C disulphide loop. Although probably related to the cystatin genes, the Cres gene is distinct in that its promoter contains consensus motifs typical of regulated genes. Finally, reverse transcriptase-mediated PCR studies and the identification of new Cres cDNA clones indicate that the Cres mRNA is alternatively spliced, resulting in two Cres mRNAs that might be involved in the regulation of Cres function.

Cornwall, G A; Hsia, N; Sutton, H G

1999-01-01

60

Alternative Splicing in Bone Following Mechanical Loading  

PubMed Central

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/?-catenin and TGF-? 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.

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

2010-01-01

61

Alternative promoter usage and splicing of the human SCN5A gene contribute to transcript heterogeneity.  

PubMed

The sodium channel isoform Na(v)1.5 mediates sodium current, excitability, and electrical conduction in the human heart. Recent studies have indicated alternative splicing within the protein-coding portion of its gene, SCN5A, as a mechanism to generate diversity in Na(v)1.5 protein structure and function. In the present study we identified several novel SCN5A transcripts in human heart, displaying distinct 5?-untranslated regions but identical protein-coding sequences. These transcripts originated from the splicing of alternative exons 1 (designated 1A, 1B, 1C, and 1D) to the translational start codon-containing exon 2, and were preferentially expressed in the heart as compared to other tissues. Comparison of their expression level between adult and fetal heart demonstrated that exon 1C- and 1D-derived sequences were more prominent in adult than in fetal heart. Two new promoters (designated P2 and P3) for the SCN5A gene were identified and functionally characterized in myocardial- and nonmyocardial-derived cell lines. Translation of the transcript containing exon 1D-derived sequences proved to be significantly impaired in these cell lines, which could be restored by mutation of an upstream translational start codon. These results implicate the usage of alternative promoters and 5?-untranslated regions as new mechanisms in the regulation of human Na(v)1.5 expression. PMID:20618077

van Stuijvenberg, Leonie; Yildirim, Cansu; Kok, Bart G J M; van Veen, Toon A B; Varró, András; Winckels, Stephan K G; Vos, Marc A; Bierhuizen, Marti F A

2010-10-01

62

Gene expression analyses implicate an alternative splicing program in regulating contractile gene expression and serum response factor activity in mice.  

PubMed

Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELF? transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an ?-myosin heavy chain promoter. MHC-CELF? mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELF? mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELF? mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELF? males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor. PMID:23437181

Dasgupta, Twishasri; Stillwagon, Samantha J; Ladd, Andrea N

2013-02-20

63

Titin Diversity--Alternative Splicing Gone Wild  

PubMed Central

Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

Guo, Wei; Bharmal, Sheila J.; Esbona, Karla; Greaser, Marion L.

2010-01-01

64

Splicing Factor Tra2B1 Is Specifically Induced in Breast Cancer and Regulates Alternative Splicing of the CD44 Gene  

Microsoft Academic Search

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C\\/A-rich exon enhancer sequence required

Dirk O. Watermann; Yesheng Tang; Markus Jager; Stefan Stamm; Elmar Stickeler

2006-01-01

65

DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees  

PubMed Central

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non–protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

Foret, Sylvain; Kucharski, Robert; Pellegrini, Matteo; Feng, Suhua; Jacobsen, Steven E.; Robinson, Gene E.; Maleszka, Ryszard

2012-01-01

66

Genomic structure and alternative splicing of 519, a gene expressed late after T cell activation  

SciTech Connect

Relatively little is known about the transcriptional control of genes expressed late after T cell activation. The authors have identified four genes expressed 3 to 5 days after T cell activation by alloantigen or mitogen. Here they report the genomic organization of 519, one of these late T cell activation Ag. Analysis of the genomic clone revealed that 519 consists of six exons. Ribonuclease protection experiments indicated that the most abundant transcript arising from this region is an alternatively spliced form of 519, referred to as 520, which lacks exon 2 and is similar in sequence to NKG5, a cDNA identified in NK cells. THese experiments also revealed the existence of two other alternatively spliced RNA transcripts, with heterogeneity in exon 2. Primer extension analysis and ribonuclease protection assays demonstrated that there are two prominent start sites for transcription; however, there is no evidence for the NKG5 transcript in T cells, indicating that NKG5 may represent a NK cell-specific form of 520. The 5[prime] flanking region of this gene contains several previously identified sequences involved in transcriptional regulation, as well as some potentially interesting novel conserved motifs. 38 refs., 7 figs., 2 tabs.

Manning, W.C.; O'Farrell, S.; Goralski, T.J.; Krensky, A.M. (Stanford Univ. School of Medicine, CA (United States))

1992-06-15

67

ASmodeler: gene modeling of alternative splicing from genomic alignment of mRNA, EST and protein sequences.  

PubMed

Alternative splicing is in important mechanism of modulating gene function and expression which greatly expands transcriptome diversity. ASmodeler is a novel web-based utility that finds gene models including alternative splicing events from genomic alignment of mRNA, EST and protein sequences. User-supplied sequences are aligned against the genome map using the BLAT and SIM4 programs. Resulting exon connectivity is analyzed by applying graph-theoretic methods to build all possible gene models including splice variants. The algorithm essentially combines the genome-based sequence clustering and transcript assembly procedures in a coherent fashion. In addition to the user-supplied sequences, UniGene clusters and many well-known gene predictions such as Genscan, Ensembl and Acembly may be included in gene modeling. The current implementation supports human, mouse and rat genomes. ASmodeler is available at http://genome.ewha.ac.kr/ECgene/ASmodeler/. PMID:15215376

Kim, Namshin; Shin, Seokmin; Lee, Sanghyuk

2004-07-01

68

Molecular cloning, expression and subcellular distribution of an alternative splice variant of the porcine Sirt2 gene  

Microsoft Academic Search

Sirt2, a NAD+-dependent histone deacetylase, plays a critical role in regulating lifespan, metabolism, mitosis and adipocyte differentiation.\\u000a Here two bands of the porcine Sirt2 protein were found by western blotting, so we speculated existence of Sirt2 isoforms.\\u000a Next, we cloned the porcine Sirt2 gene, and also found its alternative splice variant and named the novel splicing variant\\u000a Sirt2T. The complete

Bingting Liu; Fei Liu; Liang Bai; Yucheng Li; Gongshe Yang

2010-01-01

69

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

PubMed Central

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

Xia, Huiyu; Bi, Jianning; Li, Yanda

2006-01-01

70

Mechano-Regulation of Alternative Splicing  

PubMed Central

Alternative splicing contributes to the complexity of proteome by producing multiple mRNAs from a single gene. Affymetrix exon arrays and experiments in vivo or in vitro demonstrated that alternative splicing was regulated by mechanical stress. Expression of mechano-growth factor (MGF) which is the splicing isoform of insulin-like growth factor 1(IGF-1) and vascular endothelial growth factor (VEGF) splicing variants such as VEGF121, VEGF165, VEGF206, VEGF189, VEGF165 and VEGF145 are regulated by mechanical stress. However, the mechanism of this process is not yet clear. Increasing evidences showed that the possible mechanism is related to Ca2+ signal pathway and phosphorylation signal pathway. This review proposes possible mechanisms of mechanical splicing regulation. This will contribute to the biomechanical study of alternative splicing.

Liu, Huan; Tang, Liling

2013-01-01

71

Splicing and alternative splicing in rice and humans.  

PubMed

Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. [BMB Reports 2013; 46(9): 439-447]. PMID:24064058

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-09-01

72

Alternative splicing of apoptosis-related genes in imatinib-treated K562 cells identified by exon array analysis  

PubMed Central

Imatinib is the therapeutic standard for newly diagnosed patients with chronic myeloid leukemia (CML). In these patients, imatinib has been shown to induce an apoptotic response specifically in cells expressing the oncogenic fusion protein BCR-ABL. Previous studies in our lab revealed that imatinib-induced apoptosis in K562 cells involves a shift in production of Bcl-x splice isoforms towards the pro-apoptotic Bcl-xS splice variant. Here, we report the findings from our subsequent study to identify other apoptosis-related genes that are differentially spliced in response to imatinib treatment. Gene expression profiling of imatinib-treated K562 cells was performed by the Affymetrix GeneChip® Human Exon 1.0 ST array, and differences in exon-level expression and alternative splicing were analyzed using the easyExon software. Detailed analysis by reverse transcription-PCR (RT-PCR) and sequencing of key genes confirmed the experimental results of the exon array. Our results suggest that imatinib treatment of K562 cells causes a transcriptional shift towards alternative splicing in a large number of apoptotic genes. The present study provides insight into the molecular character of apoptotic leukemia cells and may help to improve the mechanism of imatinib therapy in patients with CML.

LIU, JING; XIAO, YUN; XIONG, HUO-MEI; LI, JING; HUANG, BO; ZHANG, HAI-BIN; FENG, DAN-QIN; CHEN, XI-MIN; WANG, XIAO-ZHONG

2012-01-01

73

Depolarization-Mediated Regulation of Alternative Splicing  

PubMed Central

Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

Sharma, Alok; Lou, Hua

2011-01-01

74

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

PubMed Central

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. Images

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

1991-01-01

75

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

SciTech Connect

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.

Cybulsky, M.I.; Fries, J.W.U.; Williams, A.J.; Sultan, P.; Gimbrone, M.A. Jr.; Collins, T. (Harvard Medical School, Boston, MA (United States)); Eddy, R.; Byers, M.; Shows, T. (Roswell Park Memorial Inst., Buffalo, NY (United States))

1991-09-01

76

Alternative splicing and tissue expression of CIB4 gene in sheep testis.  

PubMed

In this study, the sheep CIB4 cDNA was cloned from the small tail Han sheep by RT-PCR and RACE (rapid amplification of cDNA ends), and CIB4 cDNA and amino acid sequence were analyzed. Our results showed that the sheep CIB4 gene expressed two alternatively spliced variants L-CIB4 (long CIB4) and S-CIB4 (short CIB4). Sequence analysis indicated that the sheep CIB4 cDNA cloned (L-CIB4) was 745-bp in length (GenBank accession number: FJ039532) with 185 amino acids residues. The sheep CIB4 cDNA showed more than 72% of sequence identity, at the nucleotide level, to its equivalents in cattle, horse, chimpanzee, humans, mice and rats, while at the deduced protein level, the value increased to 79.6%. Semi-quantitative RT-PCR using total RNA from different tissues showed that CIB4 has a strong tissue-specific expression pattern in sheep. L-CIB4 expression level was shown to be no different in small tail Han sheep and the Dorset ram, but both were significantly different from the Texel (P<0.05). Surprisingly, the short spliced form, S-CIB4, could only be detected in small tail Han sheep, suggesting that CIB4 may be linked in some way to the high fecundity of this breed. PMID:20236775

Yu, Yan; Zhang, Yuan; Song, Xuemei; Jin, Mei; Guan, Qun; Zhang, Qiyao; Li, Shangang; Wei, Caihong; Lu, Guobin; Zhang, Ju; Ren, Hangxing; Sheng, Xihui; Wang, Chuduan; Du, Lixin

2010-02-12

77

Alternative Splicing for Diseases, Cancers, Drugs, and Databases  

PubMed Central

Alternative splicing is a major diversification mechanism in the human transcriptome and proteome. Several diseases, including cancers, have been associated with dysregulation of alternative splicing. Thus, correcting alternative splicing may restore normal cell physiology in patients with these diseases. This paper summarizes several alternative splicing-related diseases, including cancers and their target genes. Since new cancer drugs often target spliceosomes, several clinical drugs and natural products or their synthesized derivatives were analyzed to determine their effects on alternative splicing. Other agents known to have modulating effects on alternative splicing during therapeutic treatment of cancer are also discussed. Several commonly used bioinformatics resources are also summarized.

Lee, Jin-Ching; Hou, Ming-Feng; Wang, Chun-Lin; Chen, Chien-Chi; Huang, Hurng-Wern

2013-01-01

78

Temperature-modulated Alternative Splicing and Promoter Use in the Circadian Clock Gene frequencyD?  

PubMed Central

The expression of FREQUENCY, a central component of the circadian clock in Neurospora crassa, shows daily cycles that are exquisitely sensitive to the environment. Two forms of FRQ that differ in length by 99 amino acids, LFRQ and SFRQ, are synthesized from alternative initiation codons and the change in their ratio as a function of temperature contributes to robust rhythmicity across a range of temperatures. We have found frq expression to be surprisingly complex, despite our earlier prediction of a simple transcription unit based on limited cDNA sequencing. Two distinct environmentally regulated major promoters drive primary transcripts whose environmentally influenced alternative splicing gives rise to six different major mRNA species as well as minor forms. Temperature-sensitive alternative splicing determines AUG choice and, as a consequence, the ratio of LFRQ to SFRQ. Four of the six upstream ORFs are spliced out of the vast majority of frq mRNA species. Alternative splice site choice in the 5? UTR and relative use of two major promoters are also influenced by temperature, and the two promoters are differentially regulated by light. Evolutionary comparisons with the Sordariaceae reveal conservation of 5? UTR sequences, as well as significant conservation of the alternative splicing events, supporting their relevance to proper regulation of clock function.

Colot, Hildur V.; Loros, Jennifer J.; Dunlap, Jay C.

2005-01-01

79

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

PubMed Central

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

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

2012-01-01

80

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

PubMed Central

SUMMARY Although uridine-rich small nuclear RNAs (U-snRNAs) are essential for pre-mRNA splicing, little is known regarding their function in the regulation of alternative splicing or of the biological consequences of their dysfunction in mammals. Here, we demonstrate that mutation of Rnu2–8, one of the mouse multicopy U2 snRNA genes, causes ataxia and neurodegeneration. Coincident with the observed pathology, the level of mutant U2 RNAs was highest in the cerebellum and increased after granule neuron maturation. Furthermore, neuron loss was strongly dependent on the dosage of mutant and wild type snRNA genes. Comprehensive transcriptome analysis identified a group of alternative splicing events, including the splicing of small introns, which were disrupted in the mutant cerebellum. Our results suggest that the expression of mammalian U2 snRNA genes, previously presumed to be ubiquitious, is spatially and temporally regulated, and dysfunction of a single U2 snRNA causes neuron degeneration through distortion of pre-mRNA splicing.

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

2012-01-01

81

Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing.  

PubMed

We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1-22 aa), a transmembrane domain at the C-terminus (302-319 aa), and an extracellular Ig-like region in the middle (161-252 aa) with a putative N-linked glycosylation site (N(205)-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm-egg fusion in mammals may be under the same principle in which Izumo1 plays an important role. PMID:20963501

Xing, Wan-Jin; Han, Bao-Da; Wu, Qi; Zhao, Li; Bao, Xiao-Hong; Bou, Shorgan

2010-10-21

82

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

PubMed Central

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.

Munk, Carsten; Beck, Thomas; Zielonka, Jorg; 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

83

Global analysis of alternative splicing differences between humans and chimpanzees  

Microsoft Academic Search

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.

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

2007-01-01

84

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

PubMed Central

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.

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

85

The human homologue of the mouse Surf5 gene encodes multiple alternatively spliced transcripts.  

PubMed

Hu-Surf5 is included within the Surfeit locus, a cluster of six genes originally identified in mouse. In the present study, we have cloned and characterized the Hu-Surf5 gene and its mRNA multiple transcripts. Comparison of the most abundant cDNA and genomic sequence shows that the Hu-Surf5 is spread over a region of approximately 7.5 kb and consists of five exons separated by four introns. The nucleotide sequence of the genomic region flanking the 3'-end of the Hu-Surf5 gene revealed the presence of a processed pseudogene of human ribosomal protein L21 followed by Hu-Surf6 gene. Only 110 bp separate the transcription start site of Hu-Surf5 and Hu-Surf3/L7a gene and the transcription direction is divergent. Earlier studies defined the 110 bp region essential for promoter activity of Hu-Surf3/L7a. Here, we show that this region stimulates transcription with a slightly different efficiency in both directions. The bidirectional promoter lacks an identifiable TATA box and is characterized by a CpG island that extends through the first exon into the first intron of both genes. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for Hu-Surf5 expression. Hu-Surf5 encodes three different transcripts, Surf-5a, Surf-5b, and Surf-5c, which result from alternative splicing. Two protein products, SURF-5A and SURF-5B have been characterized. Production of chimaeras between the full-length SURF-5A or SURF-5B and the green fluorescent protein (GFP) allowed to localize both proteins in the cytoplasm. PMID:11891058

Angiolillo, Antonietta; Russo, Giulia; Porcellini, Antonio; Smaldone, Silvia; D'Alessandro, Felicia; Pietropaolo, Concetta

2002-02-01

86

The Alternative Splicing Mutation Database: a hub for investigations of alternative splicing using mutational evidence  

PubMed Central

Background Some mutations in the internal regions of exons occur within splicing enhancers and silencers, influencing the pattern of alternative splicing in the corresponding genes. To understand how these sequence changes affect splicing, we created a database of these mutations. Findings The Alternative Splicing Mutation Database (ASMD) serves as a repository for all exonic mutations not associated with splicing junctions that measurably change the pattern of alternative splicing. In this initial published release (version 1.2), only human sequences are present, but the ASMD will grow to include other organisms, (see Availability and requirements section for the ASMD web address). This relational database allows users to investigate connections between mutations and features of the surrounding sequences, including flanking sequences, RNA secondary structures and strengths of splice junctions. Splicing effects of the mutations are quantified by the relative presence of alternative mRNA isoforms with and without a given mutation. This measure is further categorized by the accuracy of the experimental methods employed. The database currently contains 170 mutations in 66 exons, yet these numbers increase regularly. We developed an algorithm to derive a table of oligonucleotide Splicing Potential (SP) values from the ASMD dataset. We present the SP concept and tools in detail in our corresponding article. Conclusion The current data set demonstrates that mutations affecting splicing are located throughout exons and might be enriched within local RNA secondary structures. Exons from the ASMD have below average splicing junction strength scores, but the difference is small and is judged not to be significant.

Bechtel, Jason M; Rajesh, Preeti; Ilikchyan, Irina; Deng, Ying; Mishra, Pankaj K; Wang, Qi; Wu, Xiaochun; Afonin, Kirill A; Grose, William E; Wang, Ye; Khuder, Sadik; Fedorov, Alexei

2008-01-01

87

Genomic organization, expression of the human CBFA1 gene, and evidence for an alternative splicing event affecting protein function  

Microsoft Academic Search

The Cbfa1 gene, which encodes the transcription factor Osf2\\/Cbfa1 required for osteoblast differentiation in mouse and human, is mutated\\u000a in cleidocranial dysplasia, a skeletal dysplasia. We describe here the isolation of the full-length human OSF2\\/ CBFA1 cDNAs,\\u000a the genomic organization of the entire CBFA1 gene, its expression, and the existence of an alternative splicing event. Nucleotide\\u000a sequence analysis of the

V. Geoffroy; D. A. Corral; L. Zhou; B. Lee; G. Karsenty

1998-01-01

88

Gene Selection, Alternative Splicing, and Posttranslational Processing Regulate Neuroligin Selectivity for ?-Neurexins †  

Microsoft Academic Search

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with ‚-neurexin. Both neuroligins and ‚-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the ‚-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of ‚-neurexin

Davide Comoletti; Robyn E. Flynn; Antony A. Boucard; Borries Demeler; Virgil Schirf; Jianxin Shi; Lori L. Jennings; Helen R. Newlin; Thomas C. Südhof; Palmer Taylor

2006-01-01

89

Alternative splicing in the human cytochrome P450IIB6 gene generates a high level of aberrant messages.  

PubMed Central

Polymorphisms within the human cytochrome P450 system can have severe clinical consequences and have been associated with adverse drug side effects and susceptibility to environmentally linked diseases such as cancer. Aberrant splicing of cytochrome P450 mRNA has been proposed as a potential mechanism for these polymorphisms. We have isolated aberrantly, as well as normally, spliced mRNAs (cDNAs) from the human P450IIB6 gene which either contain part of intron 5 and lack exon 8 or which contain a 58-bp fragment (exon 8A) instead of exon 8. Sequence analysis of the P450IIB6 gene demonstrates the presence of cryptic splice sites in intron 8 which will account for the generation of exon 8A. The mRNAs were therefore generated by alternative splicing. These data gain significance as the mRNAs will not encode a functional P450 enzyme and appear to represent a high proportion of the P450IIB6 mRNA population. Analysis of mRNA from fifteen individual human livers and cDNA libraries constructed from a variety of human tissues using the polymerase chain reaction shows that the aberrant splicing occurs in all cells and all individuals tested. This suggests a high level of infidelity in the processing of P450IIB6 mRNAs and demonstrates that the presence of abnormal transcripts does not imply the presence of a functionally inactive gene. Images

Miles, J S; McLaren, A W; Wolf, C R

1989-01-01

90

Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms  

PubMed Central

Phytochelatins (PCs) play an important role in detoxification of heavy metals in plants. PCs are synthesized from glutathione by phytochelatin synthase (PCS), a dipeptidyltransferase. Sesbania rostrata is a tropical legume plant that can tolerate high concentrations of Cd and Zn. In this study, the S. rostrata PCS gene (SrPCS) and cDNAs were isolated and characterized. Southern blot and sequence analysis revealed that a single copy of the SrPCS gene occurs in the S. rostrata genome, and produces four different SrPCS mRNAs and proteins, SrPCS1–SrPCS4, by alternative splicing of the SrPCS pre-mRNA. The SrPCS1 and SrPCS3 proteins conferred Cd tolerance when expressed in yeast cells, whereas the SrPCS2 and SrPCS4 proteins, which lack the catalytic triad and the N-terminal domains, did not. These results suggested that SrPCS1 and SrPCS3 have potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils.

