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1

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

2

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

3

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

4

Alternative-splicing-mediated gene expression  

NASA Astrophysics Data System (ADS)

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

Wang, Qianliang; Zhou, Tianshou

2014-01-01

5

Elements of the rat tropoelastin gene associated with alternative splicing.  

PubMed

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA. PMID:1572637

Pierce, R A; Alatawi, A; Deak, S B; Boyd, C D

1992-04-01

6

Alternative splicing in the fragile X gene FMR1.  

PubMed

The FMR1 gene, associated with fragile X syndrome, has recently been cloned and the sequence of partial cDNA clones is known. We have determined additional cDNA sequences both at the 5' and 3' end. We have characterized the expressed gene by means of RT-PCR in various tissues and have found that alternative splicing takes place in the FMR1 gene, which does not seem to be tissue specific. When the different alternative splicing events are combined, 12 distinct mRNA products could result from FMR1 expression in each tested tissue. In all these transcripts the open reading frame is maintained until the same stop codon. At the 3' end alternative use of polyadenylation signals is found. The alternative splicing allows functional diversity of the FMR-1 gene. Whether all the possible proteins will be synthesized and whether they will be functionally active has to be determined. PMID:8504300

Verkerk, A J; de Graaff, E; De Boulle, K; Eichler, E E; Konecki, D S; Reyniers, E; Manca, A; Poustka, A; Willems, P J; Nelson, D L

1993-04-01

7

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

PubMed

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. PMID:22123740

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

2012-03-01

8

Exon expression and alternatively spliced genes in Tourette Syndrome.  

PubMed

Tourette Syndrome (TS) is diagnosed based upon clinical criteria including motor and vocal tics. We hypothesized that differences in exon expression and splicing might be useful for pathophysiology and diagnosis. To demonstrate exon expression and alternatively spliced gene differences in blood of individuals with TS compared to healthy controls (HC), RNA was isolated from the blood of 26 un-medicated TS subjects and 23 HC. Each sample was run on Affymetrix Human Exon 1.0 ST (HuExon) arrays and on 3' biased U133 Plus 2.0 (HuU133) arrays. To investigate the differentially expressed exons and transcripts, analyses of covariance (ANCOVA) were performed, controlling for age, gender, and batch. Differential alternative splicing patterns between TS and HC were identified using analyses of variance (ANOVA) models in Partek. Three hundred and seventy-six exon probe sets were differentially expressed between TS and HC (raw P?|1.2|) that separated TS and HC subjects using hierarchical clustering and Principal Components Analysis. The probe sets predicted TS compared to HC with a >90% sensitivity and specificity using a 10-fold cross-validation. Ninety genes (transcripts) had differential expression of a single exon (raw P?alternatively spliced (raw P?

Tian, Yingfang; Liao, Isaac H; Zhan, Xinhua; Gunther, Joan R; Ander, Bradley P; Liu, Dazhi; Lit, Lisa; Jickling, Glen C; Corbett, Blythe A; Bos-Veneman, Netty G P; Hoekstra, Pieter J; Sharp, Frank R

2011-01-01

9

Gene duplication followed by exon structure divergence substitutes for alternative splicing in zebrafish.  

PubMed

In this study we report novel findings regarding the evolutionary relationship between gene duplication and alternative splicing, two processes that increase proteomic diversity. By studying teleost fish, we find that gene duplication followed by exon structure divergence between paralogs, but not gene duplication alone, leads to a significant reduction in alternative splicing, as measured by both the proportion of genes that undergo alternative splicing as well as mean number of transcripts per gene. Additionally, we show that this effect is independent of gene family size and gene function. Furthermore, we provide evidence that the reduction in alternative splicing may be due to the partitioning of ancestral splice forms among the duplicate genes - a form of subfunctionalization. Taken together these results indicate that exon structure evolution subsequent to gene duplication may be a common substitute for alternative splicing. PMID:24942242

Lambert, Matthew J; Olsen, Kyle G; Cooper, Cynthia D

2014-08-10

10

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

11

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms  

PubMed Central

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Emmert, Steffen; Schneider, Thomas D.; Khan, Sikandar G.; Kraemer, Kenneth H.

2001-01-01

12

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

13

Alternative Splicing Events Is Not a Key Event for Gene Expression Regulation in Uremia  

PubMed Central

Background The control of gene expression in the course of chronic kidney disease (CKD) is not well addressed. Alternative splicing is a common way to increase complexity of proteins. More than 90% of human transcripts are alternatively spliced. We hypothesised that CKD can induce modification of the alternative splicing machinery. Methods During mutation screening in autosomal dominant polycystic kidney disease, we identified in mononuclear cells (PBMC), an alternative splicing event on the exon 30 of PKD1 gene, the gene implicated in this disease. This alternative splice variant was not correlated with the cystic disease but with CKD. To confirm the association between this variant and CKD, a monocentric clinical study was performed with 3 different groups according to their kidney function (CKD5D, CKD3-5 and normal kidney function). An exon microarray approach was used to highlight splicing events in whole human genome in a normal cell model (fibroblasts) incubated with uremic serum. Alternative splicing variants identified were confirmed by RT-PCR. Results The splicing variant of the exon 30 of PKD1 was more frequent in PBMCs from patients with CKD compared to control. With the microarray approach, despite the analysis of more than 230 000 probes, we identified 36 genes with an abnormal splicing index evocating splicing event in fibroblasts exposed to uremic serum. Only one abnormal splicing event in one gene, ADH1B, was confirmed by RT-PCR. Conclusion We observed two alternative spliced genes in two different cell types associated with CKD. Alternative splicing could play a role in the control of gene expression during CKD but it does not seem to be a major mechanism.

Sallee, Marion; Fontes, Michel; Louis, Laurence; Cerini, Claire; Brunet, Philippe; Burtey, Stephane

2013-01-01

14

Alternative splicing of breast cancer associated gene BRCA1 from breast cancer cell line.  

PubMed

Breast cancer is the most common malignancy among women, and mutations in the BRCA1 gene produce increased susceptibility to these malignancies in certain families. In this study, the forward 1-13 exons of breast cancer associated gene BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, five splice forms of which were novel. These splice isoforms were produced from the molecular mechanism of 5' and 3' alternative splicing. All these splice forms deleting exon 11b and the locations of alternative splicing were focused on two parts:one was exons 2 and 3, and the other was exons 9 and 10. These splice forms accorded with GT-AG rule. Most these BRCA1 splice variants still kept the original reading frame. Western blot analysis indicated that some BRCA1 splice variants were expressed in ZR-75-30 cell line at the protein level. In addition, we confirmed the presence of these new transcripts of BRCA1 gene in MDA-MB-435S, K562, Hela, HLA, HIC, H9, Jurkat and human fetus samples by RT-PCR analysis. These results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants. We hypothesized that alternative splicing of BRCA1 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer. Thus, the identification of cancer-specific splice forms will provide a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention. PMID:17244477

Lixia, Miao; Zhijian, Cao; Chao, Shen; Chaojiang, Gu; Congyi, Zheng

2007-01-31

15

The Evolution of an Alternatively Spliced Exon in the ?A-Crystallin Gene  

Microsoft Academic Search

.   The evolutionary aspects of alternative splicing, as a mechanism to increase the diversity of gene products, are poorly understood.\\u000a Here we analyse the evolution of a 69-bp exon that is alternatively spliced in the primary transcript of the gene for the\\u000a mammalian eye lens protein ?A-crystallin. In rodents, the skipping of this exon 2 is attributed to the presence

Marjon A. M. van Dijk; Marcel A. M. Sweers; Wilfried W. de Jong

2001-01-01

16

Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene.  

PubMed Central

Alternative splicing is a common mechanism for regulating gene expression in different cell types. In order to understand this important process, the trans-acting factors that enforce the choice of particular splicing pathways in different environments must be identified. We have used the rat alpha-tropomyosin gene as a model system of tissue-specific alternative splicing. Exon 3 of alpha-tropomyosin is specifically inhibited in smooth muscle cells allowing the alternative inclusion of exon 2. We have used a novel gene transfer and selection strategy to detect a gene whose expression in fibroblasts is sufficient to switch them to smooth muscle-specific splicing of alpha-tropomyosin and also alpha-actinin. Extracts from the regulating fibroblasts contain an apparently novel 55 kDa protein which binds to RNA elements required for regulation of tropomyosin splicing. This protein is not detected in extracts of non-regulating cells and is therefore a strong candidate cell-specific splicing regulator. These experiments advance our understanding of smooth muscle splicing regulation as well as establishing a means for direct cloning of tissue-specific splicing regulators which have so far been refractory to biochemical analysis. Images

Roberts, G C; Gooding, C; Smith, C W

1996-01-01

17

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

PubMed Central

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

2014-01-01

18

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

PubMed Central

The mouse ?-opioid receptor gene, Oprm1, currently contains 18 recognized alternatively spliced exons (Doyle et al., 2007) that generate 27 splice variants encoding at least 11 morphine-binding isoforms of the receptor. Here, we identify five MOR variants that contain an as yet undescribed exon (exon 19) of the gene, and we provide evidence that these MOR splice variants are expressed in the C57BL/6 and DBA/2 mouse strains. Three splice variants, MOR-1Eii, MOR-1Eiii and MOR-1Eiv, encode the MOR-1E isoform and contain the newly identified exon 19 in their 3’ untranslated regions. The fourth splice variant encodes a novel ?-opioid receptor isoform, MOR-1U, and contains exon 19 in its coding region. The cytoplasmic tail of the putative MOR-1U isoform contains a putative nuclear localization signal encoded by the sequence of exon 19. Exon 19 appears to be conserved in the rat, but not in humans. In mouse and rat Oprm1, exon 19 is located between described exons 7 and 8. We also report the cloning of the “full-length” MOR-1T splice variant (Kvam et al., 2004) that encodes MOR-1 and contains the newly identified exon in its 3’ UTR. RT-PCR analysis suggests that splice variants MOR-1Eii, MOR-1Eiii, MOR-1Eiv, MOR-1T and MOR-1U are expressed in all brain regions analyzed (cortex, cerebellum, hypothalamus, thalamus and striatum). These exon 19-containing splice variants add to the growing complexity of the mouse Oprm1 gene.

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

2008-01-01

19

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

20

A subgroup of MYB transcription factor genes undergoes highly conserved alternative splicing in Arabidopsis and rice.  

PubMed

MYB transcription factor genes play important roles in many developmental processes and in various defence responses of plants. Two Arabidopsis R2R3-type MYB genes, AtMYB59 and AtMYB48, were found to undergo similar alternative splicing. Both genes have four distinctively spliced transcripts that encode either MYB-related proteins or R2R3-MYB proteins. An extensive BLAST search of the GenBank database resulted in finding and cloning two rice homologues, both of which were also found to share a similar alternative splicing pattern. In a semi-quantitative study, the expression of one splice variant of AtMYB59 was found to be differentially regulated in treatments with different phytohormones and stresses. GFP fusion protein analysis revealed that both of the two predicted nuclear localization signals (NLSs) in the R3 domain are required for localizing to the nucleus. Promoter-GUS analysis in transgenic plants showed that 5'-UTR is sufficient for the translation initiation of type 3 transcripts (encoding R2R3-MYB proteins), but not for type 2 transcripts (encoding MYB-related proteins). Moreover, a new type of non-canonical intron, with the same nucleotide repeats at the 5' and 3' splice sites, was identified. Thirty-eight Arabidopsis and rice genes were found to have this type of non-canonical intron, most of which undergo alternative splicing. These data suggest that this subgroup of transcription factor genes may be involved in multiple biological processes and may be transcriptionally regulated by alternative splicing. PMID:16531467

Li, Jigang; Li, Xiaojuan; Guo, Lei; Lu, Feng; Feng, Xiaojie; He, Kun; Wei, Liping; Chen, Zhangliang; Qu, Li-Jia; Gu, Hongya

2006-01-01

21

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

22

ECgene: Genome-based EST clustering and gene modeling for alternative splicing  

PubMed Central

With the availability of the human genome map and fast algorithms for sequence alignment, genome-based EST clustering became a viable method for gene modeling. We developed a novel gene-modeling method, ECgene (Gene modeling by EST Clustering), which combines genome-based EST clustering and the transcript assembly procedure in a coherent and consistent fashion. Specifically, ECgene takes alternative splicing events into consideration. The position of splice sites (i.e., exon–intron boundaries) in the genome map is utilized as the critical information in the whole procedure. Sequences that share any splice sites are grouped together to define an EST cluster in a manner similar to that of the genome-based version of the UniGene algorithm. Transcript assembly is achieved using graph theory that represents the exon connectivity in each cluster as a directed acyclic graph (DAG). Distinct paths along exons correspond to possible gene models encompassing all alternative splicing events. EST sequences in each cluster are subclustered further according to the compatibility with gene structure of each splice variant, and they can be regarded as clone evidence for the corresponding isoform. The reliability of each isoform is assessed from the nature of cluster members and from the minimum number of clones required to reconstruct all exons in the transcript.

Kim, Namshin; Shin, Seokmin; Lee, Sanghyuk

2005-01-01

23

Alternative splicing in regulation of cholesterol homeostasis  

PubMed Central

Purpose of review With the advent of whole-transcriptome sequencing, or RNA-seq, we now know that alternative splicing is a generalized phenomenon, with nearly all multi-exonic genes subject to alternative splicing. In this review we highlight recent studies examining alternative splicing as a modulator of cellular cholesterol homeostasis, and as an underlying mechanism of dyslipidemia. Recent findings A number of key genes involved in cholesterol metabolism are known to undergo functionally relevant alternative splicing. Recently, we have identified coordinated changes in alternative splicing in multiple genes in response to alteration in cellular sterol content. We and others have implicated several splicing factors as regulators of lipid metabolism. Furthermore, a number of cis-acting human gene variants that modulate alternative splicing have been implicated in a variety of human metabolic diseases. Summary Alternative splicing is of importance in various types of genetically influenced dyslipidemias, and in the regulation of cellular cholesterol metabolism.

Medina, Marisa W.; Krauss, Ronald M.

2013-01-01

24

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.  