Li, An-Ming; Yu, Bing-Yun; Chen, Fu-Hua; Gan, Hui-Yan; Yuan, Jian-Gang; Qiu, Rongliang; Huang, Jun-Chao; Yang, Zhong-Yi; Xu, Zeng-Fu

2009-01-01

91

Identification and expression analysis of alternative splice variants of the rat Hax-1 gene  

Microsoft Academic Search

Hax-1 protein, which has been studied in mice and humans, shows a potent anti-apoptotic activity and is involved in regulation of cell motility. Cloning of the rat Hax-1 cDNA has revealed seven alternative transcripts, which differ mostly in their 5? region. Alternative splicing concerns exon 1, skipped in 5 transcripts, intron 1 which is partially retained in these transcripts, exon

Ewa A. Grzybowska; El?bieta Sarnowska; Ryszard Konopi?ski; Anna Wilczy?ska; Tomasz J. Sarnowski; Janusz A. Siedlecki

2006-01-01

92

Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes  

PubMed Central

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.

Jimenez-Lopez, Claudia; Lorenz, Michael C.; van Hoof, Ambro

2013-01-01

93

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

Microsoft Academic Search

Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in

Jianhua Zhou; Qingming Yu; Tie Zou

2008-01-01

94

The importance of being divisible by three in alternative splicing  

Microsoft Academic Search

Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that con- served alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differ- ences between conserved and species-specific alternative

Alon Magen; Gil Ast

2005-01-01

95

Characterization and prediction of alternative splice sites  

Microsoft Academic Search

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC

Magnus Wang; Antonio Marín

2006-01-01

96

Cloning and initial characterization of an alternatively spliced transcript encoded by the bovine herpes virus 1 latency-related gene  

Microsoft Academic Search

Bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic sensory neurons of infected cattle. The latency-related\\u000a (LR) RNA is the only abundantly expressed viral transcript in sensory neurons of latently infected calves. Wild-type expression\\u000a of LR gene products is required for the latency-reactivation cycle in calves. LR RNA is alternatively spliced in trigeminal\\u000a ganglia (TG) after infection of calves, suggesting

Laxminarayana R. Devireddy; Yange Zhang; Clinton J. Jones

2003-01-01

97

Alternative splicing of the FGF antisense gene: differential subcellular localization in human tissues and esophageal adenocarcinoma  

Microsoft Academic Search

Overexpression of FGF-2 is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer,\\u000a and these risks are reduced in tumors co-expressing the FGF antisense (FGF-AS) RNA. The aim of this study was to characterize\\u000a the expression of alternatively spliced FGF-AS transcripts and encoded nudix-motif proteins in normal human tissues and in\\u000a esophageal adenocarcinoma, and to correlate

Shuo Cheng Zhang; Christie Barclay; Leigh Ann Alexander; Laurette Geldenhuys; Geoffrey A. Porter; Alan G. Casson; Paul R. Murphy

2007-01-01

98

Exon organization and novel alternative splicing of the human ANK2 gene: Implications for cardiac function and human cardiac disease  

PubMed Central

Recent findings illustrate a critical role for ankyrin-B function in normal cardiovascular physiology. Specifically, decreased expression of ankyrin-B in mice or human mutations in the ankyrin-B gene (ANK2) results in potentially fatal cardiac arrhythmias. Despite the clear role of ankyrin-B in heart, the mechanisms underlying transcriptional regulation of ANK2 are unknown. In fact, to date there is no description of ANK2 genomic organization. The aims of this study were to provide a comprehensive description of the ANK2 gene and to evaluate the relative expression of alternative splicing events associated with ANK2 transcription in heart. Using reverse-transcriptase PCR on mRNA isolated from human hearts, we identify seven new exons associated with the ANK2 gene including an alternative first exon located ~145 kb upstream of the previously-identified first exon. In addition, we identify over thirty alternative splicing events associated with ANK2 mRNA transcripts. Using real-time PCR and exon boundary-spanning primers to selectively amplify these splice variants, we demonstrate that these variants are expressed at varying levels in human heart. Finally, ankyrin-B immunoblot analysis demonstrates the expression of a heterogeneous population of ankyrin-B polypeptides in heart. ANK2 consists of 53 exons that span ~560 kb on human chromosome 4. Additionally, our data demonstrates that ANK2 is subject to complex transcriptional regulation that likely results in differential ankyrin-B polypeptide function.

Cunha, Shane R.; Le Scouarnec, Solena; Schott, Jean-Jacques; Mohler, Peter J

2008-01-01

99

Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing  

Microsoft Academic Search

We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced\\u000a by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly

Wan-Jin XingBao-Da; Bao-Da Han; Qi Wu; Li Zhao; Xiao-Hong Bao; Shorgan Bou

2011-01-01

100

Variation in alternative splicing across human tissues  

PubMed Central

Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.

Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

2004-01-01

101

Alternative splicing: increasing diversity in the proteomic world  

Microsoft Academic Search

How can the genome of Drosophila melanogaster contain fewer genes than the undoubtedly simpler organism Caenorhabditis elegans? The answer must lie within their proteomes. It is becoming clear that alternative splicing has an extremely important role in expanding protein diversity and might therefore partially underlie the apparent discrepancy between gene number and organismal complexity. Alternative splicing can generate more transcripts

Brenton R. Graveley

2001-01-01

102

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

PubMed Central

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 F1 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.

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

2012-01-01

103

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

PubMed Central

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

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

1994-01-01

104

A study of alternative splicing in the pig  

Microsoft Academic Search

BACKGROUND: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs,

Ann-Britt Nygard; Susanna Cirera; Michael J Gilchrist; Jan Gorodkin; Claus B Jørgensen; Merete Fredholm

2010-01-01

105

An alternatively spliced transcript of the PHD3 gene retains prolyl hydroxylase activity.  

PubMed

Cellular response to limiting oxygen levels is managed, in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1), and the prolyl hydroxylase (PHD) family of oxygen-requiring enzymes. In the process of analyzing the expression of PHD3, we observed the presence of two alternatively processed PHD3 transcripts, designated PHD3Delta1 and PHD3Delta4 . The expression of both PHD3 and PHD3Delta1 was observed in all tissues and cell lines tested, although the expression of the novel PHD3Delta4 appeared to be restricted to primary cancer tissues. The function of PHD3Delta4 was assessed in transfection experiments showing a preserved prolyl hydroxylase activity. We would submit that PHD3 variants generated by alternative splicing may be intrinsically involved in the complex system of oxygen sensing. PMID:16473674

Cervera, Ana M; Apostolova, Nadezda; Luna-Crespo, Francisco; Sanjuan-Pla, Alejandra; Garcia-Bou, Remedios; McCreath, Kenneth J

2005-04-22

106

Directing alternative splicing: cast and scenarios  

Microsoft Academic Search

Recent progress in the study of alternative RNA splicing indicates that the interaction of RNA-binding proteins with specific target elements modulates splice site recognition and spliceosome assembly. The identity of splicing signals, the presence of modulating elements and differences in the distribution of RNA-binding proteins are key determinants involved in the tissue-specific regulation of splice site selection.

Benoit Chabot

1996-01-01

107

Conditional knockout mice to study alternative splicing in vivo.  

PubMed

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease. PMID:16314268

Xu, Xiangdong; Fu, Xiang-Dong

2005-12-01

108

Myoadenylate deaminase deficiency caused by alternative splicing due to a novel intronic mutation in the AMPD1 gene.  

PubMed

We have examined two Caucasian brothers with myoadenylate deaminase (AMPD) deficiency who presented with exercise intolerance and muscle cramps. Allele-specific PCR amplification assays demonstrated that the common Q12X (C34T) and P48L (C143T) mutations were not found within their AMPD1 genes. Further analysis revealed that both brothers were compound heterozygotes for a previously reported K287I (A860T) mutation in exon 7 and a novel deletion within intron 2 (IVS2-(4-7)delCTTT). The intronic deletion appears to affect the splicing machinery since characterization of AMPD1 mRNA from skeletal muscle of one brother identified multiple alternatively spliced transcripts resulting in multiple deletions in exon 3, the complete deletion of either exon 3 or exons 3 and 4, and the activation of a cryptic splice site that resulted in an insertion at the 5' end of exon 4. The predominant transcript contains a 51 base deletion at the 5' end of exon 3 that is predicted to produce a functional form of AMPD containing a 17-amino acid residue deletion within its N-terminal region. Analysis of 137 Caucasian normal control patients determined that the K287I mutation is relatively frequent (5.1% carrier frequency), whereas the IVS2-(4-7)delCTTT mutation is rare and not present in 274 chromosomes. PMID:16040263

Isackson, Paul J; Bujnicki, Heather; Harding, Cary O; Vladutiu, Georgirene D

2005-07-22

109

Alternative splicing and biological heterogeneity in prostate cancer  

Microsoft Academic Search

The biological diversity of prostate cancer confounds standardization of therapy. Advances in molecular profiling suggest that differences in the genetic composition of tumors significantly contribute to the complexity of the disease. Alternative pre-mRNA splicing is a key genetic process underlying biological diversity. During alternative splicing, coding and noncoding regions of a single gene are rearranged to generate several messenger RNA

David J. Elliott; Craig N. Robson; Hing Y. Leung; Prabhakar Rajan

2009-01-01

110

A Unique, Consistent Identifier for Alternatively Spliced Transcript Variants  

Microsoft Academic Search

BackgroundAs research into alternative splicing reveals the fundamental importance of this phenomenon in the genome expression of higher organisms, there is an increasing need for a standardized, consistent and unique identifier for alternatively spliced isoforms. Such an identifier would be useful to eliminate ambiguities in references to gene isoforms, and would allow for the reliable comparison of isoforms from different

Alberto Riva; Graziano Pesole; Juan Valcarcel

2009-01-01

111

Neuronal Signaling through Alternative Splicing: Some Exons CaRRE...  

NSDL National Science Digital Library

Alternative splicing represents a mechanism by which a single gene can be used to create proteins with different functions. Neurons use alternative splicing to produce channels with different sequences and biophysical or regulatory properties. O'Donovan and Darnell discuss a mechanism by which neurons can alter channel splicing in response to neuronal activity through a signal generated by calcium and calcium/calmodulin-dependent kinase activity.

Kevin J. O'Donovan (The Rockefeller University;Laboratory of Molecular Neuro-Oncology REV); Robert B. Darnell (The Rockefeller University;Laboratory of Molecular Neuro-Oncology REV)

2001-08-07

112

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

PubMed Central

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.

2010-01-01

113

Bioinformatics of alternative splicing and its regulation  

Microsoft Academic Search

The sequencing of the human genome and ensuing wave of data generation have brought new light upon the extent and importance of alternative splicing as an RNA regulatory mechanism. Alternative splicing could potentially explain the complexity of protein repertoire during evolution, and defects in the splicing mechanism are responsible for diseases as complex as cancer. Among the challenges that rise

Liliana Florea

2006-01-01

114

Alternative splicing at the right time.  

PubMed

Alternative splicing (AS) allows the production of multiple mRNA variants from a single gene, which contributes to increase the complexity of the proteome. There is evidence that AS is regulated not only by auxiliary splicing factors, but also by components of the core spliceosomal machinery, as well as through epigenetic modifications. However, to what extent these different mechanisms contribute to the regulation of AS in response to endogenous or environmental stimuli is still unclear. Circadian clocks allow organisms to adjust physiological processes to daily changes in environmental conditions. Here we review recent evidence linking circadian clock and AS, and discuss the role of Protein Arginine Methyltransferase 5 (PRMT5) in these processes. We propose that the interactions between daily oscillations in AS and circadian rhythms in the expression of splicing factors and epigenetic regulators offer a great opportunity to dissect the contribution of these mechanisms to the regulation of AS in a physiologically relevant context. PMID:21941124

Sanchez, Sabrina E; Petrillo, Ezequiel; Kornblihtt, Alberto R; Yanovsky, Marcelo J

2011-11-01

115

Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer.  

PubMed

Alternative processing of pre-mRNA transcripts is a major source of protein diversity in eukaryotes and has been implicated in several disease processes including cancer. In this study we have performed a genome wide analysis of alternative splicing events in lung adenocarcinoma. We found that 2369 of the 17 800 core Refseq genes appear to have alternative transcripts that are differentially expressed in lung adenocarcinoma versus normal. According to their known functions the largest subset of these genes (30.8%) is believed to be cancer related. Detailed analysis was performed for several genes using PCR, quantitative RT-PCR and DNA sequencing. We found overexpression of ERG variant 2 but not variant 1 in lung tumors and overexpression of CEACAM1 variant 1 but not variant 2 in lung tumors but not in breast or colon tumors. We also identified a novel, overexpressed variant of CDH3 and verified the existence and overexpression of a novel variant of P16 transcribed from the CDKN2A locus. These findings demonstrate how analysis of alternative pre-mRNA processing can shed additional light on differences between tumors and normal tissues as well as between different tumor types. Such studies may lead to the development of additional tools for tumor diagnosis, prognosis and therapy. PMID:18927117

Xi, Liqiang; Feber, Andrew; Gupta, Vanita; Wu, Maoxin; Bergemann, Andrew D; Landreneau, Rodney J; Litle, Virginia R; Pennathur, Arjun; Luketich, James D; Godfrey, Tony E

2008-10-16

116

Alternative 5' splice site selection induced by heat shock.  

PubMed Central

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. Images

Takechi, H; Hosokawa, N; Hirayoshi, K; Nagata, K

1994-01-01

117

Allelic Variation, Alternative Splicing and Expression Analysis of Psy1 Gene in Hordeum chilense Roem. et Schult  

PubMed Central

Background The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. Methodology/Principal findings We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5? UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. Conclusions/Significance The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.

Rodriguez-Suarez, Cristina; Atienza, Sergio G.; Piston, Fernando

2011-01-01

118

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

PubMed Central

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

Read, D; Manley, J L

1992-01-01

119

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii  

PubMed Central

Background Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

2010-01-01

120

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

PubMed

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

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

2011-08-09

121

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

PubMed Central

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.

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

2011-01-01

122

Regulation of alternative splicing within the supraspliceosome  

PubMed Central

Alternative splicing is a fundamental feature in regulating the eukaryotic transcriptome, as ~95% of multi-exon human Pol II transcripts are subject to this process. Regulated splicing operates through the combinatorial interplay of positive and negative regulatory signals present in the pre-mRNA, which are recognized by trans-acting factors. All these RNA and protein components are assembled in a gigantic, 21 MDa, ribonucleoprotein splicing machine – the supraspliceosome. Because most alternatively spliced mRNA isoforms vary between different cell and tissue types, the ability to perform alternative splicing is expected to be an integral part of the supraspliceosome, which constitutes the splicing machine in vivo. Here we show that both the constitutively and alternatively spliced mRNAs of the endogenous human pol II transcripts: hnRNP A/B, survival of motor neuron (SMN) and ADAR2 are predominantly found in supraspliceosomes. This finding is consistent with our observations that the splicing regulators hnRNP G as well as all phosphorylated SR proteins are predominantly associated with supraspliceosomes. We further show that changes in alternative splicing of hnRNP A/B, affected by up regulation of SRSF5 (SRp40) or by treatment with C6-ceramide, occur within supraspliceosomes. These observations support the proposed role of the supraspliceosome in splicing regulation and alternative splicing.

Sebbag-Sznajder, Naama; Raitskin, Oleg; Angenitzki, Minna; Sato, Taka-Aki; Sperling, Joseph; Sperling, Ruth

2012-01-01

123

Alternative Splicing: Therapeutic Target and Tool  

Microsoft Academic Search

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for ther- apy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense

Mariano A. Garcia-Blanco

124

Molecular cloning of novel alternatively spliced variants of BCL2L12, a new member of the BCL2 gene family, and their expression analysis in cancer cells.  

PubMed

In the past, we identified and cloned the BCL2-like 12 (BCL2L12) gene, a novel member of the BCL2 family, which is implicated in various malignancies. The classical BCL2L12 protein isoform contains a highly conserved BH2 domain, a BH3-like motif, and a proline-rich region, and is involved in apoptosis. Most members of this apoptosis-related family are subjected to alternative splicing, thus generating multiple protein isoforms with distinct properties, and sometimes even with opposite function (pro- vs. anti-apoptotic). In the current study, we report the identification, molecular cloning, and expression pattern of novel splice variants of the human BCL2L12 gene in cancer cell lines. EST clones displaying high sequence identity (?90%) with the classical BCL2L12 transcript were aligned, in order to identify those containing at least one novel splice junction. EST database mining led to the identification of three previously unknown splice variants of this apoptotic gene. In our effort to experimentally validate these novel transcripts, we also cloned seven more, previously unidentified, BCL2L12 alternatively spliced variants. Expression analysis of all BCL2L12 splice variants in human cancer cell lines and embryonic kidney cells revealed remarkable differences between their BCL2L12 expression profiles. Interestingly, 7 out of 10 novel splice variants of BCL2L12 are predicted to encode new protein isoforms, some of which are BH3-only proteins, in contrast to the classical BCL2L12 isoform, which also contains a functional BH2 domain. The remaining three novel splice variants of BCL2L12 are nonsense-mediated mRNA decay (NMD) candidates. PMID:22664385

Kontos, Christos K; Scorilas, Andreas

2012-06-01

125

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56,419 completely sequenced and manually annotated full-length cDNAs.  

PubMed

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56,419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37,670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A; Koyanagi, Kanako O; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-08-12

126

Modulating alternative splicing by cotranscriptional cleavage of nascent intronic RNA  

PubMed Central

Cotranscriptional cleavage mediated by a hammerhead ribozyme can affect alternative splicing if interposed between an exon and its intronic regulatory elements. This has been demonstrated using two different alternative splicing systems based on ?-tropomyosin and fibronectin genes. We suggest that there is a requirement for intronic regulatory elements to be covalently attached to exons that are in turn tethered to the elongating polymerase. In the case of the alternatively spliced EDA exon of the fibronectin gene, we demonstrate that the newly identified intronic downstream regulatory element is associated with the splicing regulatory protein SRp20. Our results suggest that targeted hammerhead ribozyme cleavage within introns can be used as a tool to define splicing regulatory elements.

Gromak, Natalia; Talotti, Gabriele; Proudfoot, Nicholas J.; Pagani, Franco

2008-01-01

127

The FLOWERING LOCUS T orthologous gene of Platanus acerifolia is expressed as alternatively spliced forms with distinct spatial and temporal patterns.  

PubMed

The FLOWERING LOCUS T (FT) protein is a likely component of the 'florigen' signal that plays a crucial role in regulating the transition from vegetative to reproductive growth. Here, we report the isolation of full-length cDNAs and the corresponding genomic clones encoding orthologous FT proteins (PaFT) of London plane (Platanus acerifolia Willd). Two genes with high sequence identity were isolated (PaFT1-2), but 34 different transcript products were identified, comprising 21 splice forms produced by alternative splicing of the PaFT pre-mRNAs. Expression of the alternative splicing forms of PaFT varied according to tissue type and developmental stage. PaFT transcripts were detected throughout adult trees, whereas in juvenile trees they were only detected in dormant sub-petiolar buds. In adult trees, levels of the various alternative splicing PaFT forms were related to flower development stage and tree dormancy. Ectopic expression of the archetypal splice form of PaFT-A in tobacco and Arabidopsis developed multiple altered phenotypes, notably early flowering and attenuation of apical dominance. The evidence in this study indicates that complex alternative processing of PaFT transcripts in P. acerifolia may be associated with multiple regulatory roles, including initiation of flowering, flower development, apical dominance, tree dormancy and dormancy release. PMID:21815986

Zhang, J; Liu, G; Guo, C; He, Y; Li, Z; Ning, G; Shi, X; Bao, M

2011-02-05

128

Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms.  

PubMed

Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'. PMID:23357783

Yap, Karen; Makeyev, Eugene V

2013-01-26

129

Analysis of SRrp86-regulated alternative splicing  

PubMed Central

Previous work led to the hypothesis that SRrp86, a related member of the SR protein superfamily, can interact with and modulate the activity of other SR proteins. Here, we sought to test this hypothesis by examining the effect of changing SRrp86 concentrations on overall alternative splicing patterns. SpliceArrays were used to examine 9,854 splicing events in wild-type cells, cells overexpressing SRrp86, and cells treated with siRNAs to knockdown SRrp86. From among the 500 splicing events exhibiting altered splicing under these conditions, the splicing of c-Jun and I?B? were validated as being regulated by SRrp86 resulting in altered regulation of their downstream targets. In both cases, functionally distinct isoforms were generated that demonstrate the role SRrp86 plays in controlling alternative splicing.

Solis, Amanda S

2010-01-01

130

Alternative splicing: decoding an expansive regulatory layer.  

PubMed

Alternative splicing (AS) is the process by which splice sites in precursor (pre)-mRNA are differentially selected to produce multiple mRNA and protein isoforms. During the past few years the application of genome-wide profiling technologies coupled with bioinformatic approaches has transformed our understanding of AS complexity and regulation. These studies are further driving research directed at elucidating the functions of networks of regulated AS events in the context of normal physiology and disease. Major strides have also been made in understanding how AS is functionally integrated with- and coupled to- gene regulation at the level of chromatin and transcription. Particularly intriguing is the discovery of new AS 'switches' that control transcriptional networks required for animal development and behavior. PMID:22465326

Irimia, Manuel; Blencowe, Benjamin J

2012-03-30

131

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  

Microsoft Academic Search

XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82-882 bp) and 15 introns (0.08-5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription-polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that

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

2002-01-01

132

Adenosine deaminase-related genes: molecular identification, tissue expression pattern and truncated alternative splice isoform in adult zebrafish (Danio rerio).  

PubMed

Adenosine deaminase (ADA) is responsible for cleaving the neuromodulator adenosine to inosine. Two members of ADA subfamilies, known as ADA1 and ADA2, were described and evidence demonstrated another similar protein group named ADAL (adenosine deaminase "like"). Although the identification of ADA members seems to be consistent, the expression profile of ADA1, ADA2 and ADAL genes in zebrafish has not yet been reported. The aim of the present study was to map the expression pattern of ADA-related genes in various tissues of adult zebrafish (Danio rerio). An extensive search on zebrafish genome followed by a phylogenetic analysis confirmed the presence of distinct ADA-related genes (ADA1, ADAL and two orthologous genes of ADA2). Specific primers for each ADA member were designed, optimized semi-quantitative RT-PCR experiments were conducted and the relative amount of transcripts was determined. The tissue samples (brain, gills, heart, liver, skeletal muscle and kidney) were collected and the expression of ADA1, ADAL and ADA2 genes was characterized. ADA1 had a similar expression pattern, whereas ADAL was less expressed in the heart. The highest relative amount of ADA2-1 transcripts was observed in the brain, liver and gills and it was less expressed in the heart. RT-PCR assays revealed that the other ADA2 form (ADA2-2) was expressed ubiquitously and at comparable levels in zebrafish tissues. The strategy adopted also allowed the identification of an ADA2-1 truncated alternative splice isoform (ADA2-1/T), which was expressed at different intensities. These findings demonstrated the existence of different ADA-related genes, their distinct expression pattern and a truncated ADA2-1 isoform, which suggest a high degree of complexity in zebrafish adenosinergic system. PMID:17950365

Rosemberg, Denis Broock; Rico, Eduardo Pacheco; Guidoti, Marcus Rodrigo; Dias, Renato Dutra; Souza, Diogo Onofre; Bonan, Carla Denise; Bogo, Maurício Reis

2007-10-04

133

ASPMF: A new approach for identifying alternative splicing isoforms using peptide mass fingerprinting  

Microsoft Academic Search

Alternative splicing is generally accepted as a mechanism that explains the discrepancy between the number of genes and proteins. We used peptide mass fingerprinting with a theoretical database and scoring method to discover and identify alternative splicing isoforms. Our theoretical database was built using published alternative splicing databases such as ECgene, H-DBAS, and TISA. According to our theoretical database of

Seung-Won Lee; Jae-Pil Choi; Hyun-Jin Kim; Ji-Man Hong; Cheol-Goo Hur

2008-01-01

134

Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei  

PubMed Central

Trans-splicing of leader sequences onto the 5?ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5?splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5? splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

Nilsson, Daniel; Gunasekera, Kapila; Mani, Jan; Osteras, Magne; Farinelli, Laurent; Baerlocher, Loic; Roditi, Isabel; Ochsenreiter, Torsten

2010-01-01

135

Estimation of alternative splicing variability in human populations  

PubMed Central

DNA arrays have been widely used to perform transcriptome-wide analysis of gene expression, and many methods have been developed to measure gene expression variability and to compare gene expression between conditions. Because RNA-seq is also becoming increasingly popular for transcriptome characterization, the possibility exists for further quantification of individual alternative transcript isoforms, and therefore for estimating the relative ratios of alternative splice forms within a given gene. Changes in splicing ratios, even without changes in overall gene expression, may have important phenotypic effects. Here we have developed statistical methodology to measure variability in splicing ratios within conditions, to compare it between conditions, and to identify genes with condition-specific splicing ratios. Furthermore, we have developed methodology to deconvolute the relative contribution of variability in gene expression versus variability in splicing ratios to the overall variability of transcript abundances. As a proof of concept, we have applied this methodology to estimates of transcript abundances obtained from RNA-seq experiments in lymphoblastoid cells from Caucasian and Yoruban individuals. We have found that protein-coding genes exhibit low splicing variability within populations, with many genes exhibiting constant ratios across individuals. When comparing these two populations, we have found that up to 10% of the studied protein-coding genes exhibit population-specific splicing ratios. We estimate that ?60% of the total variability observed in the abundance of transcript isoforms can be explained by variability in transcription. A large fraction of the remaining variability can likely result from variability in splicing. Finally, we also detected that variability in splicing is uncommon without variability in transcription.

Gonzalez-Porta, Mar; Calvo, Miquel; Sammeth, Michael; Guigo, Roderic

2012-01-01

136

Characterization of tissue-specific and developmentally regulated alternative splicing of exon 64 in the COL5A1 gene.  

PubMed

The COL5A1 gene, a member of the clade B fibrillar collagen gene family, was recently shown to contain two alternatively spliced exons (64A and 64B) that encode 23 amino acids in the carboxyl-terminal propeptide. The two are identical in length, very similar in sequence, and used in a mutually exclusive fashion because of the small intron that separates them. Each COL5A1 allele uses both exons, but a given transcript will contain only one of the two exons. The sequences in other species are highly conserved at the amino acid level. The expression profile of the two isoforms was determined from analysis of RNA levels in a panel of murine tissues. While both isoforms were found in all tissues studied, actively proliferating tissues (liver, lung) used isoform B more often, while a less mitotically active tissue, brain, had a higher proportion of exon 64A. The high degree of conservation between the two exons is consistent with a regional genomic duplication. The presence of the two isoforms as far back as pufferfish (tetraodon) implies an important functional significance. The exact role determined by the two sequences is not known, but involvement in the determination of chain composition of mature type V collagen or regulation of cell activity is possible, given the differences in tissue distribution. PMID:22149965

Mitchell, Anna L; Judis, LuAnn M; Schwarze, Ulrike; Vaynshtok, Polina M; Drumm, Mitchell L; Byers, Peter H

2011-12-07

137

Multiple transcription start sites and alternative splicing in the methylenetetrahydrofolate reductase gene result in two enzyme isoforms.  

PubMed

Methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major carbon donor in the remethylation of homocysteine to methionine. Mild MTHFR deficiency, due to a common variant at nucleotide 677, has been reported to alter risk for several disorders including cardiovascular disease, neural tube defects, pregnancy complications, and certain cancers. Little is known about MTHFR regulation, since the complete cDNA and gene sequences have not been determined. In earlier work, we isolated and expressed a 2.2-kb human cDNA comprised of 11 coding exons, and we demonstrated that it encoded an active 70-kDa isoform. However, transcript sizes of approximately 7.5 kb and 9.5 kb and the presence of a second isoform of 77 kDa on Western blots suggested that cDNA sequences were incomplete. In this report, we characterized the complete cDNA and gene structure in human and mouse. Variable 5? and 3? UTR regions were identified, resulting in transcript heterogeneity. The 5? and 3? termini of the MTHFR cDNA were found to overlap with the 5? terminus of a chloride ion channel gene (CLCN-6) and the 3? terminus of an unidentified gene, respectively; this finding has resulted in finer mapping of MTHFR on Chromosome (Chr) 1p36.3. Ribonuclease protection assays identified clusters of transcriptional start sites, suggesting the existence of multiple promoters. MTHFR has several polyadenylation sites creating 3?UTR lengths of 0.2 kb-5.0 kb or 0.6 kb-4.0 kb in human and mouse, respectively. In both species, the previously reported exon 1 was redefined to approximately 3.0 kb in length and shown to be alternatively spliced. An important splice variant contains novel coding sequences; this cDNA was expressed and shown to encode the isozyme of 77 kDa. Our results, which suggest intricate regulation of MTHFR, will facilitate additional regulatory and functional studies of the different isoforms. PMID:12370778

Tran, Pamela; Leclerc, Daniel; Chan, Manuel; Pai, Aditya; Hiou-Tim, Francois; Wu, Qing; Goyette, Philippe; Artigas, Carmen; Milos, Renate; Rozen, Rima

2002-09-01

138

Genome-wide analysis of alternative splicing in Caenorhabditis elegans.  

PubMed

Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provide the most comprehensive set of annotated splice variants in C. elegans to date, and are therefore expected to facilitate focused, high resolution in vivo functional assays of AS function. PMID:21177968

Ramani, Arun K; Calarco, John A; Pan, Qun; Mavandadi, Sepand; Wang, Ying; Nelson, Andrew C; Lee, Leo J; Morris, Quaid; Blencowe, Benjamin J; Zhen, Mei; Fraser, Andrew G

2010-12-22

139

Alternative splicing networks regulated by signaling in human T cells.  

PubMed

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

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

2012-03-27

140

Alternative splicing networks regulated by signaling in human T cells  

PubMed Central

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.

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

141

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.  

PubMed Central

Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.

Konig, H; Ponta, H; Herrlich, P

1998-01-01

142

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes  

Microsoft Academic Search

Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 50 splice site (ss) serves as

Natalia N. Singh; Ravindra N. Singh; Elliot J. Androphy

2006-01-01

143

Molecular Characterization and Alternative Splicing of a Sodium Channel and DSC1 Ortholog Genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).  

PubMed

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels. PMID:24155671

Jiang, Xuan-Zhao; Wei, Dan-Dan; Yang, Wen-Jia; Dou, Wei; Chen, Shi-Chun; Wang, Jin-Jun

2013-09-26

144

Molecular Characterization and Alternative Splicing of a Sodium Channel and DSC1 Ortholog Genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)  

PubMed Central

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels.

Jiang, Xuan-Zhao; Wei, Dan-Dan; Yang, Wen-Jia; Dou, Wei; Chen, Shi-Chun; Wang, Jin-Jun

2013-01-01

145

The Alternative Splicing Gallery (ASG): bridging the gap between genome and transcriptome  

Microsoft Academic Search

Alternative splicing essentially increases the divers- ity of the transcriptome and has important implica- tions for physiology, development and the genesis of diseases. Conventionally, alternative splicing is investigated in a case-by-case fashion, but this becomes cumbersome and error prone if genes show a huge abundance of different splice variants. We use a different approach and integrate all tran- scripts derived

Jeremy Leipzig; Pavel Pevzner; Steffen Heber

2004-01-01

146

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

PubMed

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

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

2008-09-24

147

Alternative splicing in disease and therapy  

Microsoft Academic Search

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting

Andrew P Baraniak; Erika L Lasda; Mariano A Garcia-Blanco

2004-01-01

148

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

Microsoft Academic Search

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

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

2008-01-01

149

Gene organization and alternative splicing of human prohormone convertase PC8.  

PubMed Central

The mammalian Ca2+-dependent serine protease prohormone convertase PC8 is expressed ubiquitously, being transcribed as 3.5, 4.3 and 6.0 kb mRNA isoforms in various tissues. To determine the origin of these various mRNA isoforms we report the characterization of the human PC8 gene, which has been previously localized to chromosome 11q23-24. Consisting of 16 exons, the human PC8 gene spans approx. 27 kb. A comparison of the position of intron-exon junctions of the human PC8 gene with the gene structures of previously reported prohormone convertase genes demonstrated a divergence of the human PC8 from the highly conserved nature of the gene organization of this enzyme family. The nucleotide sequence of the 5'-flanking region of the human PC8 is reported and possesses putative promoter elements characteristic of a GC-rich promoter. Further supporting the potential role of a GC-rich promoter element, multiple transcriptional initiation sites within a 200 bp region were demonstrated. We propose that the various mRNA isoforms of PC8 result from the inclusion of intronic sequences within transcripts.

Goodge, K A; Thomas, R J; Martin, T J; Gillespie, M T

1998-01-01

150

Two novel transcripts for human endothelin B receptor produced by RNA editing/alternative splicing from a single gene.  

PubMed

Hirschsprung disease is a heterogeneous genetic disorder, causative genes of which include the endothelin B receptor (ETB). To investigate the mutations of ETB in Hirschsprung disease, expression of the ETB gene in lymphoblastoid cells from patients and normal healthy adults was examined, and novel mutant transcripts were found. The mutant ETB gene transcripts lacked a 134-bp nucleotide sequence corresponding to exon 5, and some also contained a substitution from A to G at position 950 in exon 4, resulting in an amino acid substitution from glutamine (Q) to arginine (R). This substitution was suspected to be the result of RNA editing because it was not present in the genomic sequence. Transfection experiments using ETB minigenes containing the editing site with or without the gene for double-strand RNA deaminases (ADAR1 and ADAR2) revealed that the deaminases were involved in RNA editing. Furthermore, a c-Myc-tagged mutant ETB protein was not detected by Western blot analysis. The present results show that the mutant ETB transcripts were novel splice variants, which might not be translated, or that the products translated from splice variants might be quickly degraded, presumably because of their instability. The preferential production of this null function ETB by RNA editing/splicing could be involved in the etiology of some cases of Hirschsprung disease. PMID:12080062

Tanoue, Akito; Koshimizu, Taka-Aki; Tsuchiya, Masako; Ishii, Kayano; Osawa, Makiko; Saeki, Morihiro; Tsujimoto, Gozoh

2002-06-21

151

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay  

Microsoft Academic Search

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to

Julie Z. Ni; Leslie Grate; John Paul Donohue; Christine Preston; Naomi Nobida; Georgeann O'Brien; Lily Shiue; Tyson A. Clark; John E. Blume; Manuel Ares Jr

2007-01-01

152

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

Microsoft Academic Search

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present

Shravan Kumar Mishra; Tim Ammon; Grzegorz M. Popowicz; Marcin Krajewski; Roland J. Nagel; Manuel Ares; Tad A. Holak; Stefan Jentsch

2011-01-01

153

Alternative splicing of the mouse embryonic poly(A) binding protein (Epab) mRNA is regulated by an exonic splicing enhancer: a model for post-transcriptional control of gene expression in the oocyte  

Microsoft Academic Search

Embryonic poly(A) binding protein (EPAB), expressed in oocytes and early embryos, binds and stabilizes maternal mRNAs, and mediates initiation of their translation. We identified an alternatively spliced form of Epab lacking exon 10 (c.Ex10del) and inves- tigated the regulation of Epab mRNA alternative splicing as a model for alternative splicing in oocytes and early preimplantation embryos. Specifically, we evaluated the

Emre Seli; Aylin Yaba; Ozlem Guzeloglu-Kayisli; Maria D. Lalioti

2008-01-01

154

The evolutionary landscape of alternative splicing in vertebrate species.  

PubMed

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

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

155

Control of alternative RNA splicing and gene expression by eukaryotic riboswitches  

Microsoft Academic Search

Bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. The most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (TPP), which is a derivative of vitamin B1. Representatives of this class have also been identified in fungi and plants, where

Ming T. Cheah; Andreas Wachter; Narasimhan Sudarsan; Ronald R. Breaker

2007-01-01

156

A starch-branching enzyme gene in wheat produces alternatively spliced transcripts  

Microsoft Academic Search

A wheat gene, denoted Sbe1, encoding a type I starch-branching enzyme (SBEI) was isolated from a genomic library and shown to comprise 14 exons distributed over a 5.7 kb DNA region. Analyses of kernel RNA by 5' rapid amplification of cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a considerable sequence variation at the 5' ends of SBEI

Monica Båga; Sarah Glaze; Clifford S. Mallard; Ravindra N. Chibbar

1999-01-01

157

Rbm20 regulates titin alternative splicing as a splicing repressor  

PubMed Central

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3? splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.

Li, Shijun; Guo, Wei; Dewey, Colin N.; Greaser, Marion L.

2013-01-01

158

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica  

PubMed Central

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3?-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.

Kabran, Philomene; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuveglise, Cecile

2012-01-01

159

AsMamDB: an alternative splice database of mammals  

PubMed Central

The objective of database AsMamDB is to facilitate the systematic study of alternatively spliced genes of mammals. Version 1.0 of AsMamDB contains 1563 alternatively spliced genes of human, mouse and rat, each associated with a cluster of nucleotide sequences. The main information provided by AsMamDB includes gene alternative splicing patterns, gene structures, locations in chromosomes, products of genes and tissues where they express. Alternative splicing patterns are represented by multiple alignments of various gene transcripts and by graphs of their topological structures. Gene structures are illustrated by exon, intron and various regulatory elements distributions. There are 4204 DNAs, 3977 mRNAs, 8989 CDSs and 126 931 ESTs in the current database. More than 130 000 GenBank entries are covered and 4443 MEDLINE records are linked. DNA, mRNA, exon, intron and relevant regulatory element sequences are provided in FASTA format. More information can be obtained by using the web-based multiple alignment tool Asalign and various category lists. AsMamDB can be accessed at http://166.111.30.6 5/ASMAM DB.html.

Ji, Hongkai; Zhou, Qing; Wen, Fang; Xia, Huiyu; Lu, Xin; Li, Yanda

2001-01-01

160

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay  

PubMed Central

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

McIlwain, David R.; Pan, Qun; Reilly, Patrick T.; Elia, Andrew J.; McCracken, Susan; Wakeham, Andrew C.; Itie-Youten, Annick; Blencowe, Benjamin J.; Mak, Tak W.

2010-01-01

161

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.  

PubMed

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome. PMID:20566848

McIlwain, David R; Pan, Qun; Reilly, Patrick T; Elia, Andrew J; McCracken, Susan; Wakeham, Andrew C; Itie-Youten, Annick; Blencowe, Benjamin J; Mak, Tak W

2010-06-21

162

Cell-to-cell variability of alternative RNA splicing  

PubMed Central

Heterogeneity in the expression levels of mammalian genes is large even in clonal populations and has phenotypic consequences. Alternative splicing is a fundamental aspect of gene expression, yet its contribution to heterogeneity is unknown. Here, we use single-molecule imaging to characterize the cell-to-cell variability in mRNA isoform ratios for two endogenous genes, CAPRIN1 and MKNK2. We show that isoform variability in non-transformed, diploid cells is remarkably close to the minimum possible given the stochastic nature of individual splicing events, while variability in HeLa cells is considerably higher. Analysis of the potential sources of isoform ratio heterogeneity indicates that a difference in the control over splicing factor activity is one origin of this increase. Our imaging approach also visualizes non-alternatively spliced mRNA and active transcription sites, and yields spatial information regarding the relationship between splicing and transcription. Together, our work demonstrates that mammalian cells minimize fluctuations in mRNA isoform ratios by tightly regulating the splicing machinery.

Waks, Zeev; Klein, Allon M; Silver, Pamela A

2011-01-01

163

Recognition of Unknown Conserved Alternatively Spliced Exons  

PubMed Central

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the ~1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.

Ohler, Uwe; Shomron, Noam; Burge, Christopher B

2005-01-01

164

Dysfunctional gene splicing as a potential contributor to neuropsychiatric disorders.  

PubMed

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

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

2011-03-22

165

Alternative splicing produces structural and functional changes in CUGBP2  

PubMed Central

Background CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. Results The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3?) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3? isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3? isoform was flexible and did not form an RRM structure. Conclusion Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

2012-01-01

166

CD28 Costimulation Regulates Genome-Wide Effects on Alternative Splicing  

PubMed Central

CD28 is the major costimulatory receptor required for activation of naïve T cells, yet CD28 costimulation affects the expression level of surprisingly few genes over those altered by TCR stimulation alone. Alternate splicing of genes adds diversity to the proteome and contributes to tissue-specific regulation of genes. Here we demonstrate that CD28 costimulation leads to major changes in alternative splicing during activation of naïve T cells, beyond the effects of TCR alone. CD28 costimulation affected many more genes through modulation of alternate splicing than by modulation of transcription. Different families of biological processes are over-represented among genes alternatively spliced in response to CD28 costimulation compared to those genes whose transcription is altered, suggesting that alternative splicing regulates distinct biological effects. Moreover, genes dependent upon hnRNPLL, a global regulator of splicing in activated T cells, were enriched in T cells activated through TCR plus CD28 as compared to TCR alone. We show that hnRNPLL expression is dependent on CD28 signaling, providing a mechanism by which CD28 can regulate splicing in T cells and insight into how hnRNPLL can influence signal-induced alternative splicing in T cells. The effects of CD28 on alternative splicing provide a newly appreciated means by which CD28 can regulate T cell responses.

Jesneck, Jonathan; Keir, Mary E.; Haining, W. Nicholas; Sharpe, Arlene H.

2012-01-01

167

The mechanism of alternative splicing of the X-linked NDUFB11 gene of the respiratory chain complex I, impact of rotenone treatment in neuroblastoma cells.  

PubMed

A study is presented on the regulation of alternative splicing (AS) of the Ndufb11 gene of complex I of the mitochondrial respiratory chain and the impact on this process of rotenone treatment in neuroblastoma cells. In physiological conditions the Ndufb11 gene produces at high level a short transcript isoform encoding for a 153 aa protein. This subunit is essential for the assembly of a functional and stable mammalian complex I. The gene produces also, at low level, a longer transcript isoform encoding for a 163 aa protein whose role is unknown. Evidence is presented here showing that the level of the two isoforms is regulated by three DGGGD ESS elements located in exon 2 which can bind the hnRNPH1 protein. In neuronal cells rotenone treatment affects the Ndufb11 alternative splicing pathway, with the increase of the 163/153 mRNAs ratio. This effect appears to be due to the down-regulation of the hnRNPH1 protein. Since rotenone induces apoptosis in neuronal cells, the post-transcriptional regulation of the Ndufb11 gene can be involved in the programmed cell death process. PMID:23246602

Panelli, Damiano; Lorusso, Francesca Paola; Papa, Francesco; Panelli, Patrizio; Stella, Alessandro; Caputi, Massimo; Sardanelli, Anna Maria; Papa, Sergio

2012-12-12

168

A procedure for identifying homologous alternative splicing events  

Microsoft Academic Search

Background: The study of the functional role of alternative splice isoforms of a gene is a very active area of research in biology. The difficulty of the experimental approach (in particular, in its high-throughput version) leaves ample room for the development of bioinformatics tools that can provide a useful first picture of the problem. Among the possible approaches, one of

David Talavera; Modesto Orozco; Xavier De La Cruz

2007-01-01

169

Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer  

PubMed Central

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

Sette, Claudio

2013-01-01

170

Finding signals that regulate alternative splicing in the post-genomic era  

PubMed Central

Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification.