PubMed

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron. PMID:10767559

Morisaki, H; Morisaki, T; Kariko, K; Genetta, T; Holmes, E W

2000-04-01

25

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

PubMed Central

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

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

2011-01-01

26

Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes  

PubMed Central

Background Several recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes. Results In a comprehensive analysis of the Arabidopsis thaliana genome, we identified 6,772 introns that exhibit a NAGNAG acceptor motif. Alternative splicing at these acceptors was assessed using available EST data, complemented by a sequence-based prediction method. Of the 36 identified introns within 30 SR and SR-related protein-coding genes that have a NAGNAG acceptor, we selected 15 candidates for an experimental analysis of alternative splicing under several conditions. We provide experimental evidence for 8 of these candidates being alternatively spliced. Quantifying the ratio of NAGNAG-derived splice variants under several conditions, we found organ-specific splicing ratios in adult plants and changes in seedlings of different ages. Splicing ratio changes were observed in response to heat shock and most strikingly, cold shock. Interestingly, the patterns of differential splicing ratios are similar for all analyzed genes. Conclusion NAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. A lack of extensive EST coverage can be compensated by using the proposed sequence-based method to predict alternative splicing at these acceptors. Our findings indicate that the differential effects on NAGNAG alternative splicing in SR and SR-related genes are organ- and condition-specific rather than gene-specific.

Schindler, Stefanie; Szafranski, Karol; Hiller, Michael; Ali, Gul Shad; Palusa, Saiprasad G; Backofen, Rolf; Platzer, Matthias; Reddy, Anireddy SN

2008-01-01

27

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

PubMed

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

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

2014-02-01

28

Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes.  

PubMed

Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53? expression and induced the expression of p53?, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3. PMID:24333670

Lu, Guan-Yu; Huang, Shih-Ming; Liu, Shu-Ting; Liu, Pei-Yao; Chou, Wei-Yuan; Lin, Wei-Shiang

2014-02-01

29

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

30

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

31

Radiolabeled Semi-quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes.  

PubMed

Alternative splicing evolved as a very efficient way to generate proteome diversity from a limited number of genes, while at the same time modulating posttranscriptional events of gene expression-such as stability, turnover, subcellular localization, binding properties, and general activity of both mRNAs and proteins. Since the vast majority of human genes undergo alternative splicing, it comes to no surprise that interleukin genes also show extensive alternative splicing. In fact, there is a growing body of evidence indicating that alternative splicing plays a central role in modulating the pleiotropic functions of cytokines, and aberrant expression of alternatively spliced interleukin mRNAs has been linked to disease. However, while several interleukin splice variants have been described, their function is still poorly understood. This is particularly relevant, since alternatively spliced cytokine isoforms can act both as disease biomarkers and as candidate entry points for therapeutic intervention. In this chapter we describe a protocol that uses radiolabeled semi-quantitative RT-PCR to efficiently detect, analyze, and quantify alternative splicing patterns of cytokine genes. PMID:24908320

Shakola, Felitsiya; Byrne, Stephen; Javed, Kainaat; Ruggiu, Matteo

2014-01-01

32

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

SciTech Connect

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

Smirnova, Anna S. [Immunogenetics Division, Pediatrics Department, Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo (Brazil); Morgun, Andrey [Immunogenetics Division, Pediatrics Department, Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo (Brazil)]. E-mail: anemorgun@hotmail.com; Shulzhenko, Natalia [Immunogenetics Division, Pediatrics Department, Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo (Brazil); Silva, Ismael D.C.G. [Gynecology Department, Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo (Brazil); Gerbase-DeLima, Maria [Immunogenetics Division, Pediatrics Department, Universidade Federal de Sao Paulo (UNIFESP-EPM), Sao Paulo (Brazil)

2005-05-13

33

Differentially expressed alternatively spliced genes in Malignant Pleural Mesothelioma identified using massively parallel transcriptome sequencing  

PubMed Central

Background Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples. Methods We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens. Results We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively). Conclusion Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

2009-01-01

34

Alternative splicing of the BSC1 gene generates tissue-specific isoforms in the German cockroach.  

PubMed

Voltage-gated sodium channels are integral transmembrane proteins responsible for the rapidly-rising phase of action potentials in most excitable cells. In mammals, the functional diversity and wide distribution of sodium channel proteins in various tissues and cell types are achieved mainly by selective expression of many distinct sodium channel genes. In the model insect, Drosophila melanogaster, however, only one confirmed sodium channel gene, para, and one putative sodium channel gene, DSC1, are known. We cloned and sequenced a DSC1 ortholog, BSC1, from the German cockroach, Blattella germanica. We found that the BSC1 transcript was present in a wide range of tissues, including nerve cord, muscle, gut, fat body and ovary, whereas the para transcript was detected only in nerve cord and muscle. Moreover, different tissues contained distinct alternatively spliced variants of BSC1, and two muscle-specific spliced variants are predicted to encode truncated proteins with only the first two of the four homologous domains. Therefore, alternative splicing and expression of distinct splicing variants in functionally different tissues may be a major mechanism by which insects increase BSC1 channel diversity in neuronal and non-neuronal tissues. PMID:11267908

Liu, Z; Chung, I; Dong, K

2001-04-27

35

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

36

MUTYH gene expression and alternative splicing in controls and polyposis patients.  

PubMed

Mutational loss of the human DNA repair gene MUTYH in the germline predisposes for colorectal polyposis and cancer, a recessively heritable disease called MUTYH-associated polyposis. The MUTYH gene shows heavy alternative splicing, but the transcripts relevant for biological function and cancer prevention have not been determined. This knowledge is required to assess the consequences that germline variants of unknown functional significance may have. We therefore quantified expression and investigated patterns of alternative splicing in control individuals, tissue samples, and carriers of two frequent germline alterations. MUTYH expression differed organ dependently, correlating with proliferative activity. Alternative first exons were used tissue specifically; transcripts for mitochondrial proteins predominated in muscle tissues, while ascending colon and testes showed the highest fractions of transcripts for nuclear proteins. Colon cancer cell lines produced predominant transcripts for nuclear protein. Exon skipping was frequent and governed by splice-site quality. Five transcripts were found to encode the biologically relevant products of the MUTYH gene. Carriers of the disease-causing mutation c.1187G>A (p.Gly396Asp) showed normal transcript composition, but the frequent single-nucleotide polymorphism rs3219468:G>C largely reduced one transcript species of MUTYH. Since this alteration decreases protein production of the gene, an increased cancer risk for compound heterozygous carriers is possible. PMID:22473953

Plotz, Guido; Casper, Markus; Raedle, Jochen; Hinrichsen, Inga; Heckel, Verena; Brieger, Angela; Trojan, Jörg; Zeuzem, Stefan

2012-07-01

37

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

38

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

39

Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.  

PubMed

This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF(65) overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF(65) alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence. PMID:24749484

Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

2014-07-01

40

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

PubMed

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

Chen, J; Patton, J R

2000-12-01

41

Key features of the two-intron Saccharomyces cerevisiae gene SUS1 contribute to its alternative splicing  

PubMed Central

Alternative pre-mRNA splicing allows dramatic expansion of the eukaryotic proteome and facilitates cellular response to changes in environmental conditions. The Saccharomyces cerevisiae gene SUS1, which encodes a protein involved in mRNA export and histone H2B deubiquitination, contains two introns; non-canonical sequences in the first intron contribute to its retention, a common form of alternative splicing in plants and fungi. Here we show that the pattern of SUS1 splicing changes in response to environmental change such as temperature elevation, and the retained intron product is subject to nonsense-mediated decay. The activities of different splicing factors determine the pattern of SUS1 splicing, including intron retention and exon skipping. Unexpectedly, removal of the 3? intron is affected by splicing of the upstream intron, suggesting that cross-exon interactions influence intron removal. Production of different SUS1 isoforms is important for cellular function, as we find that the temperature sensitivity and histone H2B deubiquitination defects observed in sus1? cells are only partially suppressed by SUS1 cDNA, but SUS1 that is able to undergo splicing complements these phenotypes. These data illustrate a role for S. cerevisiae alternative splicing in histone modification and cellular function and reveal important mechanisms for splicing of yeast genes containing multiple introns.

Hossain, Munshi Azad; Rodriguez, Caitlin M.; Johnson, Tracy L.

2011-01-01

42

Complex Alternative Splicing of the Smarca2 Gene Suggests the Importance of Smarca2-B Variants  

PubMed Central

BRM is an ATPase component of the SWI/SNF complex that regulates chromatin remodeling and cell proliferation and is considered a tumor suppressor. In this study we characterized transcripts from the Smarca2 gene that encodes the BRM protein. We found that the human Smarca2 gene (hSmarca2), like its mouse counterpart (mSmarca2), also initiated a short transcript from intron 27 of the long transcript. We name the long and short transcripts as Smarca2-a and Smarca2-b, respectively. Like its human counterpart, mSmarca2-a also underwent alternative splicing at the 54-bp exon 29. The hSmarca2-b had two alternative initiation sites and underwent alternative splicing at three different 3' sites of exon 1 and at exons 2, 3 and/or 5. We identified nine hSmarca2-b mRNA variants that might produce five different proteins. mSmarca2-b also underwent alternative splicing at exon 3 and/or exon 5, besides alternatively retaining part of intron 1 in exon 1. Smarca2-b was expressed more abundantly than Smarca2-a in many cell lines and was more sensitive to serum starvation. Moreover, cyclin D1 also regulated the expression of both Smarca2-a and Smarca2-b in a complex manner. These data suggest that the functions of the Smarca2 gene may be very complex, not just simply inhibiting cell proliferation, and in certain situations may be elicited mainly by expressing the much less known Smarca2-b, not the better studied Smarca2-a and its products BRM proteins.

Yang, Min; Sun, Yuan; Ma, Ling; Wang, Chenguang; Wu, Jian-min; Bi, Anding; Liao, D. Joshua

2011-01-01

43

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes  

PubMed Central

Background Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining. Results The human TXNRD1 gene spans 100 kb of genomic DNA organised into 16 exons and the mouse Txnrd1 gene has a similar exon/intron arrangement. We also analysed the alternative splicing patterns displayed by the mouse and human thioredoxin reductase 1 genes and mapped the different mRNA isoforms with respect to genomic organisation. These isoforms differ at the 5' end and encode putative proteins of different molecular mass. Genomic DNA sequences upstream of mouse exon 1 were compared to the human promoter to identify conserved elements. Conclusions The human and mouse thioredoxin reductase 1 gene organisation is highly conserved and both genes exhibit alternative splicing at the 5' end. The mouse and human promoters share some conserved sequences.

Osborne, Simone A; Tonissen, Kathryn F

2001-01-01

44

Alternative splicing and promoter use in the human GABRA2 gene.  

PubMed

GABA(A) receptors mediate the majority of the fast synaptic inhibition in the mammalian brain. They are the targets of several important drugs, including benzodiazepines, which are used as anxiolytics, sedatives, anti-convulsants, and in the treatment of alcohol withdrawal symptoms. Non-coding variations in GABRA2, the gene encoding the alpha2 subunit, are associated with the risk for alcoholism, suggesting that regulatory differences are important. GABRA2 mRNAs from whole human brain and from three brain regions were examined for evidence of alternative splicing using reverse transcription-PCR and DNA sequencing. A complex pattern of alternative splicing and alternative promoter use of the human GABRA2 mRNA was demonstrated. There are four major isoforms consisting of combinations of two alternative 5' and 3' exons, as well as minor isoforms lacking exon 4 or exon 8. The alternative 5' exons each lie downstream of a functional promoter sequence, as shown by transient transfection assays. The promoter activities of naturally occurring haplotypes differed, indicating genetic differences in gene expression. PMID:15950776

Tian, Huijun; Chen, Hui-Ju; Cross, Tiffeny H; Edenberg, Howard J

2005-06-13

45

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

SciTech Connect

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

Xu, Lin; Sterner, C.; Maheshwar, M.M. [and others] [and others

1995-06-10

46

Conserved RNA secondary structures promote alternative splicing  

Microsoft Academic Search

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alter- native splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexi- bility, splice-site selection in higher eukaryotes

PETER J. SHEPARD; KLEMENS J. HERTEL

2008-01-01

47

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

48

Alternative splicing regulation at tandem 3? splice sites  

PubMed Central

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ?25% are related to alternative choices of 3? and 5? splice sites. Surprisingly, half of all these events involve 3? splice sites that are exactly 3 nt apart. These tandem 3? splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ?30 bp at the intronic regions flanking the 3? splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.

Akerman, Martin; Mandel-Gutfreund, Yael

2006-01-01

49

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting.  

PubMed

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift. PMID:15623537

Levitin, Fiana; Baruch, Amos; Weiss, Mordechai; Stiegman, Keren; Hartmann, Mor-Li; Yoeli-Lerner, Merav; Ziv, Ravit; Zrihan-Licht, Sheila; Shina, Sima; Gat, Andrea; Lifschitz, Beatrice; Simha, Moshe; Stadler, Yona; Cholostoy, Alina; Gil, Benny; Greaves, David; Keydar, Iafa; Zaretsky, Joseph; Smorodinsky, Nechama; Wreschner, Daniel H

2005-03-18

50

Functional consequences of developmentally regulated alternative splicing  

PubMed Central

Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodeling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of biological processes. The functional classes of genes that are regulated by alternative splicing include both those with widespread homeostatic activities and genes with cell-type-specific functions. Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is utilized by other regulatory mechanisms.

Kalsotra, Auinash; Cooper, Thomas A.

2012-01-01

51

Alternative Splicing in Plant Immunity  

PubMed Central

Alternative splicing (AS) occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R) genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel.

Yang, Shengming; Tang, Fang; Zhu, Hongyan

2014-01-01

52

Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5) and altered Axin-1 expression during tumour progression  

PubMed Central

Background Splicing processes might play a major role in carcinogenesis and tumour progression. The Wnt pathway is of crucial relevance for cancer progression. Therefore we focussed on the Wnt/?-catenin signalling pathway in order to validate the expression of sequences predicted as alternatively spliced by bioinformatic methods. Splice variants of its key molecules were selected, which may be critical components for the understanding of colorectal tumour progression and may have the potential to act as biological markers. For some of the Wnt pathway genes the existence of splice variants was either proposed (e.g. ?-Catenin and CTNNB1) or described only in non-colon tissues (e.g. GSK3?) or hitherto not published (e.g. LRP5). Results Both splice variants – normal and alternative form – of all selected Wnt pathway components were found to be expressed in cell lines as well as in samples derived from tumour, normal and healthy tissues. All splice positions corresponded totally with the bioinformatical prediction as shown by sequencing. Two hitherto not described alternative splice forms (CTNNB1 and LRP5) were detected. Although the underlying EST data used for the bioinformatic analysis suggested a tumour-specific expression neither a qualitative nor a significant quantitative difference between the expression in tumour and healthy tissues was detected. Axin-1 expression was reduced in later stages and in samples from carcinomas forming distant metastases. Conclusion We were first to describe that splice forms of crucial genes of the Wnt-pathway are expressed in human colorectal tissue. Newly described splicefoms were found for ?-Catenin, LRP5, GSK3?, Axin-1 and CtBP1. However, the predicted cancer specificity suggested by the origin of the underlying ESTs was neither qualitatively nor significant quantitatively confirmed. That let us to conclude that EST sequence data can give adequate hints for the existence of alternative splicing in tumour tissues. That no difference in the expression of these splice forms between cancerous tissues and normal mucosa was found, may indicate that the existence of different splice forms is of less significance for cancer formation as suggested by the available EST data. The currently available EST source is still insufficient to clearly deduce colon cancer specificity. More EST data from colon (tumour and healthy) is required to make reliable predictions.