Ladd, Andrea N; Cooper, Thomas A

2002-01-01

171

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

Microsoft Academic Search

BackgroundThe vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them

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

2008-01-01

172

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

Microsoft Academic Search

Abstract Background:,The vast majority,of human,genes,(.70%) are alternatively spliced. Although,alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer

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

2008-01-01

173

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

PubMed

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

Laporta, J; Driver, A; Khatib, H

2011-08-01

174

Regulation of alternative splicing by the core spliceosomal machinery.  

PubMed

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

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

2011-02-15

175

Regulation of alternative splicing by the core spliceosomal machinery  

PubMed Central

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

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

2011-01-01

176

Alternative splicing of the mouse embryonic poly(A) binding protein (Epab) mRNA is regulated by an exonic splicing enhancer: a model for post-transcriptional control of gene expression in the oocyte.  

PubMed

Embryonic poly(A) binding protein (EPAB), expressed in oocytes and early embryos, binds and stabilizes maternal mRNAs, and mediates initiation of their translation. We identified an alternatively spliced form of Epab lacking exon 10 (c.Ex10del) and investigated the regulation of Epab mRNA alternative splicing as a model for alternative splicing in oocytes and early preimplantation embryos. Specifically, we evaluated the following mechanisms: imprinting; RNA editing and exonic splicing enhancers (ESEs). Sequence analysis led to the identification of two single nucleotide polymorphisms (SNPs): one was detected in exon 9 (rs55858A/G), and served as a marker for the parental origin of the alternatively spliced form, and the other was found in exon 10 (rs56574G/C), and co-segregated with the exon 9 SNP. We found that the presence of rs56574G in exon 10 led to the formation of an ESE, leading to efficient exclusion of exon 10. Real-time RT-PCR results revealed a 5-fold increase in the expression of the c.Ex10del alternative splicing variant in animals carrying rs56574G/G in exon 10 compared with rs56574C/C at the same locus. Our findings suggest that SNPs may alter the ratio between alternative splicing variants of oocyte-specific proteins. The role that these subtle differences play in determining individual reproductive outcome remains to be determined. PMID:18492745

Seli, Emre; Yaba, Aylin; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D

2008-05-20

177

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

PubMed Central

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5? splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M.; Krajewski, Marcin; Nagel, Roland J.; Ares, Manuel; Holak, Tad A.; Jentsch, Stefan

2013-01-01

178

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.  

PubMed

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier. PMID:21614000

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M; Krajewski, Marcin; Nagel, Roland J; Ares, Manuel; Holak, Tad A; Jentsch, Stefan

2011-05-25

179

Inferring global levels of alternative splicing isoforms using a generative model of microarray data  

Microsoft Academic Search

Motivation: Alternative splicing (AS) is a frequent step in metozoan gene expression whereby the exons of genes are spliced in different combinations to generate multiple isoforms of mature mRNA. AS func- tions to enrich an organism's proteomic complexity and regulates gene expression. Despite its importance, the mechanisms underlying AS and its regulation are not well understood, especially in the context

Ofer Shai; Quaid Morris; Benjamin J. Blencowe; Brendan J. Frey

2006-01-01

180

Modulating roles of amiloride in irradiation-induced antiproliferative effects in glioblastoma multiforme cells involving akt phosphorylation and the alternative splicing of apoptotic genes.  

PubMed

Apoptosis is a key mechanism for enhanced cellular radiosensitivity in radiation therapy. Studies suggest that Akt signaling may play a role in apoptosis and radioresistance. This study evaluates the possible modulating role of amiloride, an antihypertensive agent with a modulating effect to alternative splicing for regulating apoptosis, in the antiproliferative effects induced by ionizing radiation (IR) in glioblastoma multiforme (GBM) 8401 cells. Analysis of cell viability showed that amiloride treatment significantly inhibited cell proliferation in irradiated GBM8401 cells (p<0.05) in a time-dependent manner, especially in cells treated with amiloride with IR post-treatment. In comparison with GBM8401 cells treated with amiloride alone, with GBM8401 cells treated with IR alone, and with human embryonic lung fibroblast control cells (HEL 299), GBM8401 cells treated with IR combined with amiloride showed increased overexpression of phosphorylated Akt, regardless of whether IR treatment was performed before or after amiloride administration. The alternative splicing pattern of apoptotic protease-activating factor-1 (APAF1) in cells treated with amiloride alone, IR alone, and combined amiloride-IR treatments showed more consistent cell proliferation compared to that in other apoptosis-related genes such as baculoviral IAP repeat containing 5 (BIRC5), Bcl-X, and homeodomain interacting protein kinase-3 (HIPK3). In GBM8401 cells treated with amiloride with IR post-treatment, the ratio of prosurvival (-XL,-LC) to proapoptotic (-LN,-S) splice variants of APAF1 was lower than that seen in cells treated with amiloride with IR pretreatment, suggesting that proapoptotic splice variants of APAF1 (APAF1-LN,-S) were higher in the glioblastoma cells treated with amiloride with IR post-treatment, as compared to glioblastoma cells and fibroblast control cells that had received other treatments. Together, these results suggest that amiloride modulates cell radiosensitivity involving the Akt phosphorylation and the alternative splicing of APAF1, especially for the cells treated with amiloride with IR post-treatment. Therefore, amiloride may improve the effectiveness of radiation therapy for GBMs. PMID:23822711

Tang, Jen-Yang; Chang, Hsueh-Wei; Chang, Jan-Gowth

2013-07-03

181

Heart Failure Associated Changes in RNA Splicing of Sarcomere Genes  

PubMed Central

Background Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared to control hearts. Methods and Results We used the Affymetrix exon array to globally evaluate mRNA splicing in LV myocardial RNA from control (n=15) and ischemic cardiomyopathy (ICM) patients. We observed a broad and significant decrease in RNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ICM and control samples, suggesting distinct changes in RNA splicing between groups. RTPCR validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of four key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C gamma (FLNC), was significantly altered in ICM, as well as in dilated cardiomyopathy and aortic stenosis (AS). In AS samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with greater than 98% accuracy. Conclusions Our data indicate that RNA splicing is broadly altered in human heart disease, and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W. K.; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L.; Bisping, Egbert; Pieske, Burkert; Remedios, Cristobal G. dos; Pu, William T.

2011-01-01

182

Exon Array Analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer  

Microsoft Academic Search

BACKGROUND: Treatment of non-small cell lung cancer with novel targeted therapies is a major unmet clinical need. Alternative splicing is a mechanism which generates diverse protein products and is of functional relevance in cancer. RESULTS: In this study, a genome-wide analysis of the alteration of splicing patterns between lung cancer and normal lung tissue was performed. We generated an exon

Wolfram Langer; Florian Sohler; Gabriele Leder; Georg Beckmann; Henrik Seidel; Jörn Gröne; Michael Hummel; Anette Sommer

2010-01-01

183

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron5 of the Sheep SCF Gene: A Novel Splice Variant  

Microsoft Academic Search

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

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

2012-01-01

184

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

PubMed Central

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

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

2013-01-01

185

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

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.

Willing, M.; Deschenes, S. [Univ. of Iowa, Iowa City, IA (United States)

1994-09-01

186

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing  

Microsoft Academic Search

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5? to 3? direction.

Aaron C Goldstrohm; Arno L Greenleaf; Mariano A Garcia-Blanco

2001-01-01

187

A method for identifying alternative or cryptic donor splice sites within gene and mRNA sequences. Comparisons among sequences from vertebrates, echinoderms and other groups  

Microsoft Academic Search

BACKGROUND: As the amount of genome sequencing data grows, so does the problem of computational gene identification, and in particular, the splicing signals that flank exon borders. Traditional methods for identifying splicing signals have been created and optimized using sequences from model organisms, mostly vertebrate and yeast species. However, as genome sequencing extends across the animal kingdom and includes various

Katherine M Buckley; Liliana D Florea; L Courtney Smith

2009-01-01

188

Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing  

Microsoft Academic Search

Exon skipping by alternative splicing and circular RNA formation are proposed to be interrelated events. Since multiple patterns of alternative splicing have been demonstrated in both the 54 and 34 regions of the dystrophin gene, the dystrophin transcript in skeletal muscle cells provides a model system in which this idea is tested. Nine circular RNAs that were expected to result

Agus Surono; Yasuhiro Takeshima; Tri Wibawa; Makoto Ikezawa; Ikuya Nonaka; Masafumi Matsuo

1999-01-01

189

Regulation of Alternative Splicing by the ATP-Dependent DEAD-Box RNA Helicase p72  

Microsoft Academic Search

Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo

Arnd Honig; Didier Auboeuf; Marjorie M. Parker; Bert W. O'Malley; Susan M. Berget

2002-01-01

190

Identification of embryo specific human isoforms using a database of predicted alternative splice forms  

Microsoft Academic Search

Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from a small number of genes. Its study became one of the hot topics in computational genome analysis. The repository EASED (Extended Alternatively Spliced EST Database, http:\\/\\/eased.bioinf.mdc-berlin.de\\/) stores a large collection of splice variants predicted from comparing the human genome against

Heike Pospisil

2006-01-01

191

smg Mutants Affect the Expression of Alternatively Spliced SR Protein mRNAs in Caenorhabditis elegans  

Microsoft Academic Search

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal

Mike Morrison; Kevin S. Harris; Mark B. Roth

1997-01-01

192

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

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

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

2013-05-11

193

Cellular expression and alternative splicing of SLC25A23, a member of the mitochondrial Ca2+-dependent solute carrier gene family.  

PubMed

The transport of metabolites across the inner mitochondrial membrane is mediated by a large superfamily of mitochondrial solute carrier (MSC) proteins. A novel human member of the MSC gene family named SLC25A23, with homologs in mammalian and non-mammalian species has been recently identified together with two close paralogs, SLC25A24 and SLC25A25. These genes encode the human isoforms of the ATP-Mg/Pi carrier described in whole mitochondria. We report here the cellular expression and alternative splicing of SLC25A23. The gene encodes a 468 amino acids polypeptide, named SCaMC-3, with a bipartite structure typical of calcium-binding mitochondrial solute carrier (CaMSC) proteins. The amino-terminal portion harbors three canonical EF-hand calcium-binding domains while the carboxyl-terminal portion of SCaMC-3 has the characteristic features of the MSC superfamily. Northern blot analysis reveals the presence of the transcript in brain, heart, skeletal muscle, liver and small intestine. The SLC25A23 gene undergoes alternative splicing suggesting a modular nature of the encoded product. Three out of four putative protein isoforms lack a significant portion of the third mitochondrial carrier signature. The most common SCaMC-3 isoform shows a mitochondrial subcellular localization when transfected in HeLa cells and is able to bind calcium by Ca(2+)-dependent mobility shift assays. We believe that our study will contribute to a better knowledge of this family of mitochondrial carriers. PMID:15716113

Bassi, Maria Teresa; Manzoni, Marta; Bresciani, Roberto; Pizzo, Maria Teresa; Della Monica, Antonella; Barlati, Sergio; Monti, Eugenio; Borsani, Giuseppe

2005-01-07

194

Validation of Human Alternative Splice Forms Using the EASED Platform and Multiple Splice Site Discriminating Features  

Microsoft Academic Search

We have shown for a dataset of computationally predicted alternative splice sites how inherent information can be utilized\\u000a to validate the predictions by applying statistics on different features typical for splice sites. As a promising splice site\\u000a feature we investigated the frequencies of binding motifs in the context of exonic and intronic splice site flanks and between\\u000a the alternative and

Ralf Bortfeldt; Alexander Herrmann; Heike Pospisil; Stefan Schuster

195

The splice of life: Alternative splicing and neurological disease  

Microsoft Academic Search

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we

B. Kate Dredge; Alexandros D. Polydorides; Robert B. Darnell

2001-01-01

196

EST comparison indicates 38% of human mRNAs contain possible alternative splice forms.  

PubMed

Expressed sequence tag (EST) databases represent a large volume of information on expressed genes including tissue type, expression profile and exon structure. In this study we create an extensive data set of human alternative splicing. We report the analysis of 7867 non-redundant mRNAs, 3011 of which contained alternative splice forms (38% of all mRNAs analysed). From a total of 12572 ESTs 4560 different possible alternative splice forms were detected. Interestingly, 70% of the alternative splice forms correspond to exon deletion events with only 30% exonic insertions. We experimentally verified 19 different splice forms from 16 genes in a total subset of 20 studied; all of the respective genes are of medical relevance. PMID:10828456

Brett, D; Hanke, J; Lehmann, G; Haase, S; Delbrück, S; Krueger, S; Reich, J; Bork, P

2000-05-26

197

Model-based detection of alternative splicing signals  

PubMed Central

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

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

2010-01-01

198

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).  

PubMed

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. PMID:23474544

Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

2013-03-08

199

Epilepsy Caused by an Abnormal Alternative Splicing with Dosage Effect of the SV2A Gene in a Chicken Model  

PubMed Central

Photosensitive reflex epilepsy is caused by the combination of an individual's enhanced sensitivity with relevant light stimuli, such as stroboscopic lights or video games. This is the most common reflex epilepsy in humans; it is characterized by the photoparoxysmal response, which is an abnormal electroencephalographic reaction, and seizures triggered by intermittent light stimulation. Here, by using genetic mapping, sequencing and functional analyses, we report that a mutation in the acceptor site of the second intron of SV2A (the gene encoding synaptic vesicle glycoprotein 2A) is causing photosensitive reflex epilepsy in a unique vertebrate model, the Fepi chicken strain, a spontaneous model where the neurological disorder is inherited as an autosomal recessive mutation. This mutation causes an aberrant splicing event and significantly reduces the level of SV2A mRNA in homozygous carriers. Levetiracetam, a second generation antiepileptic drug, is known to bind SV2A, and SV2A knock-out mice develop seizures soon after birth and usually die within three weeks. The Fepi chicken survives to adulthood and responds to levetiracetam, suggesting that the low-level expression of SV2A in these animals is sufficient to allow survival, but does not protect against seizures. Thus, the Fepi chicken model shows that the role of the SV2A pathway in the brain is conserved between birds and mammals, in spite of a large phylogenetic distance. The Fepi model appears particularly useful for further studies of physiopathology of reflex epilepsy, in comparison with induced models of epilepsy in rodents. Consequently, SV2A is a very attractive candidate gene for analysis in the context of both mono- and polygenic generalized epilepsies in humans.

Douaud, Marine; Feve, Katia; Pituello, Fabienne; Gourichon, David; Boitard, Simon; Leguern, Eric; Coquerelle, Gerard; Vieaud, Agathe; Batini, Cesira; Vignal, Alain; Tixier-Boichard, Michele; Pitel, Frederique

2011-01-01

200

The evolution of alternative splicing in the Pax family: the view from the Basal chordate amphioxus.  

PubMed

Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses during vertebrate evolution has resulted in two human genes for each of the Pax1/9, Pax3/7, and Pax4/6 subfamilies and three for the Pax2/5/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate Pax transcripts from all four subfamilies, and many splice forms are known to have functional importance. To better understand the evolution of alternative splicing within the Pax family, we systematically surveyed transcripts of the four amphioxus Pax genes. We have found alternative splicing in every gene. Comparisons with vertebrates suggest that the number of alternative splicing events per gene has not decreased following duplication; there are comparable levels in the four amphioxus Pax genes as in each gene of the equivalent vertebrate families. Thus, the total number of isoforms for the nine vertebrate genes is considerably higher than for the four amphioxus genes. Most alternative splicing events appear to have arisen since the divergence of amphioxus and vertebrate lineages, suggesting that differences in alternative splicing could account for divergent functions of the highly conserved Pax genes in both lineages. However, several events predicted to dramatically alter known functional domains are conserved between amphioxus and vertebrates, suggestive of a common chordate function. Our results, together with previous studies of vertebrate Pax genes, support the theory that alternative splicing impacts functional motifs more than gene duplication followed by divergence. PMID:18473110

Short, Stephen; Holland, Linda Z

2008-05-14

201

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

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.

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

1988-11-01

202

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

PubMed Central

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.

Sheives, Paul; Lynch, Kristen W

2002-01-01

203

Technologies for the global discovery and analysis of alternative splicing.  

PubMed

During the past approximately 20 years, studies on alternative splicing (AS) have largely been directed at the identification and characterization of factors and mecha nisms responsible for the control of splice site selection, using model substrates and on a case by case basis. These studies have provided a wealth of information on the factors and interactions that control formation of the spliceosome. However, relatively little is known about the global regulatory properties of AS. Important questions that need to be addressed are: which exons are alternatively spliced and under which cellular contexts, what are the functional roles of AS events in different cellular contexts, and how are AS events controlled and coordinated with each other and with other levels of gene regulation to achieve cell- and development-specific functions. During the past several years, new technologies and experimental strategies have provided insight into these questions. For example, custom microarrays and data analysis tools are playing a prominent role in the discovery and analysis of splicing regulation. Moreover, several non-microarray-based technologies are emerging that will likely further fuel progress in this area. This review focuses on recent advances made in the development and application of high-throughput methods to study AS. PMID:18380341

Calarco, John A; Saltzman, Arneet L; Ip, Joanna Y; Blencowe, Benjamin J

2007-01-01

204

REMAS: a new regression model to identify alternative splicing events from exon array data  

Microsoft Academic Search

BACKGROUND: Alternative splicing (AS) is an important regulatory mechanism for gene expression and protein diversity in eukaryotes. Previous studies have demonstrated that it can be causative for, or specific to splicing-related diseases. Understanding the regulation of AS will be helpful for diagnostic efforts and drug discoveries on those splicing-related diseases. As a novel exon-centric microarray platform, exon array enables a

Hao Zheng; Xingyi Hang; Ji Zhu; Minping Qian; Wubin Qu; Chenggang Zhang; Minghua Deng

2009-01-01

205

Cryptic splice sites and split genes  

PubMed Central

We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes.

Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M.; Tatusova, Tatiana; Dibb, Nick J.

2011-01-01

206

Cryptic splice sites and split genes.  

PubMed

We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes. PMID:21470962

Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M; Tatusova, Tatiana; Dibb, Nick J

2011-04-05

207

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

PubMed Central

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.

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

2013-01-01

208

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

PubMed

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

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

2013-10-09

209

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

PubMed Central

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.

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

2012-01-01

210

Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, Nilaparvata lugens and Laodelphax striatellus (Hemiptera: Delphacidae).  

PubMed

Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family. Most insects possess two CHS genes (CHS1 and CHS2); however, genome and transcriptome searches showed that N. lugens possibly possess only CHS1 in both databases. Two transcript variants (CHS1a and CHS1b) resulting from exclusively alternative splicing (exon 19a or 19b in N. lugens) were identified for each of the two rice planthopper CHS1s. Gene structure comparison using the genomes that are currently sequenced showed that the CHS1 genes in all insects except Acyrthosiphon pisum have two transcript variants. Transcription of NlCHS1a reached its highest level just after molting, whereas NlCHS1b reached its highest expression level 1-2 days before molting. Injection of the N. lugens nymphs with double-strand RNA (dsRNA) of CHS1, CHS1a and CHS1b reduced the corresponding variant transcript levels and exhibited subsequent phenotypes. Silencing of CHS1 and CHS1a resulted in elongated distal wing pads and the "wasp-waisted" or crimpled cuticle phenotypes and eventually died, whereas the phenotypes caused by injection of NlCHS1b dsRNA seem not so obvious although slightly increased mortality was observed. Our results suggest that N. lugens likely lacks CHS2 and CHS1 may be efficient target gene for RNAi-based N. lugens control. PMID:22634163

Wang, Ying; Fan, Hai-Wei; Huang, Hai-Jian; Xue, Jian; Wu, Wen-Juan; Bao, Yan-Yuan; Xu, Hai-Jun; Zhu, Zeng-Rong; Cheng, Jia-An; Zhang, Chuan-Xi

2012-05-24

211

Prediction of alternatively skipped exons and splicing enhancers from exon junction arrays  

PubMed Central

Background Alternative splicing of exons in a pre-mRNA transcript is an important mechanism which contributes to protein diversity in human. Arrays for detecting alternative splicing are available using several different probe designs, including those based on exon-junctions. In this work, we introduce a new method for predicting alternatively skipped exons from exon-junction arrays. Predictions based on our method are compared against controls and their sequences are analyzed to identify motifs important for regulating alternative splicing. Results Our comparison of several alternative methods shows that an exon-skipping score based on neighboring junctions best discriminates between positive and negative controls. Sequence analysis of our predicted exons confirms the presence of known splicing regulatory sequences. In addition, we also derive a set of development-related alternatively spliced genes based on fetal versus adult tissue comparisons and find that our predictions are consistent with their functional annotations. Ab initio motif finding algorithms are applied to identify several motifs that may be relevant for splicing during development. Conclusion This work describes a new method for analyzing exon-junction arrays, identifies sequence motifs that are specific for alternative and constitutive splicing and suggests a role for several known splicing factors and their motifs in developmental regulation.

Kechris, Katerina; Yang, Yee Hwa; Yeh, Ru-Fang

2008-01-01

212

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

PubMed Central

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.

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

2011-01-01

213

APPRIS: annotation of principal and alternative splice isoforms  

PubMed Central

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L.

2013-01-01

214

New Way of Regulating Alternative Splicing in Retroviruses: the Promoter Makes a Difference?  

PubMed Central

Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.

Bohne, Jens; Schambach, Axel; Zychlinski, Daniela

2007-01-01

215

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection  

PubMed Central

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.

Takeuchi, Akihide; Hosokawa, Motoyasu; Nojima, Takayuki; Hagiwara, Masatoshi

2010-01-01

216

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

PubMed Central

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

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

2011-01-01

217

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A.  

PubMed

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed. PMID:23048131

Guerriero, Gea; Spadiut, Oliver; Kerschbamer, Christine; Giorno, Filomena; Baric, Sanja; Ezcurra, Inés

2012-10-01

218

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

PubMed

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

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

2011-06-30

219

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

PubMed Central

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

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

2011-01-01

220

Intron mis-splicing: no alternative?  

PubMed Central

A recent report reveals widespread mis-splicing of RNA transcripts in eukaryotes, with mis-spliced RNA destroyed by nonsense-mediated mRNA decay. This striking inefficiency deepens the mystery of the proliferation and persistence of introns.

Roy, Scott William; Irimia, Manuel

2008-01-01

221

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

PubMed Central

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 N279K) or destroyed (mutation ?280K), 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 (L284L), 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.

D'Souza, Ian; Poorkaj, Parvoneh; Hong, Ming; Nochlin, David; Lee, Virginia M.-Y.; Bird, Thomas D.; Schellenberg, Gerard D.

1999-01-01

222

A General Definition and Nomenclature for Alternative Splicing Events  

PubMed Central

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.

Guigo, Roderic

2008-01-01

223

Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status  

PubMed Central

Background Neuroblastoma (NB) tumors are well known for their pronounced clinical and molecular heterogeneity. The global gene expression and DNA copy number alterations have been shown to have profound differences in tumors of low or high stage and those with or without MYCN amplification. RNA splicing is an important regulatory mechanism of gene expression, and differential RNA splicing may be associated with the clinical behavior of a tumor. Methods In this study, we used exon array profiling to investigate global alternative splicing pattern of 47 neuroblastoma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4- and 4+). The ratio of exon-level expression to gene-level expression was used to detect alternative splicing events, while the gene-level expression was applied to characterize whole gene expression change. Results Principal component analysis (PCA) demonstrated distinct splicing pattern in three groups of samples. Pairwise comparison identified genes with splicing changes and/or whole gene expression changes in high stage tumors. In stage 4- compared with stage 1- tumors, alternatively spliced candidate genes had little overlap with genes showing whole gene expression changes, and most of them were involved in different biological processes. In contrast, a larger number of genes exhibited either exon-level splicing, gene-level expression or both changes in stage 4+ versus stage 1- tumors. Those biological processes involved in stage 4- tumors were disrupted to a greater extent by both splicing and transcription regulations in stage 4+ tumors. Conclusions Our results demonstrated a significant role of alternative splicing in high stage neuroblastoma, and suggested a MYCN-associated splicing regulation pathway in stage 4+ tumors. The identification of differentially spliced genes and pathways in neuroblastoma tumors of different stages and molecular subtypes may be important to the understanding of cancer biology and the discovery of diagnostic markers or therapeutic targets in neuroblastoma.

2011-01-01

224

Auto and Cross-Regulation of the hnRNP L Proteins by Alternative Splicing  

Microsoft Academic Search

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA- repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay

Oliver Rossbach; Lee-Hsueh Hung; Silke Schreiner; Inna Grishina; Monika Heiner; Jingyi Hui; Albrecht Bindereif

2009-01-01

225

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

Microsoft Academic Search

Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles

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

2010-01-01

226

Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins  

PubMed Central

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ?45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

Gurskaya, Nadya G.; Staroverov, Dmitry B.; Zhang, Lijuan; Fradkov, Arkady F.; Markina, Nadezhda M.; Pereverzev, Anton P.; Lukyanov, Konstantin A.

2012-01-01

227

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

PubMed

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

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

2013-04-05

228

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.  

PubMed Central

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5

Song, I; Brown, D R; Wiltshire, R N; Gantz, I; Trent, J M; Yamada, T

1993-01-01

229

BIPASS: BioInformatics Pipeline Alternative Splicing Services  

PubMed Central

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

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

2007-01-01

230

Evolution of exon-intron structure and alternative splicing.  

PubMed

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

Koralewski, Tomasz E; Krutovsky, Konstantin V

2011-03-25

231

RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing  

PubMed Central

Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

Guo, Wei; Schafer, Sebastian; Greaser, Marion L.; Radke, Michael H.; Liss, Martin; Govindarajan, Thirupugal; Maatz, Henrike; Schulz, Herbert; Li, Shijun; Parrish, Amanda M.; Dauksaite, Vita; Vakeel, Padmanabhan; Klaassen, Sabine; Gerull, Brenda; Thierfelder, Ludwig; Regitz-Zagrosek, Vera; Hacker, Timothy A.; Saupe, Kurt W.; Dec, G. William; Ellinor, Patrick T.; MacRae, Calum A.; Spallek, Bastian; Fischer, Robert; Perrot, Andreas; Ozcelik, Cemil; Saar, Kathrin; Hubner, Norbert; Gotthardt, Michael

2013-01-01

232

A novel computational method for the identification of plant alternative splice sites.  

PubMed

Alternative splicing (AS) increases protein diversity by generating multiple transcript isoforms from a single gene in higher eukaryotes. Up to 48% of plant genes exhibit alternative splicing, which has proven to be involved in some important plant functions such as the stress response. A hybrid feature extraction approach which combing the position weight matrix (PWM) with the increment of diversity (ID) was proposed to represent the base conservative level (BCL) near splice sites and the similarity level of two datasets, respectively. Using the extracted features, the support vector machine (SVM) was applied to classify alternative and constitutive splice sites. By the proposed algorithm, 80.8% of donor sites and 85.4% of acceptor sites were correctly classified. It is anticipated that the novel computational method is promising for the identification of AS sites in plants. PMID:23313482

Cui, Ying; Han, Jiuqiang; Zhong, Dexing; Liu, Ruiling

2013-01-09

233

An Alternative Splicing Network Links Cell Cycle Control to Apoptosis  

PubMed Central

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.

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

2010-01-01

234

Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis  

Microsoft Academic Search

A cDNA encoding a putative non-membrane bound prolactin receptor was amplified by RT-PCR from red deer (Cervus elaphus) testis. Sequence analysis suggests that the testicular cDNA is generated by alternative splicing resulting in the deletion of exons 7 and 8, which code for: (a) the final 53 aa of the extracellular domain of the receptor including the fifth conserved cysteine

H N Jabbour; L A Clarke; T Bramley; M-C Postel-Vina; P A Kelly; M Eder

1998-01-01

235

Intermolecular domain swapping induces intein-mediated protein alternative splicing.  

PubMed

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference). PMID:23974115

Aranko, A Sesilja; Oeemig, Jesper S; Kajander, Tommi; Iwaï, Hideo

2013-08-25

236

A functional alternative splicing mutation in human tryptophan hydroxylase-2  

PubMed Central

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.

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

2011-01-01

237

Genome-Wide Analysis of Alternative Splicing during Dendritic Cell Response to a Bacterial Challenge  

PubMed Central

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luis

2013-01-01

238

Alternative splicing and evolution: diversification, exon definition and function  

Microsoft Academic Search

Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how

Galit Lev-Maor; Gil Ast; Hadas Keren

2010-01-01

239

Multiple transcripts of the murine immunoglobulin ? membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites  

Microsoft Academic Search

The human C? gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed ? mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary ? chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in

Shubha Anand; Facundo D. Batista; Tatiana Tkach; Dimitar G. Efremov; Oscar R. Burrone

1997-01-01

240

Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm.  

PubMed

Alternative splicing, first discovered in the 1970s, has emerged as one of the key generators of proteomic diversity. Not surprisingly, alternative splicing is increasingly linked to the etiology of cancer. This is illustrated by vascular endothelial growth factor (VEGF), the dominant angiogenic factor. Recently, an antiangiogenic family of VEGF isoforms was discovered, and termed VEGF(xxx)b. VEGF(xxx)b isoforms arise from an alternative 3' splice site in exon 8, and differ by a mere six amino acids at the C-terminus. These alternative six amino acids radically change the functional properties of VEGF. VEGF(xxx)b isoform expression is regulated in human tissues and development, and disregulated in many pathological states including cancer. Understanding what regulates VEGF(xxx)b alternative splicing, and therefore the balance of pro- and antiangiogenic isoforms is of great importance and will be explored in detail over the next few years. PMID:17027147

Ladomery, Michael R; Harper, Steven J; Bates, David O

2006-10-05

241

Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox.  

PubMed Central

Retinoic acid (RA), the natural acidic derivative of vitamin A, can modulate the expression of specific genes and can induce some cell types, such as the murine F9 teratocarcinoma stem cell line, to differentiate in culture. As an initial step toward understanding the molecular mechanism(s) by which RA exerts these effects, we previously isolated cDNA clones for a gene, ERA-1, which has the characteristics of an early, direct target for RA. We demonstrated that RA causes a rapid, dose-dependent, and protein synthesis-independent expression of the ERA-1 gene (G. J. LaRosa and L. J. Gudas, Proc. Natl. Acad. Sci. USA 85:329-333, 1988). We now report the full-length cDNA sequence and the further characterization of this gene. The data indicate that the RA-induced 2.2- to 2.4-kilobase ERA-1 RNA species that we previously detected consists of two alternately spliced messages. One mRNA encodes a protein with a predicted mass of about 36 kilodaltons (kDa) that possesses the Hox 1.6 homeobox domain. The other mRNA encodes a truncated protein of about 15 kDa which is identical to the 36-kDa protein for 114 amino acids at the amino-terminal end but which lacks the homeobox amino acid sequence. The RA-associated increase in the ERA-1 mRNA level does not appear to be due to message stabilization, suggesting that the response is at the level of transcription. By Northern (RNA) blot analysis, the usual 2.2- to 2.4-kilobase mRNA species was also rapidly expressed in P19 teratocarcinoma cells during their differentiation to fibroblastic cells in response to RA and was detected in day 10.5 and day 13.5 mouse embryos. This result indicates that the expression of this gene is not limited to the endodermal differentiation of F9 cells. Images

LaRosa, G J; Gudas, L J

1988-01-01

242

Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.  

PubMed

Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters. PMID:3422427

Chretien, S; Dubart, A; Beaupain, D; Raich, N; Grandchamp, B; Rosa, J; Goossens, M; Romeo, P H

1988-01-01

243

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

PubMed Central

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.

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

2010-01-01

244

Riboswitch Control of Gene Expression in Plants by Splicing and Alternative 3' End Processing of mRNAs  

Microsoft Academic Search

The most widespread riboswitch class, found in organisms from all three domains of life, is responsive to the vitamin B1 derivative thiamin pyrophosphate (TPP). We have established that a TPP-sensing riboswitch is present in the 39 untranslated region (UTR) of the thiamin biosynthetic gene THIC of all plant species examined. The THIC TPP riboswitch controls the formation of transcripts with

Andreas Wachter; Meral Tunc-Ozdemir; Beth C. Grove; Pamela J. Green; David K. Shintani; R. R. Breaker

2007-01-01

245

The Apoptosis-Promoting Factor TIA-1 Is a Regulator of Alternative Pre-mRNA Splicing  

Microsoft Academic Search

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5? splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5? splice site recognition by U1 snRNP. This activity is critical for activation

Patrik Förch; Oscar Puig; Nancy Kedersha; Concepción Martínez; Sander Granneman; Bertrand Séraphin; Paul Anderson; Juan Valcárcel

2000-01-01

246

Sex Determination in Insects: a binary decision based on alternative splicing  

PubMed Central

The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks.

Salz, Helen K.

2011-01-01

247

Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants  

PubMed Central

Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions.

Chen, Ping; Lepikhova, Tatiana; Hu, Yizhou; Monni, Outi; Hautaniemi, Sampsa

2011-01-01

248

Quantitative and evolutionary biology of alternative splicing: how changing the mix of alternative transcripts affects phenotypic plasticity and reaction norms  

Microsoft Academic Search

Alternative splicing (AS) of pre-messenger RNA is a common phenomenon that creates different transcripts from a single gene, and these alternative transcripts affect phenotypes. The majority of AS research has examined tissue and developmental specificity of expression of particular AS transcripts, how this specificity affects cell function, and how aberrant AS is related to disease. Few studies have examined quantitative

J H Marden

2008-01-01

249

Differentially Expressed Messenger RNA Isoforms of the Human Estrogen Receptor  Gene Are Generated by Alternative Splicing and Promoter Usage  

Microsoft Academic Search

The isolation and characterization of several new human estrogen receptor-a (hERa) mRNAs are de- scribed. Together with those previously identified, they give rise to a total of six hERa mRNA isoforms (A-F hERa mRNAs). Produced from a single hERa gene by multiple promoter usage, all these tran- scripts encode a common protein but differ in their 5*-untranslated region as a

Gilles Flouriot; Caroline Griffin; Maryrose Kenealy; Vera Sonntag-Buck; Frank Gannon

1998-01-01

250

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

PubMed Central

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.

Barberan-Soler, Sergio; Ragle, James Matthew

2013-01-01

251

An alternatively spliced form of Met receptor is tumorigenic.  

PubMed

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type- Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer. PMID:17079873

Lee, Jae-Ho; Gao, Chong Feng; Lee, Chong Chou; Kim, Myung Deok; Vande Woude, George F

2006-10-31

252

Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.  

PubMed Central

Two overlapping human genomic clones that encode a short-chain collagen, designated alpha 1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span approximately equal to 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the alpha 1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type alpha 1(XIII)-chain gene. Images

Tikka, L; Pihlajaniemi, T; Henttu, P; Prockop, D J; Tryggvason, K

1988-01-01

253

Distinct functions of acj6 splice forms in odor receptor gene choice  

PubMed Central

Individual olfactory receptor neurons (ORNs) selectively express one or a small number of odor receptors from among a large receptor repertoire. The expression of an odor receptor dictates the odor response spectrum of the ORN. The process of receptor gene choice relies in part on a combinatorial code of transcription factors. In Drosophila, the POU domain transcription factor Acj6 is one element of the transcription factor code. In acj6 null mutants, many ORNs do not express an appropriate odor receptor gene and thus are not correctly specified. We find that acj6 is alternatively spliced to yield many structurally distinct transcripts in the olfactory organs. We generate flies that express single splice forms of acj6 in an acj6? background. We find that different splice forms are functionally distinct; they differ in their abilities to specify ORN identities. Some individual splice forms can fully rescue the specification of some ORNs. Individual splice forms can function both positively and negatively in receptor gene regulation. ORNs differ in their requirements for splice forms; some are not fully rescued by any single splice form tested, suggesting that some ORNs may require the combinatorial action of multiple splice forms. Late expression of some acj6 splice forms is sufficient to rescue some ORN classes, consistent with a direct role for Acj6 isoforms in receptor gene expression. The results indicate that alternative splicing may add another level of richness to the regulatory code that underlies the process of odor receptor gene choice.

Bai, Lei; Carlson, John R.

2010-01-01

254

Regulation of telomerase alternative splicing: a target for chemotherapy.  

PubMed

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

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

2013-04-04

255

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila  

Microsoft Academic Search

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting

Matthew L. Johnson; Alexis A. Nagengast; Helen K. Salz

2010-01-01

256

Alternative Splice Variant of ?-Calmodulin-Dependent Protein Kinase II Alters Activation by Calmodulin  

Microsoft Academic Search

Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca2+-signaling pathways. These family members are expressed from four highly homologous genes (?, ?, ?, and ?) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I–XI, are differentially expressed in various cell types.

Ann P. Kwiatkowski; James M. McGill

2000-01-01

257

Precise and Parallel Characterization of Coding Polymorphisms, Alternative Splicing, and Modifications in Human Proteins by  

Microsoft Academic Search

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high se- quence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of

Mass Spectrometry; Michael J. Roth; Andrew J. Forbes; Michael T. Boyne II; Yong-Bin Kim; Dana E. Robinson; Neil L. Kelleher

258

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.  

PubMed

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming. PMID:23739326

Han, Hong; Irimia, Manuel; Ross, P Joel; Sung, Hoon-Ki; Alipanahi, Babak; David, Laurent; Golipour, Azadeh; Gabut, Mathieu; Michael, Iacovos P; Nachman, Emil N; Wang, Eric; Trcka, Dan; Thompson, Tadeo; O'Hanlon, Dave; Slobodeniuc, Valentina; Barbosa-Morais, Nuno L; Burge, Christopher B; Moffat, Jason; Frey, Brendan J; Nagy, Andras; Ellis, James; Wrana, Jeffrey L; Blencowe, Benjamin J

2013-06-05

259

Alternative splicing modulation by a LAMMER kinase impinges on developmental and transcriptome expression.  

PubMed

Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, including signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a conserved LAMMER motif. We demonstrate in BY-2 tobacco cells a novel role for the LAMMER motif in the maintenance of proper subnuclear localization. Furthermore, high expression of the LAMMER kinase in Arabidopsis plants modulated the alternative splicing of specific endogenous genes and resulted in abnormal plant development and a novel transcriptome profile. A prominent feature was the upregulation of genes that play a role in protein turnover, suggesting a moderating function for these gene products in the control of alternative splicing events. Together, these results demonstrate alternative splicing modulation as a result of phosphorylation activity, providing an opportunity to study its global effect on the plasticity of plant development and gene expression at the organism level. PMID:12671088

Savaldi-Goldstein, Sigal; Aviv, Dvora; Davydov, Olga; Fluhr, Robert

2003-04-01

260

Functional Manipulations of Acetylcholinesterase Splice Variants Highlight Alternative Splicing Contributions to Murine Neocortical Development  

Microsoft Academic Search

Proliferation and differentiation of mammalian central nervous system progenitor cells involve concertedly controlled transcrip- tional and alternative splicing modulations. Searching for the developmental implications of this programming, we manipulated specific acetylcholinesterase (AChE) splice variants in the embry- onic mouse brain. In wild type mice, 'synaptic' AChE-S appeared in migrating neurons, whereas the C-terminus cleaved off the stress-induced AChE-R variant associated

Amir Dori; Jonathan Cohen; William F. Silverman

2005-01-01

261

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

PubMed

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

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

2010-05-04

262

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing? † ‡  

PubMed Central

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.

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

2010-01-01

263

TassDB2 - A comprehensive database of subtle alternative splicing events  

Microsoft Academic Search

BACKGROUND: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus,

Rileen Sinha; Thorsten Lenser; Niels Jahn; Ulrike Gausmann; Swetlana Friedel; Karol Szafranski; Klaus Huse; Philip Rosenstiel; Jochen Hampe; Stefan Schuster; Michael Hiller; Rolf Backofen; Matthias Platzer

2010-01-01

264

Genome-wide detection of condition-sensitive alternative splicing in Arabidopsis roots.  

PubMed

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

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

2013-06-04

265

Emetine regulates the alternative splicing of caspase 9 in tumor cells.  

PubMed

Exons 3 to 6 in the caspase 9 gene undergo alternative splicing in which the larger caspase 9 splice variant promotes apoptosis, in contrast to the dominant negative anti-apoptotic splice variant, the smaller caspase 9b. In this study, the regulation of the alternative splicing of caspase 9 pre-mRNA was examined in response to Emetine. Treatment of C33A cells, breast cancer MCF-7 cells and MCF-7/Adr cells with Emetine dihydrochloride upregulated the level of smaller caspase 9b mRNA and concomitantly decreased the mRNA level of larger caspase 9 in a dose- and time-dependent manner, indicating that Emetine desensitizes C33A, MCF-7 and MCF-7/Adr to cell death. In contrast, treatment of PC3 cells, a prostate cancer cell line, manifested an opposite effect: a greater production of the larger caspase 9 mRNA with a concomitant decrease of caspase 9b mRNA. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) blocked Emetine-induced alternative splicing in cells, in contrast to okadaic acid, a specific inhibitor of PP2A, demonstrating a PP1-mediated mechanism. These results suggest that the various splicing patterns of the caspase 9 gene that are regulated by chemotherapy reagents may contribute to the resistance or sensitization of the tumors to other cell death inducers. PMID:22848307

Pan, Danmin; Boon-Unge, Kritsanapol; Govitrapong, Piyarat; Zhou, Jianhua

2011-08-29

266

SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.  

PubMed

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5' and 3' splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C Robertson; Xu, Xiaodong; Ma, Ligeng

2012-08-31

267

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45.  

PubMed

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing. PMID:17589525

Corsini, Lorenzo; Bonnal, Sophie; Bonna, Sophie; Basquin, Jerome; Hothorn, Michael; Scheffzek, Klaus; Valcárcel, Juan; Sattler, Michael

2007-06-24

268

The Evolution of Alternative Splicing in the Pax Family: The View from the Basal Chordate Amphioxus  

Microsoft Academic Search

Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses\\u000a during vertebrate evolution has resulted in two human genes for each of the Pax1\\/9, Pax3\\/7, and Pax4\\/6 subfamilies and three for the Pax2\\/5\\/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate\\u000a Pax transcripts from all four

Stephen Short; Linda Z. Holland

2008-01-01

269

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

SciTech Connect

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.