Pospisil, Heike; Herrmann, Alexander; Butherus, Kristine; Pirson, Stefan; Reich, Jens G; Kemmner, Wolfgang

2006-01-01

53

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

54

Alternative initiation and splicing in dicer gene expression in human breast cells  

PubMed Central

Introduction Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. Method We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. Results We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. Conclusion Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells.

Irvin-Wilson, Charletha V; Chaudhuri, Gautam

2005-01-01

55

How prevalent is functional alternative splicing in the human genome?  

Microsoft Academic Search

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or ‘noise’). To address this question, we compared alternatively spliced cassette exons that are conserved

Rotem Sorek; Ron Shamir; Gil Ast

2004-01-01

56

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.  

PubMed

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development. PMID:15782199

Watahiki, Akira; Waki, Kazunori; Hayatsu, Norihito; Shiraki, Toshiyuki; Kondo, Shinji; Nakamura, Mari; Sasaki, Daisuke; Arakawa, Takahiro; Kawai, Jun; Harbers, Matthias; Hayashizaki, Yoshihide; Carninci, Piero

2004-12-01

57

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

58

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

PubMed

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

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

2013-07-30

59

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

60

E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35.  

PubMed

The transcription factor E2F1 has a key function during S phase progression and apoptosis. It has been well-demonstrated that the apoptotic function of E2F1 involves its ability to transactivate pro-apoptotic target genes. Alternative splicing of pre-mRNAs also has an important function in the regulation of apoptosis. In this study, we identify the splicing factor SC35, a member of the Ser-Rich Arg (SR) proteins family, as a new transcriptional target of E2F1. We demonstrate that E2F1 requires SC35 to switch the alternative splicing profile of various apoptotic genes such as c-flip, caspases-8 and -9 and Bcl-x, towards the expression of pro-apoptotic splice variants. Finally, we provide evidence that E2F1 upregulates SC35 in response to DNA-damaging agents and show that SC35 is required for apoptosis in response to these drugs. Taken together, these results demonstrate that E2F1 controls pre-mRNA processing events to induce apoptosis and identify the SC35 SR protein as a key direct E2F1-target in this setting. PMID:18806759

Merdzhanova, G; Edmond, V; De Seranno, S; Van den Broeck, A; Corcos, L; Brambilla, C; Brambilla, E; Gazzeri, S; Eymin, B

2008-12-01

61

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

2011-03-01

62

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

63

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

64

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

PubMed Central

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 skipped exons 4, 7 or 12. Exon 7 was skipped in ?0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in ?0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (–0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (?2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.

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

2002-01-01

65

Accurate identification of alternatively spliced exons using support vector machine  

Microsoft Academic Search

Motivation: Alternative splicing is a major component of the regulation acting on mammalian transcriptomes. It is esti- mated that over half of all human genes have more than one splice variant. Previous studies have shown that alterna- tively spliced exons possess several features that distinguish them from constitutively spliced ones. Recently, we have demonstrated that such features can be used

Gideon Dror; Rotem Sorek; Ron Shamir

2005-01-01

66

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

67

Characterization of the Sesbania rostrata phytochelatin synthase gene: alternative splicing and function of four isoforms.  

PubMed

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. PMID:20111680

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

2009-10-01

68

A novel zinc finger gene on human chromosome 1qter that is alternatively spliced in human tissues and cell lines  

SciTech Connect

DNA-binding proteins that share the conserved C[sub 2]-H[sub 2] zinc finger motif have been shown to have important roles as transcriptional regulators of gene expression and have been implicated in several hereditary human diseases. In order to define potential candidate genes for inherited disorders characterized by aberrant gene expression, the authors utilized Kruppel-related sequences to isolate zinc finger-containing cDNAs. They isolated and characterized two novel zinc finger-encoding cDNAs from a human hepatoblastoma cell line, which demonstrate DNA sequence homology to a recently described human Kruppel-related gene HZF-3 and appear to be derived from a single gene by alternate mRNA splicing. This gene, denoted HZF-16,' gives rise to at least two gene products. One cDNA (i.e., HZF-16.2) has nine zinc finger domains, while alternative splicing of the message gives rise to a smaller product (i.e., HZF-16.1) that has four domains. Despite the internal splicing event, both the 5[prime]- and 3[prime]-untranslated sequences in both cDNAs are identical, as are the first three domains. In the HZF-16.1 cDNA, the fourth zinc finger domain is a fusion product of domains four and nine of HZF-16.2 and could potentially give rise to a new DNA-binding specificity. These alternatively spliced transcripts are differentially regulated in human tissues and transformed cell lines and show a different distribution of expression between human cell lines and normal human tissue. This novel gene was mapped to human chromosome 1q44 by chromosomal in situ suppression hybridization and thus represents a candidate gene for trisomy 1q syndrome and for several other disorders. 40 refs., 6 figs.

Saleh, M.; Selleri, L.; Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

1993-01-01

69

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

PubMed Central

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

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

2012-01-01

70

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

PubMed Central

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

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

2010-01-01

71

Genomics of alternative splicing: evolution, development and pathophysiology.  

PubMed

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

Gamazon, Eric R; Stranger, Barbara E

2014-06-01

72

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

PubMed Central

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

Sheng, Juan-Juan; Jin, Jian-Ping

2014-01-01

73

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

74

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

PubMed Central

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

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

2011-01-01

75

ZEMa, a member of a novel group of MADS box genes, is alternatively spliced in maize endosperm.  

PubMed Central

The identification of a number of cis-elements which direct gene expression in maize endosperm, and the characterization of corresponding DNA binding proteins, point to the interaction of different classes of transcription factors in this tissue. To assess whether MADS box genes are also involved in maize endosperm development, cDNA and genomic MADS box clones have been isolated. The three cDNA clones ZEM1, ZEM2 and ZEM3 were cloned from a maize endosperm cDNA library using a probe based on sequences conserved in plant MADS box genes. Further transcripts were cloned by RT-PCR experiments and designated ZEM4 and ZEM5. Analysis of the corresponding genomic clones led to the identification of the ZEM2 MADS box gene family, three members of which were characterized sharing 97% sequence identity in corresponding domains. 100% sequence identities between cDNA and one of the genomic clones, conserved exon-intron boundaries and the demonstration of in vivo splicing in a maize endosperm transient expression system, show that the transcripts ZEM1-5 are derived by alternative splicing of ZEMa, one ZEM2 member. The ZEMa transcripts are present in almost all maize tissues, but specific differentially spliced forms accumulate preferentially in maturing endosperm and leaf. The function of the ZEMa gene is discussed in the light of similarities in the expression pattern with members of the human MEF2/RSRF gene family. Images

Montag, K; Salamini, F; Thompson, R D

1995-01-01

76

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

77

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

78

Human liver glucokinase gene: Cloning and sequence determination of two alternatively spliced cDNAs  

SciTech Connect

A human liver glucokinase was isolated from a liver cDNA library. This cDNA (hLGLK1) appeared to be full length as its size was consistent with a single 2.8-kilobase (kb) glucokinase mRNA on Northern blot analysis of liver poly(A){sup +}RNA. The cDNA contained an open reading frame of 1392 bp that predicted a protein of 464 amino acids and a molecular mass of 52 kDa; this protein has 97% identity to rat liver glucokinase. Fourteen residues on the amino terminus of the predicted human liver glucokinase, however, differed completely from those of the predicted rat liver enzyme and could be explained by alternative splicing of a 124-bp cassette exon in human cDNA. A second glucokinase cDNA (hLGLK2), missing the 124-bp cassette exon, was isolated by PCR amplification of human liver cDNA. These results suggested that the alternative splicing of a cassette exon in hLGLK1 resulted in an mRNA with an upstream initiator codon and reduced function. The relative biological activity of the two isoforms of human glucokinase and their possible developmental and/or metabolic regulation remain to be determined.

Tanizawa, Yukio; Koranyi, L.I.; Welling, C.M.; Permutt, M.A. (Washington Univ., St. Louis, MO (United States))

1991-08-15

79

Alternative splicing of Myb-related genes MYR1 and MYR2 may modulate activities through changes in dimerization, localization, or protein folding  

PubMed Central

Arabidopsis genes MYR1 and MYR2 are regulators of flowering time under low light intensity. These Myb-related genes are expressed as alternative splice variants affected in their coiled-coil and DNA-binding domains. We tested whether alternative splicing could affect dimerization and localization of MYR1 and MYR2, thereby potentially affecting their activity. Using MYR1 as a model for variants within the coiled-coil region, we detected 2 types of homodimers. For MYR2, alternative splicing in the DNA-binding Myb-like domain abolished the ability of MYR2 to dimerize. Alternative splicing in the coiled-coil domain did not affect nuclear localization, as determined by transient expression in tobacco, while alternative splicing in the DNA-binding domain of MYR2 yielded a distinct intranuclear localization pattern that may reflect changes in phosphorylation-dependent protein folding. Thus alternative splicing of these genes may result in changes in dimerization or protein folding resulting in changes in activity and abundance of MYR1 or MYR2 protein.

Zhao, Chengsong; Beers, Eric P

2013-01-01

80

ECgene: an alternative splicing database update  

PubMed Central

ECgene () was developed to provide functional annotation for alternatively spliced genes. The applications encompass the genome-based transcript modeling for alternative splicing (AS), domain analysis with Gene Ontology (GO) annotation and expression analysis based on the EST and SAGE data. We have expanded the ECgene's AS modeling and EST clustering to nine organisms for which sufficient EST data are available in the GenBank. As for the human genome, we have also introduced several new applications to analyze differential expression. ECprofiler is an ontology-based candidate gene search system that allows users to select an arbitrary combination of gene expression pattern and GO functional categories. DEGEST is a database of differentially expressed genes and isoforms based on the EST information. Importantly, gene expression is analyzed at three distinctive levels—gene, isoform and exon levels. The user interfaces for functional and expression analyses have been substantially improved. ASviewer is a dedicated java application that visualizes the transcript structure and functional features of alternatively spliced variants. The SAGE part of the expression module provides many additional features including SNP, differential expression and alternative tag positions.

Lee, Yeunsook; Lee, Younghee; Kim, Bumjin; Shin, Youngah; Nam, Seungyoon; Kim, Pora; Kim, Namshin; Chung, Won-Hyong; Kim, Jaesang; Lee, Sanghyuk

2007-01-01

81

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

PubMed Central

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

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

2002-01-01

82

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina  

PubMed Central

Purpose Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. Materials and Methods Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. Results PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. Conclusions The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.

DesJardin, Jacqueline Talea; Becerra, S Patricia; Subramanian, Preeti

2014-01-01

83

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

PubMed

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

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

2014-03-01

84

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

2005-01-01

85

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

86

HuR Regulates Alternative Splicing of the TRA2? Gene in Human Colon Cancer Cells under Oxidative Stress.  

PubMed

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2? gene encodes splicing factor transformer 2? (Tra2?) and generates 5 mRNA isoforms (TRA2?1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38(MAPK))-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2? exon 2, generating a TRA2?4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38(MAPK) double knockdown inhibited the arsenite-stimulated production of TRA2?4 and increased Tra2? protein, facilitating Tra2?-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2? minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2?4 interaction and TRA2?4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2?4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress. PMID:24865968

Akaike, Yoko; Masuda, Kiyoshi; Kuwano, Yuki; Nishida, Kensei; Kajita, Keisuke; Kurokawa, Ken; Satake, Yuzuru; Shoda, Katsutoshi; Imoto, Issei; Rokutan, Kazuhito

2014-08-01

87

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

88

Alternative splicing of the Drosophila melanogaster rotundRacGAP gene.  

PubMed

The rotund (rn) gene in Drosophila melanogaster codes for a RacGTPase-activating protein, RnRacGAP. Cellular studies have shown that RacGAP proteins function as negative regulators of substrate Rac proteins which, in turn, control the localization and polymerization state of actin within the cell. Previous sequence analysis of rn genomic DNA and incomplete cDNA clones suggested that at least two differentially spliced forms of the transcript exist, rnRacGAP(1) and rnRacGAP(2). Using nested reverse transcription-polymerase chain reaction (RT-PCR) methods, we have cloned missing exon and intron sequences, and detected differences between rnRacGAP(1) and rnRacGAP(2) involving 24 nucleotides (nt) of coding sequences and 119 nt of 3'UTR. This translates to a difference of seven amino acids at the C-termini of the polypeptide products. Utilization, in RT-PCR analysis, of form-specific primers provided a simple assay for the tissue specificity of expression of the two forms. rnRacGAP(1) is the predominant species in the testes and is expressed at a low level in the ovary and somatic tissues. rnRacGAP(2) is only very weakly expressed and is detectable solely in the testes. PMID:8654933

Hoemann, C D; Bergeret, E; Guichard, A; Griffin-Shea, R

1996-02-12

89

Regulation of alternative splicing within the supraspliceosome.  

PubMed

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. PMID:22100336

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

2012-01-01

90

Assessing the number of ancestral alternatively spliced exons in the human genome  

PubMed Central

Background It is estimated that between 35% and 74% of all human genes undergo alternative splicing. However, as a gene that undergoes alternative splicing can have between one and dozens of alternative exons, the number of alternatively spliced genes by itself is not informative enough. An additional parameter, which was not addressed so far, is therefore the number of human exons that undergo alternative splicing. We have previously described an accurate machine-learning method allowing the detection of conserved alternatively spliced exons without using ESTs, which relies on specific features of the exon and its genomic vicinity that distinguish alternatively spliced exons from constitutive ones. Results In this study we use the above-described approach to calculate that 7.2% (± 1.1%) of all human exons that are conserved in mouse are alternatively spliced in both species. Conclusion This number is the first estimation for the extent of ancestral alternatively spliced exons in the human genome.

Sorek, Rotem; Dror, Gideon; Shamir, Ron

2006-01-01

91

Genome-wide survey of Alternative Splicing in Sorghum Bicolor.  