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

270

Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms  

PubMed Central

Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the ?-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.

2011-01-01

271

Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors  

PubMed Central

RNA splicing is required to remove introns from pre-mRNA and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating SR splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here we tested the impact of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike to other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine, and are not simply due to reduced Top1 activity as TOP1 downregulation by siRNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by DRB blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc.

Solier, Stephanie; Barb, Jennifer; Zeeberg, Barry R.; Varma, Sudhir; Ryan, Mike C; Kohn, Kurt W.; Weinstein, John N.; Munson, Peter J.; Pommier, Yves

2010-01-01

272

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

SciTech Connect

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

Ambartsumyan, N.S. [Danish Society for Cancer Research, Copenhagen (Denmark)]|[Institute of Molecular Biology, Yerevan (Armenia); Grigorian, M.S.; Lukanidin, E.M. [Danish Society for Cancer Research, Copenhagen (Denmark)] [and others

1995-09-01

273

Fine-Scale Variation and Genetic Determinants of Alternative Splicing across Individuals  

PubMed Central

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases.

Coulombe-Huntington, Jasmin; Lam, Kevin C. L.; Dias, Christel; Majewski, Jacek

2009-01-01

274

Regulation of the Ras-MAPK and PI3K-mTOR Signalling Pathways by Alternative Splicing in Cancer  

PubMed Central

Alternative splicing is a fundamental step in regulation of gene expression of many tumor suppressors and oncogenes in cancer. Signalling through the Ras-MAPK and PI3K-mTOR pathways is misregulated and hyperactivated in most types of cancer. However, the regulation of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing is less well established. Recent studies have shown the contribution of alternative splicing regulation of these signalling pathways which can lead to cellular transformation, cancer development, and tumor maintenance. This review will discuss findings in the literature which describe new modes of regulation of components of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing. We will also describe the mechanisms by which signals from extracellular stimuli can be communicated to the splicing machinery and to specific RNA-binding proteins that ultimately control exon definition events.

Bonomi, Serena

2013-01-01

275

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

PubMed Central

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

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

2012-01-01

276

The polypyrimidine tract binding protein regulates desaturase alternative splicing and PUFA composition.  

PubMed

The ?6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling. PMID:21980057

Reardon, Holly T; Park, Woo Jung; Zhang, Jimmy; Lawrence, Peter; Kothapalli, Kumar S D; Brenna, J Thomas

2011-10-06

277

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

PubMed Central

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

2012-01-01

278

Alternative splicing caused by lentiviral integration in the human genome.  

PubMed

Gene transfer vectors derived from murine oncoretroviruses or human lentiviruses are widely used in human gene therapy. Integration of these vectors in the human genome may, however, have genotoxic effects, caused by deregulation of gene expression at the transcriptional or posttranscriptional level. In particular, integration of lentiviral vectors within transcribed genes has a significant potential to affect their expression by interfering with splicing and polyadenylation of primary transcripts. Aberrant splicing is caused by the usage of both constitutive and cryptic splice sites located in the retroviral backbone as well as in the gene expression cassettes. We describe a set of simple methods that allow the identification of chimeric transcripts generated by the insertion of a lentiviral vector within genes and the evaluation of their relative abundance. Identification of the splice sites, either constitutive or cryptic, that are frequently used by the cell splicing machinery within a given vector provides a useful resource to attempt recoding of the vector with the objective of reducing its potential genotoxicity in a clinical context. PMID:22365773

Moiani, Arianna; Mavilio, Fulvio

2012-01-01

279

Emerging Roles of Alternative Pre-mRNA Splicing Regulation in Neuronal Development and Function  

PubMed Central

Alternative pre-mRNA splicing has the potential to greatly diversify the repertoire of transcripts in multicellular organisms. Increasing evidence suggests that this expansive layer of gene regulation plays a particularly important role in the development and function of the nervous system, one of the most complex organ systems found in nature. In this review, we highlight recent studies that continue to emphasize the influence and contribution of alternative splicing regulation to various aspects of neuronal development in addition to its role in the mature nervous system.

Norris, Adam D.; Calarco, John A.

2012-01-01

280

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing.  

PubMed

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (?530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (?) located near a 5' splice site, which greatly increases use of this 5' splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches. PMID:23376932

Li, Sanshu; Breaker, Ronald R

2013-02-01

281

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing  

PubMed Central

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (?530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (?) located near a 5? splice site, which greatly increases use of this 5? splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Li, Sanshu; Breaker, Ronald R.

2013-01-01

282

cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene.  

PubMed

The human ryanodine receptor type 3 (RYR3) was cloned from a fetal brain cDNA library and its complete sequence was determined (EMBL accession number AJ001515). The sequenced cDNA spanned 15,564 bp and contained an open reading frame of 14,613 bp. The corresponding protein consisted of 4870 amino acids with a calculated molecular mass of 552 kDa. Amino acid sequence identities to the RYR3 proteins from rabbit, mink, and chicken were 96%, 95%, and 83% respectively. A previously unidentified alternative splice site was detected generating a transcript that lacked bases 11,569-11,650 and encoded a truncated protein. PMID:9515741

Leeb, T; Brenig, B

1998-02-27

283

Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing  

PubMed Central

Little is known about the processes that enable influenza A viruses to jump into new host species. Here we show that the non-structural protein1 nucleotide substitution, A374G, encoding the D125G(GAT?GGT) mutation, which evolved during the adaptation of a human virus within a mouse host, activates a novel donor splice site in the non-structural gene, hence producing a novel influenza A viral protein, NS3. Using synonymous 125G mutations that do not activate the novel donor splice site, NS3 was shown to provide replicative gain-of-function. The protein sequence of NS3 is similar to NS1 protein but with an internal deletion of a motif comprised of three antiparallel ?-strands spanning codons 126 to 168 in NS1. The NS1-125G(GGT) codon was also found in 33 natural influenza A viruses that were strongly associated with switching from avian to mammalian hosts, including human, swine and canine populations. In addition to the experimental human to mouse switch, the NS1-125G(GGT) codon was selected on avian to human transmission of the 1997 H5N1 and 1999 H9N2 lineages, as well as the avian to swine jump of 1979 H1N1 Eurasian swine influenza viruses, linking the NS1 125G(GGT) codon with host adaptation and switching among multiple species.

Selman, Mohammed; Dankar, Samar K; Forbes, Nicole E; Jia, Jian-Jun; Brown, Earl G

2012-01-01

284

b Subunits Modulate Alternatively Spliced, Large Conductance, Calcium-Activated Potassium Channels of Avian Hair Cells  

Microsoft Academic Search

Electrical tuning confers frequency selectivity onto sensory hair cells in the auditory periphery of frogs, turtles, and chicks. The resonant frequency is determined in large part by the number and kinetics of large conductance, calcium-activated potas- sium (BK) channels. BK channels in hair cells are encoded by the alternatively spliced slo gene and may include an accessory b subunit. Here

Krishnan Ramanathan; Timothy H. Michael; Paul A. Fuchs

285

Functional coordination of alternative splicing in the mammalian central nervous system  

Microsoft Academic Search

BACKGROUND: Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood. RESULTS: Using quantitative AS microarray profiling, we

Matthew Fagnani; Yoseph Barash; Joanna Y Ip; Christine Misquitta; Qun Pan; Arneet L Saltzman; Ofer Shai; Leo Lee; Aviad Rozenhek; Naveed Mohammad; Sandrine Willaime-Morawek; Tomas Babak; Wen Zhang; Timothy R Hughes; Derek van der Kooy; Brendan J Frey; Benjamin J Blencowe

2007-01-01

286

Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila  

Microsoft Academic Search

The Drosophila melanogaster transcription factor Lola (longitudinals lacking) is a pivotal regulator of neural wiring that sets the precise expression levels of proteins that execute specific axon guidance decisions. Lola has a zinc finger DNA binding domain and a BTB (for Broad-complex, Tramtrack and Bric a brac) dimerization motif. We now show that alternative splicing of the lola gene creates

Scott Goeke; Elizabeth A. Greene; Paul K. Grant; Michael A. Gates; Daniel Crowner; Toshiro Aigaki; Edward Giniger

2003-01-01

287

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged  

PubMed Central

Alternative splicing of mRNA precursors is a nearly ubiquitous and extremely flexible point of gene control in humans. It provides cells with the opportunity to create protein isoforms of differing, even opposing, functions from a single gene. Cancer cells often take advantage of this flexibility to produce proteins that promote growth and survival. Many of the isoforms produced in this manner are developmentally regulated and are preferentially re-expressed in tumors. Emerging insights into this process indicate that pathways that are frequently deregulated in cancer often play important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.

David, Charles J.; Manley, James L.

2010-01-01

288

Assessment of orthologous splicing isoforms in human and mouse orthologous genes  

PubMed Central

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

2010-01-01

289

Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5? exons of protein 4.1R gene  

PubMed Central

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3? splice sites (ss) of exon 2?/2. Here, we demonstrate that exon 1 and 2?/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3? ss and exon 1B to the proximal 3? ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2?/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2?/2 junction CAGAGAA, a sequence that overlaps the distal U2AF35-binding 3? ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3? ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3? ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

Cho, Aeri; Norton, Stephanie; Liu, Eva S.; Park, Jennie; Zhou, Anyu; Munagala, Indira D.; Ou, Alexander C.; Yang, Guang; Wickrema, Amittha; Tang, Tang K.; Benz, Edward J.

2009-01-01

290

Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program  

SciTech Connect

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.

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

291

Genome-Wide Landscape of Alternative Splicing Events in Brachypodium distachyon  

PubMed Central

Recently, Brachypodium distachyon has emerged as a model plant for studying monocot grasses and cereal crops. Using assembled expressed transcript sequences and subsequent mapping to the corresponding genome, we identified 1219 alternative splicing (AS) events spanning across 2021 putatively assembled transcripts generated from 941 genes. Approximately, 6.3% of expressed genes are alternatively spliced in B. distachyon. We observed that a majority of the identified AS events were related to retained introns (55.5%), followed by alternative acceptor sites (16.7%). We also observed a low percentage of exon skipping (5.0%) and alternative donor site events (8.8%). The ‘complex event’ that consists of a combination of two or more basic splicing events accounted for ?14.0%. Comparative AS transcript analysis revealed 163 and 39 homologous pairs between B. distachyon and Oryza sativa and between B. distachyon and Arabidopsis thaliana, respectively. In all, we found 16 AS transcripts to be conserved in all 3 species. AS events and related putative assembled transcripts annotation can be systematically browsed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/plant/).

Walters, Braden; Lum, Gengkon; Sablok, Gaurav; Min, Xiang Jia

2013-01-01

292

Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3' Splice Site Recognition  

PubMed Central

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.

Smith, Lindsay D.; Lucas, Christian M.; Eperon, Ian C.

2013-01-01

293

Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53.  

PubMed Central

Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities. Images

Will, K; Warnecke, G; Bergmann, S; Deppert, W

1995-01-01

294

Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172.  

PubMed

In Arabidopsis, the identity of perianth and reproductive organs are specified by antagonistic action of two floral homeotic genes, APETALA2 (AP2) and AGAMOUS (AG). AP2 is also negatively regulated by an evolutionary conserved interaction with a microRNA, miR172, and has additional roles in general plant development. A kiwifruit gene with high levels of homology to AP2 and AP2-like genes from other plant species was identified. The transcript was abundant in the kiwifruit flower, particularly petal, suggesting a role in floral organ identity. Splice variants were identified, all containing both AP2 domains, including a variant that potentially produces a shorter transcript without the miRNA172 targeting site. Increased AP2 transcript accumulation was detected in the aberrant flowers of the mutant ‘Pukekohe dwarf’ with multiple perianth whorls and extended petaloid features. In contrast to normal kiwifruit flowers, the aberrant flowers failed to accumulate miR172 in the developing whorls, although accumulation was detected at the base of the flower. An additional role during dormancy in kiwifruit was proposed based on AP2 transcript accumulation in axillary buds before and after budbreak. PMID:22290408

Varkonyi-Gasic, Erika; Lough, Robyn H; Moss, Sarah M A; Wu, Rongmei; Hellens, Roger P

2012-03-01

295

Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing  

Microsoft Academic Search

Alternative pre-mRNA splicing is a major cellular process by which functionally diverse proteins can be generated from the primary transcript of a single gene, often in tissue-specific patterns. The current study investigates the hypothesis that splicing of tissue-specific alternative exons is regulated in part by control sequences in adjacent introns and that such elements may be recognized via computational analysis

Michael Brudno; Mikhail S. Gelfand; Sylvia Spengler; Manfred Zorn; Inna Dubchak; John G. Conboy

2001-01-01

296

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors  

Microsoft Academic Search

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may

Agnieszka Piekielko-Witkowska; Hanna Wiszomirska; Anna Wojcicka; Piotr Poplawski; Joanna Boguslawska; Zbigniew Tanski; Alicja Nauman; Juan Valcarcel

2010-01-01

297

Gene Expression: The Close Coupling of Transcription and Splicing  

Microsoft Academic Search

Increasing evidence indicates that the transcriptional machinery can influence the efficiency of splicing as well as splice-site selection. Surprisingly, it has now been demonstrated that splicing components influence the efficiency of transcription. This mutual stimulation has important implications for the regulation of gene expression.

Emanuel Rosonina; Benjamin J Blencowe

2002-01-01

298

Alternative splicing is required for RCT1-mediated disease resistance in Medicago truncatula.  

PubMed

RCT1 is a TIR-NBS-LRR-type resistance (R) gene in Medicago truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemi-biotrophic fungal pathogen that causes anthracnose disease in Medicago and other closely related legumes. RCT1 undergoes alternative splicing at both coding and 3'-untranslated regions, thereby producing multiple transcript variants in its expression profile. Alternative splicing of RCT1 in the coding region results from the retention of intron 4. Because intron 4 lies downstream of the LRR-encoding exons and contains an in-frame stop codon, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS, and LRR domains but lacks the C-terminal domain of the full-length RCT1 protein encoded by the regular transcript. Here we provide evidence that the RCT1-mediated disease resistance requires the combined presence of the regular and alternative transcripts. Neither the regular nor the alternative RCT1 transcript alone is sufficient to confer resistance against the pathogen. This study, in addition to the reports on the tobacco N and Arabidopsis RPS4 genes, adds another significant example showing the involvement of alternative splicing in R gene-mediated plant immunity. PMID:23657790

Tang, Fang; Yang, Shengming; Gao, Muqiang; Zhu, Hongyan

2013-05-09

299

Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7\\/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms  

Microsoft Academic Search

Background  The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes\\u000a to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES),\\u000a which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes.

Marcella Vacca; Della Floriana Ragione; Andrea Carpi; Valeria Rossi; Maria Strazzullo; Nicola De Franceschi; Ornella Rossetto; Francesco Filippini; Maurizio D’Esposito

2011-01-01

300

Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense?  

Microsoft Academic Search

Alternative splicing (AS) strongly affects gene expres- sion by generating protein isoform diversity. However, up to one-third of human AS events create a premature termination codon that would cause the resulting mRNA to be degraded by nonsense-mediated mRNA decay (NMD). The extent to which such events represent func- tionally selected post-transcriptional gene control, as opposed to noise in the splicing

Nicholas J. McGlincy; Christopher W. J. Smith

2008-01-01

301

Fine Mapping of Calcineurin (PPP3CA) Gene Reveals Novel Alternative Splicing Patterns, Association of 5?UTR Trinucleotide Repeat With Addiction Vulnerability, and Differential Isoform Expression in Alzheimer's Disease  

PubMed Central

Fine mapping of calcineurin (PPP3CA) gene identified single nucleotide polymorphisms (SNPs) and simple sequence repeat polymorphisms that are associated with addiction vulnerability. A trinucleotide repeat marker, located in the 5? untranslated region (5?UTR) of the PPP3CA mRNA, exhibited significantly different genotype and allele frequencies between abusers and controls in the NIDA African–American sample. The polymorphism showed allelic-specific expression in mRNA extracted from postmortem brain specimens. Novel alternatively spliced isoforms of PPP3CA were identified and their expressions were found altered in brain regions of postmortem Alzheimer's disease patients. These data underscore the importance of calcineurin gene in the molecular mechanism of addiction and Alzheimer's diseases.

CHIOCCO, MATTHEW J.; ZHU, XUGUANG; WALTHER, DONNA; PLETNIKOVA, OLGA; TRONCOSO, JUAN C.; UHL, GEORGE R.; LIU, QING-RONG

2010-01-01

302

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function  

PubMed Central

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function.

Gallagher, Thomas L.; Arribere, Joshua A.; Geurts, Paul A.; Exner, Cameron R. T.; McDonald, Kent L.; Dill, Kariena K.; Marr, Henry L.; Adkar, Shaunak S.; Garnett, Aaron T.; Amacher, Sharon L.; Conboy, John G.

2012-01-01

303

Identifying differentially spliced genes from two groups of RNA-seq samples.  

PubMed

Recent study revealed that most human genes have alternative splicing and can produce multiple isoforms of transcripts. Differences in the relative abundance of the isoforms of a gene can have significant biological consequences. Identifying genes that are differentially spliced between two groups of RNA-sequencing samples is an important basic task in the study of transcriptomes with next-generation sequencing technology. We use the negative binomial (NB) distribution to model sequencing reads on exons, and propose a NB-statistic to detect differentially spliced genes between two groups of samples by comparing read counts on all exons. The method opens a new exon-based approach instead of isoform-based approach for the task. It does not require information about isoform composition, nor need the estimation of isoform expression. Experiments on simulated data and real RNA-seq data of human kidney and liver samples illustrated the method's good performance and applicability. It can also detect previously unknown alternative splicing events, and highlight exons that are most likely differentially spliced between the compared samples. We developed an NB-statistic method that can detect differentially spliced genes between two groups of samples without using a prior knowledge on the annotation of alternative splicing. It does not need to infer isoform structure or to estimate isoform expression. It is a useful method designed for comparing two groups of RNA-seq samples. Besides identifying differentially spliced genes, the method can highlight on the exons that contribute the most to the differential splicing. We developed a software tool called DSGseq for the presented method available at http://bioinfo.au.tsinghua.edu.cn/software/DSGseq. PMID:23228854

Wang, Weichen; Qin, Zhiyi; Feng, Zhixing; Wang, Xi; Zhang, Xuegong

2012-12-08

304

A novel functional low-density lipoprotein receptor-related protein 6 gene alternative splice variant is associated with Alzheimer's disease.  

PubMed

We previously found that single nucleotide polymorphisms in the low-density lipoprotein receptor-related protein 6 (LRP6) gene are associated with Alzheimer's disease (AD). Here, we studied the posttranscriptional metabolism of the LRP6 message scanning sequentially the 23 LRP6 exons in human tissues and found a novel LRP6 isoform that completely skips exon 3 (LRP6?3) in all tissues examined and was also conserved in mice. Expression levels of the LRP6 isoforms were determined in 47 cortical brain messenger (m)RNA samples including 22 AD cases, 11 control subjects, and 14 individuals with other neurological disorders. LRP6?3 mRNA levels were significantly augmented in AD brains compared with controls (1.6-fold; p = 0.037) or other pathological samples (2-fold; p = 0.007). Functional analysis in Wnt/?-catenin signaling assays revealed that skipping of exon 3 reduced significantly the signaling activity of the LRP6 coreceptor. We conclude that the LRP6?3 isoform is a novel splice variant, which shows diminished Wnt/?-catenin signaling activity and might have a functional role in individuals with AD. PMID:23218566

Alarcón, Marcelo A; Medina, Matías A; Hu, Qubai; Avila, Miguel E; Bustos, Bernabé I; Pérez-Palma, Eduardo; Peralta, Alexis; Salazar, Paulina; Ugarte, Giorgia D; Reyes, Ariel E; Martin, George M; Opazo, Carlos; Moon, Randall T; De Ferrari, Giancarlo V

2012-12-06

305

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types  

Microsoft Academic Search

Alternative splicing of a-tropomyosin (a-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitu- tions of the

Natalia Gromak; Christopher W. J. Smith

2002-01-01

306

Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.  

PubMed

Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system. PMID:19737518

Calarco, John A; Superina, Simone; O'Hanlon, Dave; Gabut, Mathieu; Raj, Bushra; Pan, Qun; Skalska, Ursula; Clarke, Laura; Gelinas, Danielle; van der Kooy, Derek; Zhen, Mei; Ciruna, Brian; Blencowe, Benjamin J

2009-09-01

307

Exon-level microarray analyses identify alternative splicing programs in breast cancer  

PubMed Central

Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays (HJAY). We analyzed these data using a computational pipeline specifically designed to detect AS with a low false positive rate. This identified 181 splice events representing 156 genes as candidates for AS. RT-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype-specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. siRNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype specific AS. The subtype specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues.

Lapuk, Anna; Marr, Henry; Jakkula, Lakshmi; Pedro, Helder; Bhattacharya, Sanchita; Purdom, Elizabeth; Hu, Zhi; Simpson, Ken; Pachter, Lior; Durinck, Steffen; Wang, Nicholas; Parvin, Bahram; Fontenay, Gerald; Speed, Terence; Garbe, James; Stampfer, Martha; Bayandorian, Hovig; Dorton, Shannon; Clark, Tyson A.; Schweitzer, Anthony; Wyrobek, Andrew; Feiler, Heidi; Spellman, Paul; Conboy, John; Gray, Joe W.

2010-01-01

308

Correction of alternative splicing of tau in frontotemporal dementia and parkinsonism linked to chromosome 17.  

PubMed

Mutations in the human tau gene cause frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17). One of the major disease mechanisms in FTDP-17 is the increased inclusion of tau exon 10 during pre-mRNA splicing. Here we show that modified oligonucleotides directed against the tau exon 10 splice junctions suppress inclusion of tau exon 10. The effect is mediated by the formation of a stable pre-mRNA-oligonucleotide hybrid, which blocks access of the splicing machinery to the pre-mRNA. Correction of tau splicing occurs in a tau minigene system and in endogenous tau RNA in neuronal pheochromocytoma cells and is specific to exon 10 of the tau gene. Antisense oligonucleotide-mediated exclusion of exon 10 has a physiological effect by increasing the ratio of protein lacking the microtubule-binding domain encoded by exon 10. As a consequence, the microtubule cytoskeleton becomes destabilized and cell morphology is altered. Our results demonstrate that alternative splicing defects of tau as found in FTDP-17 patients can be corrected by application of antisense oligonucleotides. These findings provide a tool to study specific tau isoforms in vivo and might lead to a novel therapeutic strategy for FTDP-17. PMID:11560926

Kalbfuss, B; Mabon, S A; Misteli, T

2001-09-17

309

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA  

PubMed Central

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5? and 3? untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

310

Intracellular calcium activates TRPM2 and its alternative spliced isoforms  

PubMed Central

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca2+ ([Ca2+]i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca2+]i, indicating that [Ca2+]i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca2+]i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca2+-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca2+]i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca2+ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca2+ release is capable of activating TRPM2. These results indicate that [Ca2+]i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca2+]i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca2+]i.

Du, Jianyang; Xie, Jia; Yue, Lixia

2009-01-01

311

Anti-proliferative and pro-apoptotic actions of a novel human and mouse ovarian tumor-associated gene OTAG-12: downregulation, alternative splicing and drug sensitization.  

PubMed

In studying the age dependence and chronology of ovarian tumors in follicle stimulating hormone receptor knockout mice, we identified a novel ovarian tumor associated gene-12 (OTAG-12), which is progressively downregulated and maps to Chr. 8B3.3. OTAG-12 protein overexpression in mouse ovarian and mammary tumor cells suggested powerful anti-proliferative effects. In human epithelial ovarian cancers (OCs) and OC cell lines, OTAG-12 mRNA expression is downregulated in comparison with normal ovaries. Cloning and identification revealed that human OTAG-12 mapping to gene-rich Chr. 19p13.12 is expressed in three spliced forms: hOTAG-12a, hOTAG-12b and hOTAG-12c, of which b is predominant in the normal ovary. Functionally active hOTAG-12b is a simple protein with no disulfide bonds and a nuclear localization signal is present in all variants. Transfection of OTAG-12 variants in OC and tumorigenic HEK293 cells confirmed nuclear localization. hOTAG-12b overexpression in OC and HEK293 cells effectively suppressed cell growth, anchorage-dependent and independent colony formation followed by apoptosis, whereas hOTAG-12a and hOTAG-12c had no such effects. Deletion mutants identified the critical importance of carboxyl terminus for hOTAG-12b function. Doxycycline-inducible growth inhibition of HEK293 cells by hOTAG-12a was associated with effects on G2 cell cycle arrest and apoptosis induction. hOTAG-12b expression rendered tumorigenic cells more sensitive to four apoptotic stimuli including etoposide-a topoisomerase-II inhibitor. Doxycycline-induced hOTAG-12b expression blocked xenograft tumor growth in nude mice, whereas hOTAG-12a was ineffective. Although p53-pathway-dependent apoptotic agents could upregulate endogenous hOTAG-12b and p53 in UCI-101/107 OC cells, hOTAG-12b could also induce apoptosis in p53-null and platinum-resistant SKOV3 OC cells and Doxycycline-induced hOTAG-12b did not alter p53. Further study showed that hOTAG-12b increases mRNAs of pro-apoptotic genes such as BAD, GADD45? and CIEDB, while inhibiting anti-apoptotic NAIP and Akt1 expression, suggesting that hOTAG-12b-induced apoptosis might be p53-independent. These results indicate that hOTAG-12b is a putative ovarian tumor suppressor gene warranting further studies. PMID:21339736

Chen, X; Zhang, H; Aravindakshan, J P; Gotlieb, W H; Sairam, M R

2011-02-21

312

Microenvironment Changes (in pH) Affect VEGF Alternative Splicing  

Microsoft Academic Search

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix\\u000a proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied\\u000a in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested\\u000a microenvironment cues that might regulate

Ana Paula Elias; Sergio Dias

2008-01-01

313

Ca 2+ Signaling, Alternative Splicing and Endoplasmic Reticulum Stress Responses  

Microsoft Academic Search

Ca2+-signaling, alternative splicing, and stress responses by the endoplasmic reticulum are three important cellular activities\\u000a which can be strongly interconnected to alter the expression of protein isoforms in a tissue dependent manner or during development\\u000a depending on the environmental conditions. This integrated network of signaling pathways permits a high degree of versatility\\u000a and adaptation to metabolic, developmental and stress processes.

Joachim Krebs; Jody Groenendyk; Marek Michalak

2011-01-01

314

A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples  

Microsoft Academic Search

One important goal of genomics is to explore the extent of alternative splicing in the transcriptome and generate a comprehensive catalog of splice forms. New computational and experimental approaches have led to an increase in the number of predicted alternatively spliced tran- scripts; however, validation of these predictions has not kept pace. In this work, we systematically explore different methods

German Gaston Leparc; Robi David Mitra

2007-01-01

315

Complex changes in alternative pre-mRNA splicing play a central role in the Epithelial-Mesenchymal Transition (EMT)  

PubMed Central

The epithelial to mesenchymal transition (EMT) is an important developmental process that is also implicated in disease pathophysiology, such as cancer progression and metastasis. A wealth of literature in recent years has identified important transcriptional regulators and large-scale changes in gene expression programs that drive the phenotypic changes that occur during the EMT. However, in the past couple of years it has become apparent that extensive changes in alternative splicing also play a profound role in shaping the changes in cell behavior that characterize the EMT. While long known splicing switches in FGFR2 and p120-catenin provided hints of a larger program of EMT-associated alternative splicing, the recent identification of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) began to reveal this genome-wide post-transcriptional network. Several studies have now demonstrated the truly vast extent of this alternative splicing program. The global switches in splicing associated with the EMT add an important additional layer of post-transcriptional control that works in harmony with transcriptional and epigenetic regulation to effect complex changes in cell shape, polarity, and behavior that mediate transitions between epithelial and mesenchymal cell states. Future challenges include the need to investigate the functional consequences of these splicing switches at both the individual gene as well as systems level.

Warzecha, Claude C.; Carstens, Russ P.

2012-01-01

316

Alternative splicing in spinal muscular atrophy underscores the role of an intron definition model  

PubMed Central

Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene: SMN1 and SMN2. The two SMN genes code for identical proteins; however, SMN2 predominantly generates a shorter transcript due to skipping of exon 7, the last coding exon. Skipping of SMN2 exon 7 leads to production of a truncated SMN protein that is highly unstable. The inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), the second most prevalent genetic cause of infant mortality. Since SMN2 is almost universally present in SMA patients, correction of SMN2 exon 7 splicing holds the promise for cure. Consistently, SMN2 exon 7 splicing has emerged as one of the best studied splicing systems in humans. The vast amount of recent literature provides a clue that SMN2 exon 7 splicing is regulated by an intron definition mechanism, which does not require cross-exon communication as prerequisite for exon inclusion. Our conclusion is based on the prominent role of intronic cis-elements, some of them have emerged as the frontrunners among potential therapeutic targets of SMA. Further, the widely expressed T-cell-restricted intracellular antigen-1 (TIA1), a member of the glutamine rich domain containing RNA-binding proteins, has recently been found to regulate SMN exon 7 splicing by binding to intron 7 sequences away from the 5? splice site (ss). These findings make a strong argument for an “intron definition model,” according to which regulatory sequences within a downstream intron are capable of enforcing exon inclusion even in the absence of a defined upstream 3? ss of an alternatively spliced exon.

Singh, Natalia N

2011-01-01

317

The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.  

PubMed

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression. PMID:20797886

Tripathi, Vidisha; Ellis, Jonathan D; Shen, Zhen; Song, David Y; Pan, Qun; Watt, Andrew T; Freier, Susan M; Bennett, C Frank; Sharma, Alok; Bubulya, Paula A; Blencowe, Benjamin J; Prasanth, Supriya G; Prasanth, Kannanganattu V

2010-09-24

318

The PTB interacting protein raver1 regulates ?-tropomyosin alternative splicing  

PubMed Central

Regulated switching of the mutually exclusive exons 2 and 3 of ?-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

Gromak, Natalia; Rideau, Alexis; Southby, Justine; Scadden, A.D.J.; Gooding, Clare; Huttelmaier, Stefan; Singer, Robert H.; Smith, Christopher W.J.

2003-01-01

319

Alternative splice acceptor utilization during human immunodeficiency virus type 1 infection of cultured cells.  

PubMed Central

The utilization of alternative splice acceptors for excision of the 5' major intron of human immunodeficiency virus type 1 RNA was observed after infection in vitro. Specific splice events were monitored by a cDNA-polymerase chain reaction. These splice events shared a common splice donor but utilized several alternative splice acceptors. In addition to identifying the previously documented splice acceptors for tat and nef (S. K. Arya, C. Guo, S. F. Josephs, and F. Wong-Staal, Science 229:69-73, 1985), nucleotide sequence analysis of cDNA-polymerase chain reaction fragments also revealed the following: (i) two splice acceptors 15 and 9 nucleotides upstream from the rev start codon, which are utilized to create transcripts suitable for specific rev expression; and (ii) use of the splice acceptor previously attributed to nef to generate a singly spliced, env-encoding transcript. Hybridization signals representing the nef/env, tat, and rev splice events increased in intensity between 6 and 12 h after infection of CEM cells with the LAV-1BRU strain of human immunodeficiency virus type 1. In contrast, the signal for utilization of the nef/env splice acceptor for the singly spliced env transcript appeared first at 12 h and increased to maximum intensity by 24 h. The nef/env splice acceptor was dominant at all time points examined. We propose that this dominance ensures efficient downstream splicing proximal to the env initiation codon in singly spliced transcripts. However, early after infection, the dominance of the nef/env splice acceptor appears to divert primary transcripts away from tat- and rev-specific processing paths. The relative proportions of hybridization signals representing these alternative splice events remained constant throughout the viral replicative cycle. This result suggests that trans-acting factors that might influence splice choices are not induced during infection, but rather that cis-acting, sequence-specific splice preferences determine the relative efficiency of alternative acceptor utilization. Images

Guatelli, J C; Gingeras, T R; Richman, D D

1990-01-01

320

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B  

PubMed Central

Modulation of alternative pre-mRNA splicing is a potential approach to therapeutic targeting for a variety of human diseases. We investigated the mechanism by which digitoxin, a member of the cardiotonic steroid class of drugs, regulates alternative splicing. Transcriptome-wide analysis identified a large set of alternative splicing events that change after digitoxin treatment. Within and adjacent to these regulated exons, we identified enrichment of potential binding sites for the splicing factors SRp20 (SRSF3/SFRS3) and Tra2-? (SFRS10/TRA2B). We further find that both of these proteins are depleted from cells by digitoxin treatment. Characterization of SRp20 and Tra2-? splicing targets revealed that many, but not all, digitoxin-induced splicing changes can be attributed to the depletion of one or both of these factors. Re-expression of SRp20 or Tra2-? after digitoxin treatment restores normal splicing of their targets, indicating that the digitoxin effect is directly due to these factors. These results demonstrate that cardiotonic steroids, long prescribed in the clinical treatment of heart failure, have broad effects on the cellular transcriptome through these and likely other RNA binding proteins. The approach described here can be used to identify targets of other potential therapeutics that act as alternative splicing modulators.

Anderson, Erik S.; Lin, Chia-Ho; Xiao, Xinshu; Stoilov, Peter; Burge, Christopher B.; Black, Douglas L.

2012-01-01

321

Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells  

PubMed Central

Purpose. We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context. Methods. Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis. Results. Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement. Conclusions. Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.

Joo, Jeong-Hoon; Correia, Greg P.; Li, Jian-Liang; Lopez, Maria-Cecilia; Baker, Henry V.; Sugrue, Stephen P.

2013-01-01

322

Regulation of Multiple Core Spliceosomal Proteins by Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay  

Microsoft Academic Search

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets

Arneet L. Saltzman; Yoon Ki Kim; Qun Pan; Matthew M. Fagnani; Lynne E. Maquat; Benjamin J. Blencowe

2008-01-01

323

Support vector machines-based identification of alternative splicing in Arabidopsis thaliana from whole-genome tiling arrays  

Microsoft Academic Search

Background  Alternative splicing (AS) is a process which generates several distinct mRNA isoforms from the same gene by splicing different\\u000a portions out of the precursor transcript. Due to the (patho-)physiological importance of AS, a complete inventory of AS is\\u000a of great interest. While this is in reach for human and mammalian model organisms, our knowledge of AS in plants has remained

Johannes Eichner; Georg Zeller; Sascha Laubinger; Gunnar Rätsch

2011-01-01

324

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation  

PubMed Central

Precise 5? splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5? splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.

Rosel, Tanja Dorothe; Hung, Lee-Hsueh; Medenbach, Jan; Donde, Katrin; Starke, Stefan; Benes, Vladimir; Ratsch, Gunnar; Bindereif, Albrecht

2011-01-01

325

FIRMA: a method for detection of alternative splicing from exon array data  

PubMed Central

Motivation: Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays. Results: We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRMA has been developed for Affymetrix exon arrays, but could in principle be extended to other exon arrays, tiling arrays or splice junction arrays. We have evaluated the method using simulated data, and have also applied it to two datasets: a panel of 11 human tissues and a set of 10 pairs of matched normal and tumor colon tissue. FIRMA is able to detect exons in several genes confirmed by reverse transcriptase PCR. Availability: R code implementing our methods is contributed to the package aroma.affymetrix. Contact: epurdom@stat.berkeley.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Purdom, E.; Simpson, K. M.; Robinson, M. D.; Conboy, J. G.; Lapuk, A. V.; Speed, T.P.

2008-01-01

326

Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing  

PubMed Central

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs. 32.6% in controls, P = 0.016, OR = 0.90 [0.82-0.98]). Two of these SNPs are in exon 10, directly 5? of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs as well as a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

Douglas, Katherine B.; Windels, Daniel C.; Zhao, Jian; Gadeliya, Agnessa V.; Wu, Hui; Kaufman, Kenneth M.; Harley, John B.; Merrill, Joan; Kimberly, Robert P.; Alarcon, Graciela S.; Brown, Elizabeth E.; Edberg, Jeffrey C.; Ramsey-Goldman, Rosalind; Petri, Michelle; Reveille, John D.; Vila, Luis M.; Gaffney, Patrick M.; James, Judith A.; Moser, Kathy L.; Alarcon-Riquelme, Marta E.; Vyse, Timothy J.; Gilkeson, Gary S.; Jacob, Chaim O.; Ziegler, Julie T.; Langefeld, Carl D.; Ulgiati, Daniela; Tsao, Betty P.; Boackle, Susan A.

2009-01-01

327

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

PubMed Central

Alternative pre-mRNA splicing predominates in the nervous systems of complex organisms including humans dramatically expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type CaV2.2 calcium channels in nociceptors. Using an exon replacement strategy in mice, we show that mutually exclusive splicing in the CaV2.2 gene modulates N-type channel function in nociceptors leading to a change in morphine analgesia. Exon 37a enhances ?-opioid receptor mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a spinal morphine analgesia is weakened in vivo without influencing the basal response to noxious thermal stimuli. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

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

2010-01-01

328

fruitless alternative splicing and sex behaviour in insects: an ancient and unforgettable love story?  

Microsoft Academic Search

Courtship behaviours are common features of animal species that reproduce sexually. Typically, males are involved in courting\\u000a females. Insects display an astonishing variety of courtship strategies primarily based on innate stereotyped responses to\\u000a various external stimuli. In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila

Marco Salvemini; Catello Polito; Giuseppe Saccone

2010-01-01

329

Alternative splicing of hMSH2 in normal human tissues  

Microsoft Academic Search

hMSH2 is a homolog of bacterial mutS and yeast Msh2, a member of the group of mismatch repair genes whose products bind to mismatched regions of double-stranded DNA. We analyzed\\u000a expression of hMSH2 in normal human organs by the polymerase chain reaction coupled with reverse transcription and found two novel types of alternatively\\u000a spliced mRNAs that were expressed in normal

Yuriko Mori; Hiromi Shiwaku; Shinichi Fukushige; Shigeru Wakatsuki; Masami Sato; Toshihiro Nukiwa; A. Horii

1997-01-01

330

The alternative splicing of tau exon 10 and its regulatory proteins CLK2 and TRA2-BETA1 changes in sporadic Alzheimer's disease.  

PubMed

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in tauopathies, which include Alzheimer's disease (AD). Tau is a microtubule-associated protein whose transcript undergoes alternative splicing in the brain. Exon 10 encodes one of four microtubule-binding repeats. Exon 10 inclusion gives rise to tau protein isoforms containing four microtubule-binding repeats (4R) whereas exclusion leads to isoforms containing only three repeats (3R). The ratio between 3R and 4R isoforms is tightly controlled via alternative splicing in the human adult nervous system and distortion of this balance results in neurodegeneration. Previous studies showed that several splicing regulators, among them hTRA2-beta1 and CLK2, regulate exon 10 alternative splicing. Like most splicing factors, htra2-beta and clk2 pre-mRNAs are regulated by alternative splicing. Here, we investigated whether human postmortem brain tissue of AD patients reveal differences in alternative splicing patterns of the tau, htra2-beta, presenilin 2 and clk2 genes when compared with age-matched controls. We found that the splicing patterns of all four genes are altered in affected brain areas of sporadic AD patients. In these affected areas, the amount of mRNAs of tau isoforms including exon 10, the htra2-beta1 isoform and an inactive form of clk2 are significantly increased. These findings suggest that a misregulation of alternative splicing seems to contribute to sporadic AD. PMID:16371011

Glatz, Daniela C; Rujescu, Dan; Tang, Yesheng; Berendt, Frank J; Hartmann, Annette M; Faltraco, Frank; Rosenberg, Carlyn; Hulette, Christine; Jellinger, Kurt; Hampel, Harald; Riederer, Peter; Möller, Hans-J; Andreadis, Athena; Henkel, Kerstin; Stamm, Stefan

2005-12-20

331

Evidence that RNA editing modulates splice site selection in the 5HT2C receptor gene  

Microsoft Academic Search

Adenosine to inosine editing of mRNA from the human 5-HT2C receptor gene (HTR2C) occurs at five exonic positions (A-E) in a stable stem-loop that includes the normal 5¢ splice site of intron 5 and is flanked by two alternative splice sites. Using in vitro editing, we identified a novel editing site (F) located in the intronic part of the stem-loop

Rachel Flomen; Joanne Knight; Pak Sham; Robert Kerwin; Andrew Makoff

2004-01-01

332

Gene set enrichment analysis of RNA-Seq data: integrating differential expression and splicing  

PubMed Central

Background RNA-Seq has become a key technology in transcriptome studies because it can quantify overall expression levels and the degree of alternative splicing for each gene simultaneously. To interpret high-throughout transcriptome profiling data, functional enrichment analysis is critical. However, existing functional analysis methods can only account for differential expression, leaving differential splicing out altogether. Results In this work, we present a novel approach to derive biological insight by integrating differential expression and splicing from RNA-Seq data with functional gene set analysis. This approach designated SeqGSEA, uses count data modelling with negative binomial distributions to first score differential expression and splicing in each gene, respectively, followed by two strategies to combine the two scores for integrated gene set enrichment analysis. Method comparison results and biological insight analysis on an artificial data set and three real RNA-Seq data sets indicate that our approach outperforms alternative analysis pipelines and can detect biological meaningful gene sets with high confidence, and that it has the ability to determine if transcription or splicing is their predominant regulatory mechanism. Conclusions By integrating differential expression and splicing, the proposed method SeqGSEA is particularly useful for efficiently translating RNA-Seq data to biological discoveries.

2013-01-01

333

Dynamic integration of splicing within gene regulatory pathways.  

PubMed

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 crosstalk 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

Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex M; Graveley, Brenton R; Blencowe, Benjamin J

2013-03-14

334

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.  

PubMed Central

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images

Harper, J E; Manley, J L

1991-01-01

335

Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.  

PubMed

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5' splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3' splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing. PMID:18086893

Zhu, Hui; Hinman, Melissa N; Hasman, Robert A; Mehta, Priyesh; Lou, Hua

2007-12-17

336

Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts  

PubMed Central

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

Grohmann, Maik; Hammer, Paul; Walther, Maria; Paulmann, Nils; Buttner, Andreas; Eisenmenger, Wolfgang; Baghai, Thomas C.; Schule, Cornelius; Rupprecht, Rainer; Bader, Michael; Bondy, Brigitta; Zill, Peter

2010-01-01

337

Co-transcriptional regulation of alternative pre-mRNA splicing  

PubMed Central

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.