PubMed

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

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

2014-07-01

92

Analysis of Alternative Splicing in Plants with Bioinformatics Tools  

Microsoft Academic Search

Alternative splicing is a molecular mechanism utilized by a broad range of eukaryotes to extend the repertoire of functions\\u000a encoded by single genes and to posttranscriptionally regulate gene expression. Recent analyses of expressed transcript sequences\\u000a aligned to the complete genomes of Arabidopsis and rice indicate that alternative splicing in plants is prevalent and exhibits\\u000a several features similar to other higher

B. J. Haas

93

Myocardial Alternative RNA Splicing and Gene Expression Profiling in Early Stage Hypoplastic Left Heart Syndrome  

Microsoft Academic Search

Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is

Marco Ricci; Yanji Xu; Harriet L. Hammond; David A. Willoughby; Lubov Nathanson; Maria M. Rodriguez; Matteo Vatta; Steven E. Lipshultz; Joy Lincoln

2012-01-01

94

Human aging is characterized by focused changes in gene expression and deregulation of alternative splicing  

PubMed Central

Summary Aging is a major risk factor for chronic disease in the human population, but there is little human data on gene expression alterations that accompany the process. We examined human peripheral blood leucocyte in-vivo RNA in a large-scale transcriptomic microarray study (subjects aged 30 to 104 years). We tested associations between probe expression intensity and advancing age (adjusting for confounding factors), initially in a discovery set (n = 458), following-up findings in a replication set (n=240). We confirmed expression of key results by real-time PCR. Of 16,571 expressed probes, only 295 (2%) were robustly associated with age. Just six probes were required for a highly efficient model for distinguishing between young and old (Area Under the Curve in replication set; 95%). The focussed nature of age-related gene expression may therefore provide potential biomarkers of aging. Similarly, only 7 of 1065 biological or metabolic pathways were age-associated, in Gene Set Enrichment Analysis (GSEA), notably including the processing of messenger RNAs (mRNAs); (p<0.002, FDR q<0.05). This is supported by our observation of age-associated disruption to the balance of alternatively-expressed isoforms for selected genes, suggesting that modification of mRNA processing may be a feature of human aging.

Harries, Lorna W.; Hernandez, Dena; Henley, William; Wood, Andrew; Holly, Alice C.; Bradley-Smith, Rachel M.; Yaghootkar, Hanieh; Dutta, Ambarish; Murray, Anna; Frayling, Timothy M.; Guralnik, Jack M.; Bandinelli, Stefania; Singleton, Andrew; Ferrucci, Luigi; Melzer, David

2011-01-01

95

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

2011-09-01

96

Comparative analysis of alternative splicing, alternative polyadenylation and the expression of the two KIN genes from cytoplasmic male sterility cabbage (Brassica oleracea L. var. capitata L.).  

PubMed

The KIN genes are crucial members of the cold-regulated gene family. They play exclusive roles during the developmental processes of many organs and respond to various abiotic stresses in plants. However, little is known about the regulation of KIN gene expression in cytoplasmic male sterility (CMS) cabbages (Brassica oleracea L. var. capitata L.). We carried out a genome-wide analysis to identify the KIN genes in the CMS cabbage. Two non-redundant KIN genes, named BoKIN1 (Bol021262) and BoKIN2 (Bol030498), were identified. Reverse transcriptase PCR detected alternative splicing (AS) products of BoKIN1 (four AS products) and BoKIN2 (three AS products). In addition, alternative polyadenylation (APA) was observed for BoKIN1 and BoKIN2 in the CMS cabbage, resulting in variable 3'UTRs in their transcripts. Furthermore, the transcription levels of BoKIN1-0 and BoKIN2-0, the introns of which were spliced completely, were analyzed in various organs and young leaves treated by abiotic stresses. Our data indicated that BoKIN1-0 is highly expressed in various organs, whereas BoKIN2-0 is expressed exclusively in the stamen. Our study also suggested that BoKIN1-0 was upregulated significantly in young leaves of plants exposed to abscisic acid treatment, and cold and heat stress. BoKIN1 and BoKIN2 had differential AS and APA patterns in pre-mRNA processing, and showed differences in their expression patterns and transcript levels. BoKIN1 participates widely in organ development and responds to diverse abiotic stresses, whereas BoKIN2 plays a main role in stamen development in the CMS cabbage. PMID:24488150

Tao, Peng; Huang, Xiaoyun; Li, Biyuan; Wang, Wuhong; Yue, Zhichen; Lei, Juanli; Zhong, Xinmin

2014-06-01

97

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

PubMed Central

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

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

2003-01-01

98

Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts  

PubMed Central

Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0). The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1) and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin) and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand) transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin. This study reveals several novel aspects of the ghrelin gene and suggests that the ghrelin locus is far more complex than previously recognised.

Seim, Inge; Collet, Chris; Herington, Adrian C; Chopin, Lisa K

2007-01-01

99

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections  

Microsoft Academic Search

Background  In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific\\u000a splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a

Paul H Smith; Jonathan M Mwangi; Yaw A Afrane; Guiyun Yan; Darren J Obbard; Lisa C Ranford-Cartwright; Tom J Little

2011-01-01

100

Genome-wide mapping of alternative splicing in Arabidopsis thaliana  

PubMed Central

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

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

2010-01-01

101

Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W  

PubMed Central

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-01-01

102

Identification of Common Genetic Variation That Modulates Alternative Splicing  

PubMed Central

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

2007-01-01

103

Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches  

Microsoft Academic Search

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity

Mo Chen; James L. Manley

2009-01-01

104

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

PubMed Central

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

Philippou, Anastassios; Armakolas, Athanasios; Koutsilieris, Michael

2013-01-01

105

Regulation of alternative splicing in obesity and weight loss  

PubMed Central

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

Kaminska, Dorota; Pihlajamaki, Jussi

2013-01-01

106

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

107

Alternative splicing: multiple control mechanisms and involvement in human disease  

Microsoft Academic Search

Alternative splicing is an important mechanism for controlling gene expression. It allows large proteomic complexity from a limited number of genes. An interplay of cis-acting sequences and trans-acting factors modulates the splicing of regulated exons. Here, we discuss the roles of the SR and hnRNP families of proteins in this process. We also focus on the role of the transcriptional

Javier F Cáceres; Alberto R Kornblihtt

2002-01-01

108

Alternative splicing in human tumour viruses: a therapeutic target?  

PubMed

Persistent infection with cancer risk-related viruses leads to molecular, cellular and immune response changes in host organisms that in some cases direct cellular transformation. Alternative splicing is a conserved cellular process that increases the coding complexity of genomes at the pre-mRNA processing stage. Human and other animal tumour viruses use alternative splicing as a process to maximize their transcriptomes and proteomes. Medical therapeutics to clear persistent viral infections are still limited. However, specific lessons learned in some viruses [e.g. HIV and HCV (hepatitis C virus)] suggest that drug-directed inhibition of alternative splicing could be useful for this purpose. The present review describes the basic mechanisms of constitutive and alternative splicing in a cellular context and known splicing patterns and the mechanisms by which these might be achieved for the major human infective tumour viruses. The roles of splicing-related proteins expressed by these viruses in cellular and viral gene regulation are explored. Moreover, we discuss some currently available drugs targeting SR (serine/arginine-rich) proteins that are the main regulators of constitutive and alternative splicing, and their potential use in treatment for so-called persistent viral infections. PMID:22738337

Hernandez-Lopez, Hegel R; Graham, Sheila V

2012-07-15

109

Functional characterization of alternatively spliced human SECISBP2 transcript variants  

PubMed Central

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

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

2008-01-01

110

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

111

Alternative Splicing of the Tuberous Sclerosis 2 ( TSC2) Gene in Human and Mouse Tissues  

Microsoft Academic Search

The recently isolated gene for tuberous sclerosis 2 (TSC2) encodes a 5.5-kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. We have detected a novel variant of the TSC2 mRNA lacking 129 nucleotides, predicting an in-frame deletion of 43 amino

Lin Xu; Christopher Sterner; Magitha M. Maheshwar; Peter J. Wilson; Mark Nellist; Priscilla M. Short; Jonathan L. Haines; Julian R. Sampson; Vijaya Ramesh

1995-01-01

112

Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing  

Microsoft Academic Search

BACKGROUND: Human sex hormone-binding globulin (SHBG) regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (PL). The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (PT), and two

Atif M Nakhla; Daniel J Hryb; William Rosner; Nicholas A Romas; Zhaoying Xiang; Scott M Kahn

2009-01-01

113

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

114

Regulation of Chemoresistance Via Alternative Messenger RNA Splicing  

PubMed Central

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

Eblen, Scott T.

2012-01-01

115

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

SciTech Connect

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

Sampaio, S.O.; Mei, C.; Butcher, E.C. [Stanford Univ. School of Medicine, CA (United States)] [and others

1995-09-01

116

Expression Map of the Human Exome in CD34+ Cells and Blood Cells: Increased Alternative Splicing in Cell Motility and Immune Response Genes  

PubMed Central

Background Hematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC) residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS) to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells. Principal Findings Using Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php). At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi), a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes. Conclusion The relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function.

Tondeur, Sylvie; Pangault, Celine; Le Carrour, Tanguy; Lannay, Yoann; Benmahdi, Rima; Cubizolle, Aurelie; Assou, Said; Pantesco, Veronique; Klein, Bernard; Hamamah, Samir; Schved, Jean-Francois; Fest, Thierry; De Vos, John

2010-01-01

117

Hypoxia-Induced Alternative Splicing in Endothelial Cells  

PubMed Central

Background Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. Methodology/Principal findings Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O2) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). Conclusions/Significance For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.

Weigand, Julia E.; Boeckel, Jes-Niels; Gellert, Pascal; Dimmeler, Stefanie

2012-01-01

118

Genomewide comparative analysis of alternative splicing in plants.  

PubMed

Alternative splicing (AS) has been extensively studied in mammalian systems but much less in plants. Here we report AS events deduced from EST/cDNA analysis in two model plants: Arabidopsis and rice. In Arabidopsis, 4,707 (21.8%) of the genes with EST/cDNA evidence show 8,264 AS events. Approximately 56% of these events are intron retention (IntronR), and only 8% are exon skipping. In rice, 6,568 (21.2%) of the expressed genes display 14,542 AS events, of which 53.5% are IntronR and 13.8% are exon skipping. The consistent high frequency of IntronR suggests prevalence of splice site recognition by intron definition in plants. Different AS events within a given gene occur, for the most part, independently. In total, 36-43% of the AS events produce transcripts that would be targets of the non-sense-mediated decay pathway, if that pathway were to operate in plants as in humans. Forty percent of Arabidopsis AS genes are alternatively spliced also in rice, with some examples strongly suggesting a role of the AS event as an evolutionary conserved mechanism of posttranscriptional regulation. We created a comprehensive web-interfaced database to compile and visualize the evidence for alternative splicing in plants (Alternative Splicing in Plants, available at www.plantgdb.org/ASIP). PMID:16632598

Wang, Bing-Bing; Brendel, Volker

2006-05-01

119

Alternative splicing of type II procollagen: IIB or not IIB?  

PubMed

Abstract Over two decades ago, two isoforms of the type II procollagen gene (COL2A1) were discovered. These isoforms, named IIA and IIB, are generated in a developmentally-regulated manner by alternative splicing of exon 2. Chondroprogenitor cells synthesize predominantly IIA isoforms (containing exon 2) while differentiated chondrocytes produce mainly IIB transcripts (devoid of exon 2). Importantly, this IIA-to-IIB alternative splicing switch occurs only during chondrogenesis. More recently, two other isoforms have been reported (IIC and IID) that also involve splicing of exon 2; these findings highlight the complexities involving regulation of COL2A1 expression. The biological significance of why different isoforms of COL2A1 exist within the context of skeletal development and maintenance is still not completely understood. This review will provide current knowledge on COL2A1 isoform expression during chondrocyte differentiation and what is known about some of the mechanisms that control exon 2 alternative splicing. Utilization of mouse models to address the biological significance of Col2a1 alternative splicing in vivo will also be discussed. From the knowledge acquired to date, some new questions and concepts are now being proposed on the importance of Col2a1 alternative splicing in regulating extracellular matrix assembly and how this may subsequently affect cartilage and endochondral bone quality and function. PMID:24669942

McAlinden, Audrey

2014-06-01

120

Alternatively spliced domains interact to regulate BK potassium channel gating.  

PubMed

Most human genes contain multiple alternative splice sites believed to extend the complexity and diversity of the proteome. However, little is known about how interactions among alternative exons regulate protein function. We used the Caenorhabditis elegans slo-1 large-conductance calcium and voltage-activated potassium (BK) channel gene, which contains three alternative splice sites (A, B, and C) and encodes at least 12 splice variants, to investigate the functional consequences of alternative splicing. These splice sites enable the insertion of exons encoding part of the regulator of K(+) conductance (RCK)1 Ca(2+) coordination domain (exons A1 and A2) and portions of the RCK1-RCK2 linker (exons B0, B1, B2, C0, and C1). Exons A1 and A2 are used in a mutually exclusive manner and are 67% identical. The other exons can extend the RCK1-RCK2 linker by up to 41 residues. Electrophysiological recordings of all isoforms show that the A1 and A2 exons regulate activation kinetics and Ca(2+) sensitivity, but only if alternate exons are inserted at site B or C. Thus, RCK1 interacts with the RCK1-RCK2 linker, and the effect of exon variation on gating depends on the combination of alternate exons present in each isoform. PMID:22049343

Johnson, Brandon E; Glauser, Dominique A; Dan-Glauser, Elise S; Halling, D Brent; Aldrich, Richard W; Goodman, Miriam B

2011-12-20

121

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome  

PubMed Central

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye_retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10–30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.

Xu, Qiang; Modrek, Barmak; Lee, Christopher

2002-01-01

122

Systematic analysis of human kinase genes: a large number of genes and alternative splicing events result in functional and structural diversity  

PubMed Central

Background Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosys. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. Results A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was perfomed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. Conclusion The predicted human kinome was extended by identifiying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set.

Milanesi, Luciano; Petrillo, Mauro; Sepe, Leandra; Boccia, Angelo; D'Agostino, Nunzio; Passamano, Myriam; Di Nardo, Salvatore; Tasco, Gianluca; Casadio, Rita; Paolella, Giovanni

2005-01-01

123

Global analysis of alternative splicing regulation by insulin and wingless signaling in Drosophila cells  

PubMed Central

Background Despite the prevalence and biological relevance of both signaling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signaling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis using splicing-sensitive microarrays of changes in alternative splicing induced by activation of two distinct signaling pathways, insulin and wingless, in Drosophila cells in culture. Results Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and post-transcriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related to carbohydrate metabolism and cellular signaling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5' splice sites. Conclusions Taken together, our data indicate that signaling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for crosstalk between different signaling pathways.