Shukla, Sanjeev; Oberdoerffer, Shalini

2012-01-01

338

Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein  

SciTech Connect

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.

Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. (Columbia Univ., New York, NY (United States))

1992-03-03

339

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes.  

PubMed

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. PMID:20052763

Le Guédard-Méreuze, Sandie; Vaché, Christel; Baux, David; Faugère, Valérie; Larrieu, Lise; Abadie, Caroline; Janecke, Andreas; Claustres, Mireille; Roux, Anne-Françoise; Tuffery-Giraud, Sylvie

2010-03-01

340

Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation  

PubMed Central

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5? alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Allemand, Eric; Gattoni, Renata; Bourbon, Henri-Marc; Stevenin, James; Caceres, Javier F.; Soret, Johann; Tazi, Jamal

2001-01-01

341

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.  

PubMed Central

Many cellular and viral genes are parts of complex transcription units containing multiple splicing choices. During the course of an adenoviral replicative cycle, different spliced versions of a single gene predominate, depending on the stage of infection. This is true for several adenoviral genes. In this paper we show for the viral E1B transcription unit that splice site usage regulates this process. The change in alternative splicing in this system does not depend on the sequence of the transcribed genes. Non-adenoviral genes, such as the SV40 early region and the polyoma early region, which normally show little or no regulation of spliced RNA product formation, become regulated for mRNA production after insertion into the adenoviral genome. Additional studies show that E1B splicing regulation in adenovirus is a cis effect. Staggered infections using two discernable viral genomes resulted in a situation where both early and late genomes exist in the same nucleus. Neither genome was able to impose its regulated splicing pattern on the other, indicating that the cue for the switch in viral gene splicing is not directly dependent on global changes in trans-acting splicing factors. This suggests a model where the signal for changes in RNA processing for the E1B gene is linked to the state of the DNA template or its localization within nuclear subcompartments. Images

Adami, G; Babiss, L E

1991-01-01

342

Highlights of Alternative Splicing Regulation Session: Yes, No, Maybe--A History of Paradigm Shifts  

NSDL National Science Digital Library

Cooper summarizes the discussions and presentations from the session entitled "Control of Splice Site Selection" held at the Sixth Annual Meeting of the RNA Society. Paradigms are shifting as experiments show that some of the proteins involved in regulating splicing can act as splicing enhancers or repressors, depending on the cellular context. The complex interactions among the ribonucleoproteins (RNPs) and proteins, and the role of cis elements, in controlling cell-specific splicing are highlighted. The importance of properly regulated splicing is emphasized by examples of disease pathologies in which alternative splicing is aberrant.

Thomas A. Cooper (Baylor College of Medicine;Departments of Pathology and Molecular and Cellular Biology REV)

2001-10-23

343

Alternative splicing and the progesterone receptor in breast cancer  

PubMed Central

Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions.

Cork, David MW; Lennard, Thomas WJ; Tyson-Capper, Alison J

2008-01-01

344

ASPicDB: a database of annotated transcript and protein variants generated by alternative splicing  

PubMed Central

Alternative splicing is emerging as a major mechanism for the expansion of the transcriptome and proteome diversity, particularly in human and other vertebrates. However, the proportion of alternative transcripts and proteins actually endowed with functional activity is currently highly debated. We present here a new release of ASPicDB which now provides a unique annotation resource of human protein variants generated by alternative splicing. A total of 256?939 protein variants from 17?191 multi-exon genes have been extensively annotated through state of the art machine learning tools providing information of the protein type (globular and transmembrane), localization, presence of PFAM domains, signal peptides, GPI-anchor propeptides, transmembrane and coiled-coil segments. Furthermore, full-length variants can be now specifically selected based on the annotation of CAGE-tags and polyA signal and/or polyA sites, marking transcription initiation and termination sites, respectively. The retrieval can be carried out at gene, transcript, exon, protein or splice site level allowing the selection of data sets fulfilling one or more features settled by the user. The retrieval interface also enables the selection of protein variants showing specific differences in the annotated features. ASPicDB is available at http://www.caspur.it/ASPicDB/.

Martelli, Pier L.; D'Antonio, Mattia; Bonizzoni, Paola; Castrignano, Tiziana; D'Erchia, Anna M.; D'Onorio De Meo, Paolo; Fariselli, Piero; Finelli, Michele; Licciulli, Flavio; Mangiulli, Marina; Mignone, Flavio; Pavesi, Giulio; Picardi, Ernesto; Rizzi, Raffaella; Rossi, Ivan; Valletti, Alessio; Zauli, Andrea; Zambelli, Federico; Casadio, Rita; Pesole, Graziano

2011-01-01

345

Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila.  

PubMed

The Drosophila melanogaster transcription factor Lola (longitudinals lacking) is a pivotal regulator of neural wiring that sets the precise expression levels of proteins that execute specific axon guidance decisions. Lola has a zinc finger DNA binding domain and a BTB (for Broad-complex, Tramtrack and Bric a brac) dimerization motif. We now show that alternative splicing of the lola gene creates a family of 19 transcription factors. All lola isoforms share a common dimerization domain, but 17 have their own unique DNA-binding domains. Seven of these 17 isoforms are present in the distantly-related Dipteran Anopheles gambiae, suggesting that the properties of specific isoforms are likely to be critical to lola function. Analysis of the expression patterns of individual splice variants and of the phenotypes of mutants lacking single isoforms supports this idea and establishes that the alternative forms of lola are responsible for different functions of this gene. Thus, in this system, the alternative splicing of a key transcription factor helps to explain how a small genome encodes all the information that is necessary to specify the enormous diversity of axonal trajectories. PMID:12897787

Goeke, Scott; Greene, Elizabeth A; Grant, Paul K; Gates, Michael A; Crowner, Daniel; Aigaki, Toshiro; Giniger, Edward

2003-09-01

346

SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W  

PubMed Central

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5? and 3? splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

2012-01-01

347

PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells  

PubMed Central

Background PCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. Results We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. Conclusions We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

2010-01-01

348

Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing.  

PubMed

The crux of current RNA-based therapeutics relies on association of synthetic nucleic acids with cellular RNA targets. Antisense oligonucleotide binding to mature microRNA and splicing junctions on pre-mRNA represent methods of gene therapy that respectively inhibit microRNA-mediated gene regulation and induce alternative splicing. We have developed biodegradable polymer nanoparticles, which are coated with cell-penetrating peptides, that can effectively deliver chemically modified oligonucleotide analogues to achieve these forms of gene regulation. We found that this nanoparticle system could block the activity of the oncogenic microRNA, miR-155, as well as modulate splicing to attenuate the expression of the proto-oncogene, Mcl-1. Regulation of these genes in human cancer cells reduced cell viability and produced pro-apoptotic effects. These findings establish polymer nanoparticles as delivery vectors for nonconventional forms of gene therapy activated by cellular delivery of RNA-targeted molecules, which have strong therapeutic implications. PMID:22482958

Cheng, Christopher J; Saltzman, W Mark

2012-04-18

349

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization  

PubMed Central

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource—available at —is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility.

Castrignano, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

2006-01-01

350

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization.  

PubMed

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource-available at http://www.caspur.it/ASPIC/--is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. PMID:16845044

Castrignanò, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

2006-07-01

351

beta subunits modulate alternatively spliced, large conductance, calcium-activated potassium channels of avian hair cells.  

PubMed

Electrical tuning confers frequency selectivity onto sensory hair cells in the auditory periphery of frogs, turtles, and chicks. The resonant frequency is determined in large part by the number and kinetics of large conductance, calcium-activated potassium (BK) channels. BK channels in hair cells are encoded by the alternatively spliced slo gene and may include an accessory beta subunit. Here we examine the origins of kinetic variability among BK channels by heterologous expression of avian cochlear slo cDNAs. Four alternatively spliced forms of the slo-alpha gene from chick hair cells were co-expressed with accessory beta subunits (from quail cochlea) by transient transfection of human embryonic kidney 293 cells. Addition of the beta subunit increased steady-state calcium affinity, raised the Hill coefficient for calcium binding, and slowed channel deactivation rates, resulting in eight functionally distinct channels. For example, a naturally occurring splice variant containing three additional exons deactivated 20-fold more slowly when combined with beta. Deactivation kinetics were used to predict tuning frequencies and thus tonotopic location if hair cells were endowed with each of the expressed channels. All beta-containing channels were predicted to lie within the apical (low-frequency) 30% of the epithelium, consistent with previous in situ hybridization studies. Individual slo-alpha exons would be found anywhere within the apical 70%, depending on the presence of beta, and other alternative exons. Alternative splicing of the slo-alpha channel message provides intrinsic variability in gating kinetics that is expanded to a wider range of tuning by modulation with beta subunits. PMID:10684869

Ramanathan, K; Michael, T H; Fuchs, P A

2000-03-01

352

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells  

Microsoft Academic Search

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an expla- nation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using

Laura Borsi; Barbara C; Patrizia Castellani; Claudio Rosellini; Daniela Vecchio; Giorgio Allemanni; Sidney E. Chang; Joyce Taylor-Papadimitriou; Hema Pande; Luciano Zardi

1987-01-01

353

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

PubMed Central

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.

Corrionero, Anna; Minana, Belen; Valcarcel, Juan

2011-01-01

354

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

PubMed

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

Corrionero, Anna; Miñana, Belén; Valcárcel, Juan

2011-03-01

355

Processing sites involved in intron splicing of Armillaria natural product genes.  

PubMed

We analysed the structure of four genes whose transcriptional products are likely to be involved in the small molecule metabolism of the homobasidiomycete Armillaria mellea with the aim of verifying splice sites. To this end we experimentally validated in silico predicted intron/exon junctions for accuracy. Based on 78 verified junctions, a consensus for donor and acceptor sites in Armillaria is presented, along with experimental evidence for non-canonical splice sites, introns with alternative donor or acceptor junctions, and allele-selective splicing. The investigated reading frames show significant homologies to: (1) antibiotic and other small molecule efflux transporter genes; (2) phenoloxidase/laccase genes; (3) genes for dual Cys2His2/Zn(II)2Cys6 transcriptional regulators. For all of these gene categories, this is the first report on examples from the genus Armillaria. PMID:18280725

Misiek, Mathias; Hoffmeister, Dirk

2007-11-01

356

Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms.  

PubMed

The molecular mechanisms that direct splice-site selection and assure orderly exon juxtaposition have not been fully clarified. The extraordinary nature of the dystrophin gene points to several hurdles in the processing of transcripts. In this study, dystrophin statistical and thermodynamic splicing parameters have been evaluated providing the first comprehensive description for a single human gene. We show that concomitant use of consensus values (CV) and DeltaDG degrees 37 values for U1snRNA annealing better discriminates between real donor sites and donor-like sequences. Evidence is also provided that, on average, out-of-frame dystrophin exons have significantly stronger CVs and more favorable DeltaDG degrees 37 values; this feature has never been reported and might reflect evolutionary-driven minimization of out-of-frame exon misplicing. Dystrophin splicing mutations have been reported to determine either Duchenne or Becker Muscular Dystrophy, but no comprehensive genotypic/phenotypic correlation has ever been investigated. We have analyzed splicing affecting single base-pair substitutions in the dystrophin gene with respect to their effect on splicing parameters; functional and clinical consequences are also reported. We have found 5'-splice-site mutation occurrence to be statistically related to mutability quotients and propose the use of DeltaDG degrees 37 values as a more effective tool than CV alone to describe donor site mutation consequences. Our analysis also indicates a nearly 100% correlation between clinical phenotype and the reading-frame rule determined at the RNA level. We consider that elucidation of the relative importance of splicing determinants might help to clarify the molecular mechanisms that direct correct splicing in complex genes and might be useful in the validation of predictive models. PMID:11479738

Sironi, M; Pozzoli, U; Cagliani, R; Comi, G P; Bardoni, A; Bresolin, N

2001-07-01

357

PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.  

PubMed

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL-mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance. PMID:20221253

Johnson, Matthew L; Nagengast, Alexis A; Salz, Helen K

2010-03-05

358

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila  

PubMed Central

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

Johnson, Matthew L.; Nagengast, Alexis A.; Salz, Helen K.

2010-01-01

359

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.  

PubMed

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region approximately 18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1-exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression. PMID:19965599

Cheng, Dongmei; MacArthur, Philip S; Rong, Shunxing; Parks, John S; Shelness, Gregory S

2009-10-27

360

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation.  

PubMed

Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules. PMID:21624952

Schmidt, Ute; Basyuk, Eugenia; Robert, Marie-Cécile; Yoshida, Minoru; Villemin, Jean-Philippe; Auboeuf, Didier; Aitken, Stuart; Bertrand, Edouard

2011-05-30

361

ANOSVA: a statistical method for detecting splice variation from expression data  

Microsoft Academic Search

Motivation: Many or most mammalian genes undergo altern- ative splicing, generating a variety of transcripts from a single gene. New information on splice variation is becoming avail- able through technology for measuring expression levels of several exons or splice junctions per gene. We have developed a statistical method, ANalysis Of Splice VAriation (ANOSVA) to detect alternative splicing from expression data.

Melissa S. Cline; John Blume; Simon Cawley; Tyson Clark; Jing-shan Hu; Gang Lu; Nathan Salomonis; Hui Wang; Alan Williams

2005-01-01

362

Poliovirus 2A Protease Triggers a Selective Nucleo-Cytoplasmic Redistribution of Splicing Factors to Regulate Alternative Pre-mRNA Splicing  

PubMed Central

Poliovirus protease 2A (2Apro) obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2Apro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2Apro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2Apro-expressing cells. Therefore, poliovirus 2Apro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

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

2013-01-01

363

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer.  

PubMed

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism. PMID:23707073

Babic, Ivan; Anderson, Erik S; Tanaka, Kazuhiro; Guo, Deliang; Masui, Kenta; Li, Bing; Zhu, Shaojun; Gu, Yuchao; Villa, Genaro R; Akhavan, David; Nathanson, David; Gini, Beatrice; Mareninov, Sergey; Li, Rui; Camacho, Carolina Espindola; Kurdistani, Siavash K; Eskin, Ascia; Nelson, Stanley F; Yong, William H; Cavenee, Webster K; Cloughesy, Timothy F; Christofk, Heather R; Black, Douglas L; Mischel, Paul S

2013-05-23

364

Alternative Splicing Regulates Prdm1/Blimp-1 DNA Binding Activities and Corepressor Interactions  

PubMed Central

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-1?exon7) predicted to have distinct functions. Here we demonstrate that the short Blimp-1?exon7 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-1?exon7 die at around embryonic day 10.5 (E10.5) due to placental defects. In heterozygous ?exon7 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-1?exon7 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.

Morgan, Marc A. J.; Mould, Arne W.; Li, Li; Robertson, Elizabeth J.

2012-01-01

365

Identification of an Alternate Splice Form of Tapasin in Human Melanoma  

PubMed Central

Assembly of MHC class I molecules with peptide in the endoplasmic reticulum requires the assistance of tapasin. Alternative splicing, which is known to regulate many genes, has been reported for tapasin only in the context of mutations. Here, we report on an alternate splice form of tapasin (tpsn?Ex3) derived from a human melanoma cell line that does not appear to be due to mutations. Excision of exon 3 results in deletion of amino acids 70 to 156 within the beta barrel region, but the membrane proximal Ig domain, the transmembrane domain and cytoplasmic tail of tapasin are intact. Introduction of tpsn?Ex3 into a tapasin deficient cell line does not restore MHC class I expression at the cell surface. Similar to a previously described tapasin mutant (tpsn?N50), tpsn?Ex3 interacts with TAP. Therefore we utilized these altered forms of tapasin to test the importance of MHC class I interaction with TAP. In the presence of wild type tapasin, transfection of tpsn?N50, but not tpsn?Ex3, reduced MHC class I expression at the cell surface likely due its ability to compete MHC class I molecules from TAP. Together these findings suggest that tumor cells may contain alternate splice forms of tapasin which may regulate MHC class I antigen presentation.

Belicha-Villanueva, Alan; Golding, Michelle; McEvoy, Sarah; Sarvaiya, Nilofar; Cresswell, Peter; Gollnick, Sandra O.; Bangia, Naveen

2010-01-01

366

Alternative splicing regulates Prdm1/Blimp-1 DNA binding activities and corepressor interactions.  

PubMed

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-1?exon7) predicted to have distinct functions. Here we demonstrate that the short Blimp-1?exon7 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-1?exon7 die at around embryonic day 10.5 (E10.5) due to placental defects. In heterozygous ?exon7 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-1?exon7 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

Morgan, Marc A J; Mould, Arne W; Li, Li; Robertson, Elizabeth J; Bikoff, Elizabeth K

2012-06-25

367

Eye development under the control of SRp55/B52-mediated alternative splicing of eyeless.  

PubMed

The genetic programs specifying eye development are highly conserved during evolution and involve the vertebrate Pax-6 gene and its Drosophila melanogaster homolog eyeless (ey). Here we report that the SR protein B52/SRp55 controls a novel developmentally regulated splicing event of eyeless that is crucial for eye growth and specification in Drosophila. B52/SRp55 generates two isoforms of eyeless differing by an alternative exon encoding a 60-amino-acid insert at the beginning of the paired domain. The long isoform has impaired ability to trigger formation of ectopic eyes and to bind efficiently Eyeless target DNA sequences in vitro. When over-produced in the eye imaginal disc, this isoform induces a small eye phenotype, whereas the isoform lacking the alternative exon triggers eye over-growth and strong disorganization. Our results suggest that B52/SRp55 splicing activity is used during normal eye development to control eye organogenesis and size through regulation of eyeless alternative splicing. PMID:17327915

Fic, Weronika; Juge, François; Soret, Johann; Tazi, Jamal

2007-02-28

368

Auto- and cross-regulation of the hnRNP L proteins by alternative splicing.  

PubMed

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This "poison exon" is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins. PMID:19124611

Rossbach, Oliver; Hung, Lee-Hsueh; Schreiner, Silke; Grishina, Inna; Heiner, Monika; Hui, Jingyi; Bindereif, Albrecht

2009-01-05

369

Auto- and Cross-Regulation of the hnRNP L Proteins by Alternative Splicing? ‡  

PubMed Central

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This “poison exon” is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

Rossbach, Oliver; Hung, Lee-Hsueh; Schreiner, Silke; Grishina, Inna; Heiner, Monika; Hui, Jingyi; Bindereif, Albrecht

2009-01-01

370

The Ewing sarcoma protein regulates DNA damage-induced alternative splicing.  

PubMed

The Ewing sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA- and DNA-binding proteins whose expression is altered in cancer. We report that EWS depletion results in alternative splicing changes of genes involved in DNA repair and genotoxic stress signaling, including ABL1, CHEK2, and MAP4K2. Chromatin and RNA crosslinking immunoprecipitation results indicate that EWS cotranscriptionally binds to its target RNAs. This association is reduced upon irradiation of cells with ultraviolet light, concomitant with transient enrichment of EWS in nucleoli and with alternative splicing changes that parallel those induced by EWS depletion and that lead to reduced c-ABL protein expression. Consistent with the functional relevance of EWS-mediated alternative splicing regulation in DNA damage response, EWS depletion reduces cell viability and proliferation upon UV irradiation, effects that are attenuated by restoring c-ABL expression. These results provide insights into posttranscriptional mechanisms of DNA damage response by a TET protein. PMID:21816343

Paronetto, Maria Paola; Miñana, Belén; Valcárcel, Juan

2011-08-01

371

SAM68 regulates neuronal activity-dependent alternative splicing of Neurexin-1  

PubMed Central

Summary The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA binding protein SAM68 which associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Iijima, Takatoshi; Wu, Karen; Witte, Harald; Hanno-Iijima, Yoko; Glatter, Timo; Richard, Stephane; Scheiffele, Peter

2011-01-01

372

Periostin shows increased evolutionary plasticity in its alternatively spliced region  

PubMed Central

Background Periostin (POSTN) is a secreted extracellular matrix protein of poorly defined function that has been related to bone and heart development as well as to cancer. In human and mouse, it is known to undergo alternative splicing in its C-terminal region, which is devoid of known protein domains. Differential expression of periostin, sometimes of specific splicing isoforms, is observed in a broad range of human cancers, including breast, pancreatic, and colon cancer. Here, we combine genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C-terminal region. Results We found that the C-terminal part of periostin is markedly more variable among vertebrates than the rest of periostin in terms of exon count, length, and splicing pattern, which we interpret as a consequence of neofunctionalization after the split between periostin and its paralog transforming growth factor, beta-induced (TGFBI). We also defined periostin's sequential 13-amino acid repeat units - well conserved in teleost fish, but more obscure in higher vertebrates - whose secondary structure is predicted to be consecutive beta strands. We suggest that these beta strands may mediate binding interactions with other proteins through an extended beta-zipper in a manner similar to the way repeat units in bacterial cell wall proteins have been reported to bind human fibronectin. Conclusions Our results, obtained with the help of the increasingly large collection of complete vertebrate genomes, document the evolutionary plasticity of periostin's C-terminal region, and for the first time suggest a basis for its functional role.

2010-01-01

373

In Vivo Regulation of Alternative Pre-mRNA Splicing by the Clk1 Protein Kinase  

Microsoft Academic Search

truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing

PETER I. DUNCAN; DAVID F. STOJDL; RICARDO M. MARIUS; JOHN C. BELL