Hartmann, Britta; Castelo, Robert; Blanchette, Marco; Boue, Stephanie; Rio, Donald C; Valcarcel, Juan

2009-01-01

124

Alu-Containing Exons are Alternatively Spliced  

Microsoft Academic Search

Alu repetitive elements are found in ?1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned

Rotem Sorek; Gil Ast; Dan Graur

2002-01-01

125

Genetic Variation of Pre-mRNA Alternative Splicing in Human Populations  

PubMed Central

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

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

2011-01-01

126

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing  

PubMed Central

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ER?) and their ligands promote alternative splicing. The endogenous targets of ER?:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ER? on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ER? binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ER? cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ER? binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ER? and splicing factors, influenced ER?:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ER?-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ER?-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

2013-01-01

127

SASD: the Synthetic Alternative Splicing Database for identifying novel isoform from proteomics  

PubMed Central

Background Alternative splicing is an important and widespread mechanism for generating protein diversity and regulating protein expression. High-throughput identification and analysis of alternative splicing in the protein level has more advantages than in the mRNA level. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, based on the peptidomic database of human protein isoforms for proteomics experiments, our objective is to design a new alternative splicing database to 1) provide more coverage of genes, transcripts and alternative splicing, 2) exclusively focus on the alternative splicing, and 3) perform context-specific alternative splicing analysis. Results We used a three-step pipeline to create a synthetic alternative splicing database (SASD) to identify novel alternative splicing isoforms and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. First, we extracted information on gene structures of all genes in the Ensembl Genes 71 database and incorporated the Integrated Pathway Analysis Database. Then, we compiled artificial splicing transcripts. Lastly, we translated the artificial transcripts into alternative splicing peptides. The SASD is a comprehensive database containing 56,630 genes (Ensembl gene IDs), 95,260 transcripts (Ensembl transcript IDs), and 11,919,779 Alternative Splicing peptides, and also covering about 1,956 pathways, 6,704 diseases, 5,615 drugs, and 52 organs. The database has a web-based user interface that allows users to search, display and download a single gene/transcript/protein, custom gene set, pathway, disease, drug, organ related alternative splicing. Moreover, the quality of the database was validated with comparison to other known databases and two case studies: 1) in liver cancer and 2) in breast cancer. Conclusions The SASD provides the scientific community with an efficient means to identify, analyze, and characterize novel Exon Skipping and Intron Retention protein isoforms from mass spectrometry and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing.

2013-01-01

128

Tumor microenvironment-associated modifications of alternative splicing  

PubMed Central

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ?20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Brosseau, Jean-Philippe; Lucier, Jean-Francois; Nwilati, Hanad; Thibault, Philippe; Garneau, Daniel; Gendron, Daniel; Durand, Mathieu; Couture, Sonia; Lapointe, Elvy; Prinos, Panagiotis; Klinck, Roscoe; Perreault, Jean-Pierre; Chabot, Benoit; Abou-Elela, Sherif

2014-01-01

129

A chloroplast retrograde signal regulates nuclear alternative splicing.  

PubMed

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

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

2014-04-25

130

The Coupling of Alternative Splicing and Nonsense?Mediated mRNA Decay  

Microsoft Academic Search

M ost human genes exhibit alternative splicing, but not all alternatively spliced transcripts produce functional proteins. Computational and experimental results indicate that a substantial fraction of alternative splicing events in humans result in mRNA isoforms that harbor a premature termination codon (PTC). These transcripts are predicted to be degraded by the nonsense?mediated mRNA decay (NMD) pathway. One explanation for the

Liana F. Lareau; Angela N. Brooks; David A. W. Soergel; Qi Meng; Steven E. Brenner

2007-01-01

131

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

132

SRp54 (SFRS11), a Regulator for tau Exon 10 Alternative Splicing Identified by an Expression Cloning Strategy  

Microsoft Academic Search

The tau gene encodes a microtubule-associated protein that is critical for neuronal survival and function. Splicing defects in the human tau gene lead to frontotemporal dementia with Parkinsonism linked to chro- mosome 17 (FTDP-17), an autosomal dominant neurodegenerative disorder. Genetic mutations associated with FTDP-17 often affect tau exon 10 alternative splicing. To investigate mechanisms regulating tau exon 10 alternative splicing,

Jane Y. Wu; Amar Kar; David Kuo; Bing Yu; Necat Havlioglu

2006-01-01

133

Implicit alternative splicing for genetic algorithms  

Microsoft Academic Search

In this paper we present a new nature-inspired variation operator for binary encodings in genetic algorithms (GAs). Our method, called implicit alternative splicing (iAS), is repeatedly applied to the individual encodings in the algorithm's population and inverts randomly chosen segments of decreasing size in a systematic fashion. Its goal is to determine the largest possible segment the inversion of which

Philipp Rohlfshagen; John A. Bullinaria

2007-01-01

134

Gene Structure, Chromosomal Location, and Basis for Alternative mRNA Splicing of the Human VCAM1 Gene  

Microsoft Academic Search

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

Myron I. Cybulsky; Jochen W. U. Fries; Amy J. Williams; Parvez Sultan; Roger Eddy; Mary Byers; Thomas Shows; Michael A. Gimbrone Jr.; Tucker Collins

1991-01-01

135

hnRNP A1 and hnRNP F Modulate the Alternative Splicing of Exon 11 of the Insulin Receptor Gene  

PubMed Central

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5? GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3? end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5? splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U?C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

Urbano, Rodolfo; Thompson, James; Yates, John R.; Webster, Nicholas J. G.

2011-01-01

136

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

137

Does distance matter? Variations in alternative 3? splicing regulation  

PubMed Central

Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5? or 3? splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3? splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3? splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3? splice sites from constitutive 3? splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites.

Akerman, Martin; Mandel-Gutfreund, Yael

2007-01-01

138

Given Dimensions of Neoplastic Events as Aberrantly Operative Alternative Splicing  

PubMed Central

The provision of dynamic splicing events constitutes the reflected nature of neoplasia that locally infiltrates and systemically spreads in terms of evolutionary attributes of the primary and various secondary pathways in malignant transformation. The significant diversity in molecular characterization of the given tumor lesion would adaptively conform to dynamics of splicing as enhanced or silenced exons of the premessenger RNA molecule. The proteins synthesized are in turn potential modifiers in further gene expression within such contexts as RNA:protein and RNA:DNA binding events. The recognition of pathways of incremental scope would underline the development of lesions, such as tumors, as multiple alternative splicing phenomena primarily affecting molecular physicochemical identity. It is within contexts of operative intervention and modification that the real identity of the malignant neoplastic process arises, within terms of reference of contextual splicing events. Disrupted gene expression is thus a referential pathway in the modification of splicing that may prove constitutive or alternative, in first instance, but also aberrant as the lesion progresses locally and systemically.

Agius, Lawrence M.

2010-01-01

139

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

PubMed Central

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

2014-01-01

140

Gene recognition via spliced sequence alignment.  

PubMed Central

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

Gelfand, M S; Mironov, A A; Pevzner, P A

1996-01-01

141

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

142

Variable window binding for mutually exclusive alternative splicing  

PubMed Central

Background Genes of advanced organisms undergo alternative splicing, which can be mutually exclusive, in the sense that only one exon is included in the mature mRNA out of a cluster of alternative choices, often arranged in a tandem array. In many cases, however, the details of the underlying biologic mechanisms are unknown. Results We describe 'variable window binding' - a mechanism used for mutually exclusive alternative splicing by which a segment ('window') of a conserved nucleotide 'anchor' sequence upstream of the exon 6 cluster in the pre-mRNA of the fruitfly Dscam gene binds to one of the introns, thereby activating selection of the exon directly downstream from the binding site. This mechanism is supported by the fact that the anchor sequence can be inferred solely from a comparison of the intron sequences using a genetic algorithm. Because the window location varies for each exon choice, regulation can be achieved by obstructing part of that sequence. We also describe a related mechanism based on competing pre-mRNA stem-loop structures that could explain the mutually exclusive choice of exon 17 of the Dscam gene. Conclusion On the basis of comparative sequence analysis, we propose efficient biologic mechanisms of alternative splicing of the Drosophila Dscam gene that rely on the inherent structure of the pre-mRNA. Related mechanisms employing 'locus control regions' could be involved on other occasions of mutually exclusive choices of exons or genes.

Anastassiou, Dimitris; Liu, Hairuo; Varadan, Vinay

2006-01-01

143

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

PubMed

Alternative splicing essentially increases the diversity of the transcriptome and has important implications 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 transcripts derived from a gene into a single splicing graph. Each transcript corresponds to a path in the graph, and alternative splicing is displayed by bifurcations. This representation preserves the relationships between different splicing variants and allows us to investigate systematically all possible putative transcripts. We built a database of splicing graphs for human genes, using transcript information from various major sources (Ensembl, RefSeq, STACK, TIGR and UniGene). A Web interface allows users to display the splicing graphs, to interactively assemble transcripts and to access their sequences as well as neighboring genomic regions. We also provide for each gene an exhaustive pre-computed catalog of putative transcripts--in total more than 1.2 million sequences. We found that approximately 65% of the investigated genes show evidence for alternative splicing, and in 5% of the cases, a single gene might produce over 100 transcripts. PMID:15292448

Leipzig, Jeremy; Pevzner, Pavel; Heber, Steffen

2004-01-01

144

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

145

Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.  

PubMed Central

The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7.

Buvoli, M; Cobianchi, F; Bestagno, M G; Mangiarotti, A; Bassi, M T; Biamonti, G; Riva, S

1990-01-01

146

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

PubMed Central

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

2013-01-01

147

WebScipio: reconstructing alternative splice variants of eukaryotic proteins  

PubMed Central

Accurate exon–intron structures are essential prerequisites in genomics, proteomics and for many protein family and single gene studies. We originally developed Scipio and the corresponding web service WebScipio for the reconstruction of gene structures based on protein sequences and available genome assemblies. WebScipio also allows predicting mutually exclusive spliced exons and tandemly arrayed gene duplicates. The obtained gene structures are illustrated in graphical schemes and can be analysed down to the nucleotide level. The set of eukaryotic genomes available at the WebScipio server is updated on a daily basis. The current version of the web server provides access to ?3400 genome assembly files of >1100 sequenced eukaryotic species. Here, we have also extended the functionality by adding a module with which expressed sequence tag (EST) and cDNA data can be mapped to the reconstructed gene structure for the identification of all types of alternative splice variants. WebScipio has a user-friendly web interface, and we believe that the improved web server will provide better service to biologists interested in the gene structure corresponding to their protein of interest, including all types of alternative splice forms and tandem gene duplicates. WebScipio is freely available at http://www.webscipio.org.

Hatje, Klas; Hammesfahr, Bjorn; Kollmar, Martin

2013-01-01

148

Muscle-specific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans.  

PubMed

Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript. PMID:23071450

Ohno, Genta; Ono, Kanako; Togo, Marina; Watanabe, Yohei; Ono, Shoichiro; Hagiwara, Masatoshi; Kuroyanagi, Hidehito

2012-01-01

149

Alternative splicing in the NF-?B signaling pathway  

PubMed Central

Activation of transcription factor NF-?B can affect the expression of several hundred genes, many of which are involved in inflammation and immunity. The proper NF-?B transcriptional response is primarily regulated by post-translational modification of NF-?B signaling constituents. Herein, we review the accumulating evidence suggesting that alternative splicing of NF-?B signaling components is another means of controlling NF-?B signaling. Several alternative splicing events in both the tumor necrosis factor and Toll/interleukin-1 NF-?B signaling pathways can inhibit the NF-?B response, whereas others enhance NF-?B signaling. Alternative splicing of mRNAs encoding some NF-?B signaling components can be induced by prolonged exposure to an NF-?B-activating signal, such as lipopolysaccharide, suggesting a mechanism for negative feedback to dampen excessive NF-?B signaling. Moreover, some NF-?B alternative splicing events appear to be specific for certain diseases, and could serve as therapeutic targets or biomarkers.

Leeman, Joshua R.; Gilmore, Thomas D.

2008-01-01

150

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

PubMed Central

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

2012-01-01

151

Characterization of the Six Zebrafish Clade B Fibrillar Procollagen Genes, with Evidence for Evolutionarily Conserved Alternative Splicing within the pro-?1(V) C-propeptide  

PubMed Central

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I–III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-?1(V), pro-?3(V), pro-?1(XI) and pro-?2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-?1(V), pro?3(V)a and b, pro-?1(XI)a and b, and pro-?2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH2-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-?1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

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

2010-01-01

152

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

153

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

PubMed Central

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.

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

2013-01-01

154

PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing and Up-regulated in Basal Cell Carcinomas1  

Microsoft Academic Search

By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, PTCH2 has been localized to chromosome 1p33-34, a region often lost in a variety of tumors. Several alternatively

Peter G. Zaphiropoulos; Anne Birgitte Unden; Fahimeh Rahnama; Robert E. Hollingsworth; Rune Toftgård

155

Genomewide comparative analysis of alternative splicing in plants  

Microsoft Academic Search

Alternative splicing (AS) has been extensively studied in mammalian systems but much less in plants. Here we report AS events deduced from EST\\/cDNA analysis in two model plants: Arabidopsis and rice. In Arabidopsis, 4,707 (21.8%) of the genes with EST\\/cDNA evidence show 8,264 AS events. Approximately 56% of these events are intron retention (IntronR), and only 8% are exon skipping.

Bing-Bing Wang; Volker Brendel

2006-01-01

156

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

PubMed

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

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

2007-02-01

157

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

Microsoft Academic Search

BackgroundAlternative 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 FindingsThe purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using

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

2011-01-01

158

Sudemycin E influences alternative splicing and changes chromatin modifications  

PubMed Central

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

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

2014-01-01

159

Sudemycin E influences alternative splicing and changes chromatin modifications.  

PubMed

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

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

2014-04-01

160

The Birth of an Alternatively Spliced Exon: 3' Splice-Site Selection in Alu Exons  

Microsoft Academic Search

Alu repetitive elements can be inserted into mature messenger RNAs via a splicing-mediated process termed exonization. To understand the molecular basis and the regulation of the process of turning intronic Alus into new exons, we compiled and analyzed a data set of human exonized Alus. We revealed a mechanism that governs 3' splice-site selection in these exons during alternative splicing.

Galit Lev-Maor; Rotem Sorek; Noam Shomron; Gil Ast

2003-01-01

161

Role ofBMPR2 Alternative Splicing in HPAH Penetrance  

PubMed Central

Background Bone morphogenic protein receptor 2 (BMPR2) gene mutations are the most common cause of heritable PAH (HPAH). However only 20% of mutation carriers get clinical disease. Here we explored the hypothesis that this reduced penetrance is in part due to an alteration in BMPR2 alternative splicing. Methods and Results Our data showed that BMPR2 has multiple alternatively spliced variants. Two of these, isoform-A (full-length) and isoform-B (missing exon 12), were expressed in all tissues analyzed. Analysis of cultured lymphocytes (CLs) of 47 BMPR2 mutation-positive HPAH-patients and 35 BMPR2 mutation-positive unaffected-carriers showed that patients had higher levels of isoform-B compared to isoform-A (B/A ratio) than carriers (P=0.002). Furthermore compared to cells with low B/A ratio, cells with high B/A ratio had lower levels of unphosphorylated cofilin following BMP stimulation. Analysis of exon 12 sequences identified an exonic splice enhancer, which binds serine arginine splicing factor 2 (SRSF2). Because SRSF2 promotes exon inclusion, reduced SRSF2 expression would mean that exon 12 would not be included in final BMPR2 mRNA (thus promoting increased isoform-B formation). Western blot analysis showed that SRSF2 expression was lower in cells from patients compared to carriers; and, siRNA-mediated knockdown of SRSF2 in pulmonary microvascular endothelial cells resulted in elevated levels of isoform-B compared to isoform-A, i.e. elevated B/A ratio. Conclusions Alterations in BMPR2 isoform ratios may provide an explanation of the reduced penetrance among BMPR2 mutation carriers. This ratio is controlled by an exonic splice enhancer in exon 12 and its associated splicing factor SRSF2.

Cogan, Joy; Austin, Eric; Hedges, Lora; Womack, Bethany; West, James; Loyd, James; Hamid, Rizwan

2012-01-01

162

An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype  

PubMed Central

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

2011-01-01

163

ESTGenes: Alternative Splicing From ESTs in Ensembl  

PubMed Central

We describe a novel algorithm for deriving the minimal set of nonredundant transcripts compatible with the splicing structure of a set of ESTs mapped on a genome. Sets of ESTs with compatible splicing are represented by a special type of graph. We describe the algorithms for building the graphs and for deriving the minimal set of transcripts from the graphs that are compatible with the evidence. These algorithms are part of the Ensembl automatic gene annotation system, and its results, using ESTs, are provided at www.ensembl.org as ESTgenes for the mosquito, Caenorhabditis briggsae, C. elegans, zebrafish, human, mouse, and rat genomes. Here we also report on the results of this method applied to the human and mouse genomes.

Eyras, Eduardo; Caccamo, Mario; Curwen, Val; Clamp, Michele

2004-01-01

164

Alternative splicing, expression, and genomic structure of the 3' region of the gene encoding the sarcolemmal-associated proteins (SLAPs) defines a novel class of coiled-coil tail-anchored membrane proteins.  

PubMed

The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOP(AP), a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic alpha-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3' region of the SLAP gene indicated that SLAP1 and TOP(AP) are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOP(AP) were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOP(AP), and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOP(AP) transmembrane domains mutually exclusive. These data reveal that TOP(AP) and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins. PMID:10986292

Wielowieyski, P A; Sevinc, S; Guzzo, R; Salih, M; Wigle, J T; Tuana, B S

2000-12-01

165

Drosha Promotes Splicing of a Pre-microRNA-like Alternative Exon  

PubMed Central

The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.

Havens, Mallory A.; Reich, Ashley A.; Hastings, Michelle L.

2014-01-01

166

Drosha promotes splicing of a pre-microRNA-like alternative exon.  

PubMed

The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing. PMID:24786770

Havens, Mallory A; Reich, Ashley A; Hastings, Michelle L

2014-05-01

167

The coupling of alternative splicing and nonsense-mediated mRNA decay.  

PubMed

Most human genes exhibit alternative splicing, but not all alternatively spliced transcripts produce functional proteins. Computational and experimental results indicate that a substantial fraction of alternative splicing events in humans result in mRNA isoforms that harbor a premature termination codon (PTC). These transcripts are predicted to be degraded by the nonsense-mediated mRNA decay (NMD) pathway. One explanation for the abundance of PTC-containing isoforms is that they represent splicing errors that are identified and degraded by the NMD pathway. Another potential explanation for this startling observation is that cells may link alternative splicing and NMD to regulate the abundance of mRNA transcripts. This mechanism, which we call "Regulated Unproductive Splicing and Translation" (RUST), has been experimentally shown to regulate expression of a wide variety of genes in many organisms from yeast to human. It is frequently employed for autoregulation of proteins that affect the splicing process itself. Thus, alternative splicing and NMD act together to play an important role in regulating gene expression. PMID:18380348

Lareau, Liana F; Brooks, Angela N; Soergel, David A W; Meng, Qi; Brenner, Steven E

2007-01-01

168

Correcting for Differential Transcript Coverage Reveals a Strong Relationship between Alternative Splicing and Organism Complexity.  

PubMed

What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species-an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced Ne in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome's functional information capacity. PMID:24682283

Chen, Lu; Bush, Stephen J; Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Urrutia, Araxi O

2014-06-01

169

Correcting for Differential Transcript Coverage Reveals a Strong Relationship between Alternative Splicing and Organism Complexity  

PubMed Central

What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species—an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced Ne in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome’s functional information capacity.

Chen, Lu; Bush, Stephen J.; Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Urrutia, Araxi O.

2014-01-01

170

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

PubMed Central

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

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

2012-01-01

171

Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.  

PubMed

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway. PMID:22573328

Sugitani, Hideki; Hirano, Eiichi; Knutsen, Russell H; Shifren, Adrian; Wagenseil, Jessica E; Ciliberto, Christopher; Kozel, Beth A; Urban, Zsolt; Davis, Elaine C; Broekelmann, Thomas J; Mecham, Robert P

2012-06-22

172

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

PubMed Central

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

Ren, Shancheng; Peng, Zhiyu; Mao, Jian-Hua; Yu, Yongwei; Yin, Changjun; Gao, Xin; Cui, Zilian; Zhang, Jibin; Yi, Kang; Xu, Weidong; Chen, Chao; Wang, Fubo; Guo, Xinwu; Lu, Ji; Yang, Jun; Wei, Min; Tian, Zhijian; Guan, Yinghui; Tang, Liang; Xu, Chuanliang; Wang, Linhui; Gao, Xu; Tian, Wei; Wang, Jian; Yang, Huanming; Wang, Jun; Sun, Yinghao

2012-01-01

173

Alternative Splicing and Tissue-specific Elastin Misassembly Act as Biological Modifiers of Human Elastin Gene Frameshift Mutations Associated with Dominant Cutis Laxa*  

PubMed Central

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Sugitani, Hideki; Hirano, Eiichi; Knutsen, Russell H.; Shifren, Adrian; Wagenseil, Jessica E.; Ciliberto, Christopher; Kozel, Beth A.; Urban, Zsolt; Davis, Elaine C.; Broekelmann, Thomas J.; Mecham, Robert P.

2012-01-01

174

Alternative Splicing of Spg7, a Gene Involved in Hereditary Spastic Paraplegia, Encodes a Variant of Paraplegin Targeted to the Endoplasmic Reticulum  

PubMed Central

Background Hereditary spastic paraplegia defines a group of genetically heterogeneous diseases characterized by weakness and spasticity of the lower limbs owing to retrograde degeneration of corticospinal axons. One autosomal recessive form of the disease is caused by mutation in the SPG7 gene. Paraplegin, the product of SPG7, is a component of the m-AAA protease, a high molecular weight complex that resides in the mitochondrial inner membrane, and performs crucial quality control and biogenesis functions in mitochondria. Principal Findings Here we show the existence in the mouse of a novel isoform of paraplegin, which we name paraplegin-2, encoded by alternative splicing of Spg7 through usage of an alternative first exon. Paraplegin-2 lacks the mitochondrial targeting sequence, and is identical to the mature mitochondrial protein. Remarkably, paraplegin-2 is targeted to the endoplasmic reticulum. We find that paraplegin-2 exposes the catalytic domains to the lumen of the endoplasmic reticulum. Moreover, endogenous paraplegin-2 accumulates in microsomal fractions prepared from mouse brain and retina. Finally, we show that the previously generated mouse model of Spg7-linked hereditary spastic paraplegia is an isoform-specific knock-out, in which mitochondrial paraplegin is specifically ablated, while expression of paraplegin-2 is retained. Conclusions/Significance These data suggest a possible additional role of AAA proteases outside mitochondria and open the question of their implication in neurodegeneration.

Mancuso, Giuseppe; Barth, Esther; Crivello, Pietro; Rugarli, Elena I.

2012-01-01

175

Transgene regulation in plants by alternative splicing of a suicide exon  

PubMed Central

Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.

Hickey, Scott F.; Sridhar, Malathy; Westermann, Alexander J.; Qin, Qian; Vijayendra, Pooja; Liou, Geoffrey; Hammond, Ming C.

2012-01-01

176

Alternative splicing of rat tropoelastin mRNA is tissue-specific and developmentally regulated.  

PubMed

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1811166

Heim, R A; Pierce, R A; Deak, S B; Riley, D J; Boyd, C D; Stolle, C A

1991-11-01

177

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

PubMed

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

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

2014-05-01

178

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

179

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene.  

PubMed Central

We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IVPDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 microM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 microM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.

Bolger, G B; Erdogan, S; Jones, R E; Loughney, K; Scotland, G; Hoffmann, R; Wilkinson, I; Farrell, C; Houslay, M D

1997-01-01

180

TMEM16A alternative splicing coordination in breast cancer  

PubMed Central

Background TMEM16A, also known as Anoctamin-1, is a calcium-activated chloride channel gene overexpressed in many tumors. The role of TMEM16A in cancer is not completely understood and no data are available regarding the potential tumorigenic properties of the multiple isoforms generated by alternative splicing (AS). Methods We evaluated TMEM16A AS pattern, isoforms distribution and Splicing Coordination (SC), in normal tissues and breast cancers, through a semi-quantitative PCR-assay that amplifies transcripts across three AS exons, 6b, 13 and 15. Results In breast cancer, we did not observe an association either to AS of individual exons or to specific TMEM16A isoforms, and induced expression of the most common isoforms present in tumors in the HEK293 Flp-In Tet-ON system had no effect on cellular proliferation and migration. The analysis of splicing coordination, a mechanism that regulates AS of distant exons, showed a preferential association of exon 6b and 15 in several normal tissues and tumors: isoforms that predominantly include exon 6b tend to exclude exon 15 and vice versa. Interestingly, we found an increase in SC in breast tumors compared to matched normal tissues. Conclusions As the different TMEM16A isoforms do not affect proliferation or migration and do not associate with tumors, our results suggest that the resulting channel activities are not directly involved in cell growth and motility. Conversely, the observed increase in SC in breast tumors suggests that the maintenance of the regulatory mechanism that coordinates distant alternative spliced exons in multiple genes other than TMEM16A is necessary for cancer cell viability.

2013-01-01

181

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

2011-08-01

182

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

183

Computational Evidence of NAGNAG Alternative Splicing in Human Large Intergenic Noncoding RNA  

PubMed Central

NAGNAG alternative splicing plays an essential role in biological processes and represents a highly adaptable system for posttranslational regulation of gene function. NAGNAG alternative splicing impacts a myriad of biological processes. Previous studies of NAGNAG largely focused on messenger RNA. To the best of our knowledge, this is the first study testing the hypothesis that NAGNAG alternative splicing is also operative in large intergenic noncoding RNA (lincRNA). The RNA-seq data sets from recent deep sequencing studies were queried to test our hypothesis. NAGNAG alternative splicing of human lincRNA was identified while querying two independent RNA-seq data sets. Within these datasets, 31 NAGNAG alternative splicing sites were identified in lincRNA. Notably, most exons of lincRNA containing NAGNAG acceptors were longer than those from protein-coding genes. Furthermore, presence of CAG coding appeared to participate in the splice site selection. Finally, expression of the isoforms of NAGNAG lincRNA exhibited tissue specificity. Together, this study improves our understanding of the NAGNAG alternative splicing in lincRNA.

Sun, Xiaoyong; Lin, Simon M.; Yan, Xiaoyan

2014-01-01

184

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

PubMed Central

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

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

1991-01-01

185

Long noncoding RNA modulates alternative splicing regulators in Arabidopsis.  

PubMed

Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear AS regulators to modulate AS patterns during development. PMID:25073154

Bardou, Florian; Ariel, Federico; Simpson, Craig G; Romero-Barrios, Natali; Laporte, Philippe; Balzergue, Sandrine; Brown, John W S; Crespi, Martin

2014-07-28

186

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer  

Microsoft Academic Search

BACKGROUND: Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. RESULTS: The array consisted of

Ruben Pio; David Blanco; Maria Jose Pajares; Elena Aibar; Olga Durany; Teresa Ezponda; Jackeline Agorreta; Javier Gomez-Roman; Miguel Angel Anton; Angel Rubio; Maria D Lozano; Jose M López-Picazo; Francesc Subirada; Tamara Maes; Luis M Montuenga

2010-01-01

187

Selective secretion of alternatively spliced fibronectin variants  

PubMed Central

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0- V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein- protein interactions in FN dimer formation.

1989-01-01

188

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution  

PubMed Central

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3? splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ?15-fold increase in the frequency of three base pair gaps at 3? splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.

Bradley, Robert K.; Merkin, Jason; Lambert, Nicole J.; Burge, Christopher B.

2012-01-01

189

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

190

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

Microsoft Academic Search

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 12?572

David Brett; Jens Hanke; Gerrit Lehmann; Sabine Haase; Sebastian Delbrück; Steffen Krueger; Jens Reich; Peer Bork

2000-01-01

191

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

PubMed Central

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.

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

2013-01-01

192

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

PubMed Central

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

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

2014-01-01

193

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

194

Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family  

PubMed Central

Background The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules. Proteins containing this domain are implicated in diverse biological activities and may be important for chronic host/parasite interactions. Results We report the first description of an SCP/TAPS gene family (Schistosoma mansoni venom allergen-like (SmVALs)) in the medically important Platyhelminthes (class Trematoda) and describe individual members' phylogenetic relationships, genomic organization and life cycle expression profiles. Twenty-eight SmVALs with complete SCP/TAPS domains were identified and comparison of their predicted protein features and gene structures indicated the presence of two distinct sub-families (group 1 & group 2). Phylogenetic analysis demonstrated that this group 1/group 2 split is zoologically widespread as it exists across the metazoan sub-kingdom. Chromosomal localisation and PCR analysis, coupled to inspection of the current S. mansoni genomic assembly, revealed that many of the SmVAL genes are spatially linked throughout the genome. Quantitative lifecycle expression profiling demonstrated distinct SmVAL expression patterns, including transcripts specifically associated with lifestages involved in definitive host invasion, transcripts restricted to lifestages involved in the invasion of the intermediate host and transcripts ubiquitously expressed. Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing. Conclusion Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa. The extensive lifecycle expression analysis indicates several SmVAL transcripts are upregulated in infective stages of the parasite, suggesting that these particular protein products may be linked to the establishment of chronic host/parasite interactions.

Chalmers, Iain W; McArdle, Andrew J; Coulson, Richard MR; Wagner, Marissa A; Schmid, Ralf; Hirai, Hirohisa; Hoffmann, Karl F

2008-01-01

195

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

196

The splicing fate of plant SPO11 genes.  

PubMed

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

Sprink, Thorben; Hartung, Frank

2014-01-01

197

The splicing fate of plant SPO11 genes  

PubMed Central

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

Sprink, Thorben; Hartung, Frank

2014-01-01

198

Transcription regulation and alternative splicing of an early zygotic gene encoding two structurally distinct zinc finger proteins in Xenopus laevis  

Microsoft Academic Search

We describe the structural organization of a gene, termed XFDL 141\\/156, that is transiently activated during early Xenopus development. XFDL 141\\/156 is first transcribed at the midblastula transition (MBT) and during early gastrulation events. A roughly 200 nucleotide fragment immediately 5? to the transcription start site is sufficient for transient, early zygotic activation of gene expression. The primary transcript is

Eric Bellefroid; Catherine Bourguignon; Tewis Bouwmeester; Oliver Rausch; Bruce Blumberg; Tomas Pieler

1997-01-01

199

The importance of identifying alternative splicing in vertebrate genome annotation.  

PubMed

While alternative splicing (AS) can potentially expand the functional repertoire of vertebrate genomes, relatively few AS transcripts have been experimentally characterized. We describe our detailed manual annotation of vertebrate genomes, which is generating a publicly available geneset rich in AS. In order to achieve this we have adopted a highly sensitive approach to annotating gene models supported by correctly mapped, canonically spliced transcriptional evidence combined with a highly cautious approach to adding unsupported extensions to models and making decisions on their functional potential. We use information about the predicted functional potential and structural properties of every AS transcript annotated at a protein-coding or non-coding locus to place them into one of eleven subclasses. We describe the incorporation of new sequencing and proteomics technologies into our annotation pipelines, which are used to identify and validate AS. Combining all data sources has led to the production of a rich geneset containing an average of 6.3?AS transcripts for every human multi-exon protein-coding gene. The datasets produced have proved very useful in providing context to studies investigating the functional potential of genes and the effect of variation may have on gene structure and function. DATABASE URL: http://www.ensembl.org/index.html, http://vega.sanger.ac.uk/index.html. PMID:22434846

Frankish, Adam; Mudge, Jonathan M; Thomas, Mark; Harrow, Jennifer

2012-01-01

200

Opportunities and methods for studying alternative splicing in cancer with RNA-Seq.  

PubMed

The biogenesis, development and metastases of cancer are associated with many variations in the transcriptome. Alternative splicing of genes is a major post-transcriptional regulation mechanism that is involved in many types of cancer. The next-generation sequencing applied on RNAs (RNA-Seq) provides a new technology for studying transcriptomes. It provides an unprecedented opportunity for quantitatively studying alternative splicing in a systematic way. This mini-review summarizes the current RNA-Seq studies on cancer transcriptomes especially studies on cancer-related alternative splicing, and discusses the strategy for quantitative study of alternative splicing in cancers with RNA-Seq, the bioinformatics methods available and existing questions. PMID:23196057

Feng, Huijuan; Qin, Zhiyi; Zhang, Xuegong

2013-11-01

201

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

202

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing.  

PubMed

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

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

2014-02-01

203

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

Microsoft Academic Search

BACKGROUND: The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for

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

2003-01-01

204

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

PubMed

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 190,529 isoforms, 37% of human genes have multiple isoforms. The isoforms produced from a gene partially share common peptide fragments because they have common exons, making it difficult to distinguish isoforms. Therefore, we developed a new method that effectively distinguishes a true isoform among multiple isoforms in a gene. In order to evaluate our algorithm, we made test sets for 4226 protein isoforms extracted from our theoretical database randomly. Consequently, 94% of true isoforms were identified by our scoring algorithm. PMID:18835246

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

2008-12-01

205

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

SciTech Connect

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

Thiede, M.A.; Strewler, G.J.; Nissenson, R.A.; Rosenblatt, M.; Rodan, G.A. (Merck Sharp Dohme Research Labs., West Point, PA (USA))

1988-07-01

206

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

207

RBM5, 6, and 10 differentially regulate NUMB alternative splicing to control cancer cell proliferation.  

PubMed

RBM5, a regulator of alternative splicing of apoptotic genes, and its highly homologous RBM6 and RBM10 are RNA-binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation, and its modulation recapitulates or antagonizes the effects of RBM5, 6, and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation. PMID:24332178

Bechara, Elias G; Sebestyén, Endre; Bernardis, Isabella; Eyras, Eduardo; Valcárcel, Juan

2013-12-12

208

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

209

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

PubMed Central

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

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

2012-01-01

210

Functional regulation of alternatively spliced Na+/Ca2+ exchanger (NCX1) isoforms.  

PubMed

Alternative splicing of RNA transcripts is a general characteristic for NCX genes in mammals, mollusks, and arthropods. Among the family of three NCX genes in mammals, the NCX1 gene contains six exons, namely, A, B, C, D, E, and F, that make up the alternatively spliced region. Studies of the NCX1 gene transcripts suggested that 16 distinct gene products can be produced from the NCX1 gene. The exons A and B are mutually exclusive when expressed. Generally, exon A-containing transcripts are predominantly found in excitable cells like cardiomyoctes and neurons, whereas exon B-containing transcripts are mostly found in nonexcitable cells like astrocytes and kidney cells. Other alternatively spliced exons (C-F) appear to be cassette-type exons and are found in various combinations. Interestingly, exon D is present in all characterized transcripts. The alternatively spliced isoforms of NCX1 show tissue-specific expression patterns, suggesting functional adaptation to tissues. To investigate functional differences among alternatively spliced isoforms of NCX1, we expressed an exon A-containing transcript present in cardiac tissue (NCX1.1) and an exon B-containing transcript found in the kidney (NCX1.3) in Xenopus oocytes. We demonstrated that the Na(+)/Ca(2+) exchangers expressed by exon A- and exon B-containing transcripts display differences in activation by PKA and by [Ca(2+)](i). We also observed that these two isoforms show differences in voltage dependence. Surprisingly, the alternatively spliced isoforms of NCX1 display greater functional differences among themselves than the products of different gene loci, NCX1, NCX2, and NCX3. PMID:12502560

Schulze, D H; Polumuri, S K; Gille, T; Ruknudin, A

2002-11-01

211

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

PubMed Central

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

2014-01-01

212

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

PubMed Central

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

2012-01-01

213

Predicting the Impact of Alternative Splicing on Plant MADS Domain Protein Function  

Microsoft Academic Search

Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function.

Edouard I. Severing; Aalt D. J. van Dijk; Giuseppa Morabito; Jacqueline Busscher-Lange; Richard G. H. Immink; Roeland C. H. J. van Ham

2012-01-01

214

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

215

Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene.  

PubMed Central

The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures. Images

Libri, D; Goux-Pelletan, M; Brody, E; Fiszman, M Y

1990-01-01

216

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

PubMed Central

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

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

2012-01-01

217

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.  

PubMed

The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage. PMID:22684629

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

2012-09-01

218

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

PubMed Central

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

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

2008-01-01

219

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

PubMed Central

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

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

2010-01-01

220

CAG repeats mimic CUG repeats in the misregulation of alternative splicing  

PubMed Central

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

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

2011-01-01

221

Alternative mRNA splice variants of the rat ClC-2 chloride channel gene are expressed in lung: genomic sequence and organization of ClC-2.  

PubMed Central

The ClC-2 epithelial cell chloride channel is a voltage-, tonicity- and pH-regulated member of the ClC super family. We have previously shown that rat lung ClC-2 (rClC-2) is down-regulated at birth, and molecular diversity is generated by alternative splicing [Murray et al. (1995) Am. J. Respir. Cell Mol. Biol. 12, 597-604; Murray et al. (1996) Am. J. Physiol. 271, L829-L837; Chu et al . (1996) Nucleic Acids Res. 24, 3453-3457]. To investigate other possible mRNA splice variations, we sequenced the entire rClC-2 gene and found that ClC-2Sa (formerly ClC-2S) results from the deletion of exon 20. The preceding intron 19 has an unusually high CT content and a rare AAG acceptor site. Because both features were also found in intron 13, we next tested the hypothesis that intron 13 would be involved in alternative splicing. As predicted, a second splice product, ClC-2Sb, was found by RT-PCR, but only in lung. When we compared the genomic maps of rClC-2 and human ClC-1 (hClC-1), striking similarities were found in each exon except for rClC-2 exon 20, which is absent in hClC-1. These observations suggest that ClC-1 and ClC-2 may have evolved by gene duplication, mutation and DNA rearrangement.

Chu, S; Zeitlin, P L

1997-01-01

222

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

223

Alternatively Spliced Tissue Factor Is Not Sufficient for Embryonic Development  

PubMed Central

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the ?-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.

Sluka, Susanna H. M.; Akhmedov, Alexander; Vogel, Johannes; Unruh, Dusten; Bogdanov, Vladimir Y.; Camici, Giovanni G.; Luscher, Thomas F.; Ruf, Wolfram; Tanner, Felix C.

2014-01-01

224

TFPI? is an Active Alternatively Spliced Form of TFPI Present in Mice but not in Humans  

PubMed Central

Background Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPI? and TFPI?, which differ in domain structure and mechanism for cell surface association. 3’ RACE was used to search for new TFPI isoforms. TFPI?, a new alternatively spliced form of TFPI was identified and characterized. Methods The tissue expression, cell surface association and anticoagulant activity of TFPI? were characterized and compared to TFPI? and TFPI? through studies of mouse and human tissues and expression of recombinant proteins in CHO cells. Results TFPI? is produced by alternative splicing using the same 5’ splice donor site as TFPI? and a 3’ splice acceptor site 187 nucleotides beyond the stop codon of TFPI? in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPI?. TFPI? mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPI? is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. Conclusions TFPI? is a third alternatively spliced form of TFPI widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface associated protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor mediated models of disease.

Maroney, Susan A.; Ferrel, Josephine P.; Collins, Maureen L.; Mast, Alan E.

2013-01-01

225

A protocol for visual analysis of alternative splicing in RNA-Seq data using integrated genome browser.  

PubMed

Ultrahigh-throughput sequencing of cDNA (RNA-Seq) is an invaluable resource for investigating alternative splicing in an organism. Alternative splicing is a form of posttranscriptional regulation in which primary RNA transcripts from a single gene can be spliced in multiple ways leading to different RNA and protein products. In plants and other species, it has been shown that many genes involved in circadian regulation are alternatively spliced. As new RNA-Seq data sets become available, these data will lead to new insights into links between regulation RNA splicing and the circadian system. Analyzing RNA-Seq data sets requires software tools that can display RNA-Seq read alignments alongside gene models, enabling assessment of how treatments or developmental stages affect splicing patterns and production of novel variants. The Integrated Genome Browser (IGB) software program is a free and flexible desktop tool that enables discovery and quantification of alternative splicing. In this protocol, we use IGB and a cold-stress RNA-Seq data set to examine alternative splicing of Arabidopsis thaliana LHY, a circadian clock regulator. IGB is freely available from http://www.bioviz.org . PMID:24792048

Gulledge, Alyssa A; Vora, Hiral; Patel, Ketan; Loraine, Ann E

2014-01-01

226

A protocol for visual analysis of alternative splicing in RNA-Seq data using Integrated Genome Browser  

PubMed Central

Summary Ultra-high throughput sequencing of cDNA (RNA-Seq) is an invaluable resource for investigating alternative splicing in an organism. Alternative splicing is a form of post-transcriptional regulation in which primary RNA transcripts from a single gene can be spliced in multiple ways leading to different RNA and protein products. In plants and other species, it has been shown that many genes involved in circadian regulation are alternatively spliced. As new RNA-Seq data sets become available, these data will lead to new insights into links between regulation RNA splicing and the circadian system. Analyzing RNA-Seq data sets requires software tools that can display RNA-Seq read alignments alongside gene models, enabling assessment of how treatments or developmental stages affect splicing patterns and production of novel variants. The Integrated Genome Browser software program (IGB) is a free and flexible desktop tool that enables discovery and quantification of alternative splicing. In this protocol, we use IGB and a cold-stress RNA-Seq data set to examine alternative splicing of Arabidopsis thaliana LHY, a circadian clock regulator. Integrated Genome Browser is freely available from http://www.bioviz.org.

Gulledge, Alyssa A.; Vora, Hiral; Patel, Ketan; Loraine, Ann E.

2014-01-01

227

Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors  

PubMed Central

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

Salomonis, Nathan; Nelson, Brandon; Vranizan, Karen; Pico, Alexander R.; Hanspers, Kristina; Kuchinsky, Allan; Ta, Linda; Mercola, Mark; Conklin, Bruce R.

2009-01-01

228

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

PubMed Central

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

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

2010-01-01

229

Identification and characterization of alternative splicing in parasitic nematode transcriptomes  

PubMed Central

Background Alternative splicing (AS) of mRNA is a vital mechanism for enhancing genomic complexity in eukaryotes. Spliced isoforms of the same gene can have diverse molecular and biological functions and are often differentially expressed across various tissues, times, and conditions. Thus, AS has important implications in the study of parasitic nematodes with complex life cycles. Transcriptomic datasets are available from many species, but data must be revisited with splice-aware assembly protocols to facilitate the study of AS in helminthes. Methods We sequenced cDNA from the model worm Caenorhabditis elegans using 454/Roche technology for use as an experimental dataset. Reads were assembled with Newbler software, invoking the cDNA option. Several combinations of parameters were tested and assembled transcripts were verified by comparison with previously reported C. elegans genes and transcript isoforms and with Illumina RNAseq data. Results Thoughtful adjustment of program parameters increased the percentage of assembled transcripts that matched known C. elegans sequences, decreased mis-assembly rates (i.e., cis- and trans-chimeras), and improved the coverage of the geneset. The optimized protocol was used to update de novo transcriptome assemblies from nine parasitic nematode species, including important pathogens of humans and domestic animals. Our assemblies indicated AS rates in the range of 20-30%, typically with 2-3 transcripts per AS locus, depending on the species. Transcript isoforms from the nine species were translated and searched for similarity to known proteins and functional domains. Some 21 InterPro domains, including several involved in nucleotide and chromatin binding, were statistically correlated with AS genetic loci. In most cases, the Roche/454 data explored in this study are the only sequences available from the species in question; however, the recently published genome of the human hookworm Necator americanus provided an additional opportunity to validate our results. Conclusions Our optimized assembly parameters facilitated the first survey of AS among parasitic nematodes. The nine transcriptome assemblies, their protein translations, and basic annotations are available from Nematode.net as a resource for the research community. These should be useful for studies of specific genes and gene families of interest as well as for curating draft genome assemblies as they become available.

2014-01-01

230

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform  

Microsoft Academic Search

Background Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a

Sandhya P. Koushika; Michael J. Lisbin; Kalpana White

1996-01-01

231

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

232

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

233

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

234

Expression of Two Novel Alternatively Spliced COL2A1 Isoforms During Chondrocyte Differentiation  

PubMed Central

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

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

2008-01-01

235

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation.  

PubMed

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

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

2008-04-01

236

Discovery and expression analysis of alternative splicing events conserved among plant SR proteins.  

PubMed

The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. In this article, we have searched and manually annotated all the SR proteins in the genomes of maize and sorghum. The experimental validation of gene structure by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed, with few exceptions, that SR genes produced multiple isoforms of transcripts by alternative splicing. Despite sharing high structural similarity and conserved positions of the introns, the profile of alternative splicing diverged significantly between maize and sorghum for the vast majority of SR genes. These include many transcript isoforms discovered by RT-PCR and not represented in extant expressed sequence tag (EST) collection. However, we report the occurrence of various maize and sorghum SR mRNA isoforms that display evolutionary conservation of splicing events with their homologous SR genes in Arabidopsis and moss. Our data also indicate an important role of both 5' and 3' untranslated regions in the regulation of SR gene expression. These observations have potentially important implications for the processes of evolution and adaptation of plants to land. PMID:24356560

Rauch, Hypaitia B; Patrick, Tara L; Klusman, Katarina M; Battistuzzi, Fabia U; Mei, Wenbin; Brendel, Volker P; Lal, Shailesh K

2014-03-01

237

Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.  

PubMed

Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions. PMID:24244129

Eksi, Ridvan; Li, Hong-Dong; Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S; Kretzler, Matthias; Guan, Yuanfang

2013-11-01

238

Factors influencing alternative splice site utilization in vivo.  

PubMed Central

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images

Fu, X Y; Manley, J L

1987-01-01

239

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes  

PubMed Central

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfap? is the most predominant isoform. The Gfap? isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfap? mRNA and Gfap? protein. RT-qPCR analysis showed that Gfap? mRNA and Gfap? mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfap? is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfap? into the Gfap intermediate filament network and overlap in Gfap? and Gfap? subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfap? and Gfap? mRNA in mouse primary astrocytes. A larger fraction of Gfap? mRNA showed mRNA localization to astrocyte protrusions compared to Gfap? mRNA. The differential mRNA localization patterns were dependent on the different 3?-exon sequences included in Gfap? and Gfap? mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.

Thomsen, Rune; Daugaard, Tina F.; Holm, Ida E.; Nielsen, Anders Lade

2013-01-01

240

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

241

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

242

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

243

Alternative splicing during Arabidopsis flower development results in constitutive and stage-regulated isoforms  

PubMed Central

Alternative splicing (AS) is a process in eukaryotic gene expression, in which the primary transcript of a multi-exon gene is spliced into two or more different mature transcripts, thereby increasing proteome diversity. AS is often regulated differentially between different tissues or developmental stages. Recent studies suggested that up to 60% of intron-containing genes in Arabidopsis thaliana undergo AS. Yet little is known about this complicated and important process during floral development. To investigate the preferential expression of different isoforms of individual alternatively spliced genes, we used high throughput RNA-Seq technology to explore the transcriptomes of three floral development stages of Arabidopsis thaliana and obtained information of various AS events. We identified approximately 24,000 genes that were expressed at one or more of these stages, and found that nearly 25% of multi-exon genes had two or more spliced variants. This is less frequent than the previously reported 40–60% for multiple organs and stages of A. thaliana, indicating that many genes expressed in floral development function with a single predominant isoform. On the other hand, 1716 isoforms were differentially expressed between the three stages, suggesting that AS might still play important roles in stage transition during floral development. Moreover, 337 novel transcribed regions were identified and most of them have a single exon. Taken together, our analyses provide a comprehensive survey of AS in floral development and facilitate further genomic and genetic studies.

Wang, Haifeng; You, Chenjiang; Chang, Fang; Wang, Yingxiang; Wang, Lei; Qi, Ji; Ma, Hong

2014-01-01

244

Microenvironment Changes (in pH) Affect VEGF Alternative Splicing  

PubMed Central

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p?splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process. Electronic supplementary material The online version of this article (doi:10.1007/s12307-008-0013-4) contains supplementary material, which is available to authorized users.

Elias, Ana Paula

2008-01-01

245

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

246

Epithelial splicing regulator protein 1 and alternative splicing in somatotroph adenomas.  

PubMed

Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously hypothesized that epithelial mesenchymal transition (EMT) may play a central role in the progression of these adenomas and that epithelial splicing regulator 1 (ESRP1) may function prominently as a master regulator of the EMT process in pituitary adenomas causing acromegaly. To further elucidate the role of ESRP1 in somatotroph adenomas and in EMT progression, we used RNA sequencing (RNAseq) to sequence somatotroph adenomas characterized by high and low ESRP1 levels. Transcripts identified by RNAseq were analyzed in 65 somatotroph adenomas and in GH-producing pituitary rat cells with a specific knockdown of Esrp1. The clinical importance of the transcripts was further investigated by correlating mRNA expression levels with clinical indices of disease activity and treatment response. Many of the transcripts and isoforms identified by RNAseq and verified by quantitative PCR were involved in vesicle transport and calcium signaling and were associated with clinical outcomes. Silencing Esrp1 in GH3 cells resulted in changes of gene expression overlapping the data observed in human somatotroph adenomas and revealed a decreased granulation pattern and attenuated GH release. We observed an alternative splicing pattern for F-box and leucine-rich repeat protein 20, depending on the ESPR1 levels and on changes in circulating IGF-I levels after somatostatin analog treatment. Our study indicates that ESRP1 in somatotroph adenomas regulates transcripts that may be essential in the EMT progression and in the response to somatostatin analog treatment. PMID:23825128

Lekva, Tove; Berg, Jens Petter; Lyle, Robert; Heck, Ansgar; Ringstad, Geir; Olstad, Ole Kristoffer; Michelsen, Annika Elisabet; Casar-Borota, Olivera; Bollerslev, Jens; Ueland, Thor

2013-09-01

247

Ancient nature of alternative splicing and functions of introns  

SciTech Connect

Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

2011-03-21

248

Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development  

PubMed Central

Alternative premRNA splicing is a major mechanism to generate diversity of gene products. However, the biological roles of alternative splicing during development remain elusive. Here, we focus on a neuron-specific RNA-binding protein, Rbfox3, recently identified as the antigen of the widely used anti-NeuN antibody. siRNA-mediated loss-of-function studies using the developing chicken spinal cord revealed that Rbfox3 is required to promote neuronal differentiation of postmitotic neurons. Numb premRNA encoding a signaling adaptor protein was found to be a target of Rbfox3 action, and Rbfox3 repressed the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform reproduced similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform was sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development.

Kim, Kee K.; Nam, Joseph

2013-01-01

249

Regulation of Dscam exon 17 alternative splicing by steric hindrance in combination with RNA secondary structures.  

PubMed

The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38 016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. Here we reveal a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activated exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppressed exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes. PMID:24448213

Yue, Yuan; Li, Guoli; Yang, Yun; Zhang, Wenjing; Pan, Huawei; Chen, Ran; Shi, Feng; Jin, Yongfeng

2013-12-01

250

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

251

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

252

Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium.  

PubMed

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (?1Aq, ?5, ?5q, ?8p, ?9, ?(9,10), ?9_11, ?11q, ?13p and ?14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms. PMID:24569164

Colombo, Mara; Blok, Marinus J; Whiley, Phillip; Santamariña, Marta; Gutiérrez-Enríquez, Sara; Romero, Atocha; Garre, Pilar; Becker, Alexandra; Smith, Lindsay Denise; De Vecchi, Giovanna; Brandão, Rita D; Tserpelis, Demis; Brown, Melissa; Blanco, Ana; Bonache, Sandra; Menéndez, Mireia; Houdayer, Claude; Foglia, Claudia; Fackenthal, James D; Baralle, Diana; Wappenschmidt, Barbara; Díaz-Rubio, Eduardo; Caldés, Trinidad; Walker, Logan; Díez, Orland; Vega, Ana; Spurdle, Amanda B; Radice, Paolo; De La Hoya, Miguel

2014-07-15

253

The ASRG database: identification and survey of Arabidopsis thaliana genes involved in pre-mRNA splicing  

Microsoft Academic Search

A total of 74 small nuclear RNA (snRNA) genes and 395 genes encoding splicing-related proteins were identified in the Arabidopsis genome by sequence comparison and motif searches, including the previously elusive U4atac snRNA gene. Most of the genes have not been studied experimentally. Classification of these genes and detailed information on gene structure, alternative splicing, gene duplications and phylogenetic relationships

Bing-Bing Wang; Volker Brendel

2004-01-01

254

Alternatively spliced isoforms of the human constitutive androstane receptor  

PubMed Central

The nuclear receptor CAR (NR1I3) regulates transcription of genes encoding xenobiotic- and steroid-metabolizing enzymes. Regulatory processes that are mediated by CAR are modulated by a structurally diverse array of chemicals including common pharmaceutical and environmental agents. Here we describe four in-frame splice variants of the human CAR receptor gene. The variant mRNA splice transcripts were expressed in all human livers evaluated. Molecular modeling of the splice variant proteins predicts that the structural effects are localized within the receptor’s ligand-binding domain. Assays to assess function indicate that the variant proteins, when compared with the reference protein isoform, exhibit compromised activities with respect to DNA binding, transcriptional activation and coactivator recruitment.

Auerbach, Scott S.; Ramsden, Richard; Stoner, Matthew A.; Verlinde, Christophe; Hassett, Christopher; Omiecinski, Curtis J.

2003-01-01

255

Alternative Splicing and Transcriptome Profiling of Experimental Autoimmune Encephalomyelitis Using Genome-Wide Exon Arrays  

PubMed Central

Background Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease. Methodology/Principal Findings We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility. Conclusions/Significance Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE.

Gillett, Alan; Maratou, Klio; Fewings, Chris; Harris, Robert A.; Jagodic, Maja; Aitman, Tim; Olsson, Tomas

2009-01-01

256

RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing  

PubMed Central

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.

2014-01-01

257

DBIRD integrates alternative mRNA splicing with RNA polymerase II transcript elongation  

PubMed Central

Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation1,2,3. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle which is its functional form and determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles. This led to the identification of Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD as subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.

Close, Pierre; East, Philip; Dirac-Svejstrup, A. Barbara; Hartmann, Holger; Heron, Mark; Maslen, Sarah; Chariot, Alain; Soding, Johannes; Skehel, Mark; Svejstrup, Jesper Q.

2012-01-01

258

Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements.  

PubMed

The TAF6? pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6? is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6? has been shown to be a pivotal event in triggering death via the TAF6? pathway, yet nothing is currently known about the mechanisms that promote TAF6? splicing. Furthermore the transcriptome impact of the gain of function of TAF6? versus the loss of function of the major TAF6? splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6? drives a transcriptome profile distinct from that resulting from depletion of TAF6?. To define the cis-acting RNA elements responsible for TAF6? alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6? and also reveal a role for RNA secondary structure in the selection of TAF6?. PMID:25025302

Kamtchueng, Catherine; Stébenne, Marie-Éve; Delannoy, Aurélie; Wilhelm, Emmanuelle; Léger, Hélène; Benecke, Arndt G; Bell, Brendan

2014-01-01

259

Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements  

PubMed Central

The TAF6? pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6? is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6? has been shown to be a pivotal event in triggering death via the TAF6? pathway, yet nothing is currently known about the mechanisms that promote TAF6? splicing. Furthermore the transcriptome impact of the gain of function of TAF6? versus the loss of function of the major TAF6? splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6? drives a transcriptome profile distinct from that resulting from depletion of TAF6?. To define the cis-acting RNA elements responsible for TAF6? alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6? and also reveal a role for RNA secondary structure in the selection of TAF6?.

Kamtchueng, Catherine; Stebenne, Marie-Eve; Delannoy, Aurelie; Wilhelm, Emmanuelle; Leger, Helene; Benecke, Arndt G.; Bell, Brendan

2014-01-01

260

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

261

Alternative transcription exceeds alternative splicing in generating the transcriptome diversity of cerebellar development  

PubMed Central

Despite our growing knowledge that many mammalian genes generate multiple transcript variants that may encode functionally distinct protein isoforms, the transcriptomes of various tissues and their developmental stages are poorly defined. Identifying the transcriptome and its regulation in a cell/tissue is the key to deciphering the cell/tissue-specific functions of a gene. We built a genome-wide inventory of noncoding and protein-coding transcripts (transcriptomes), their promoters (promoteromes) and histone modification states (epigenomes) for developing, and adult cerebella using integrative massive-parallel sequencing and bioinformatics approach. The data consists of 61,525 (12,796 novel) distinct mRNAs transcribed by 29,589 (4792 novel) promoters corresponding to 15,669 protein-coding and 7624 noncoding genes. Importantly, our results show that the transcript variants from a gene are predominantly generated using alternative transcriptional rather than splicing mechanisms, highlighting alternative promoters and transcriptional terminations as major sources of transcriptome diversity. Moreover, H3K4me3, and not H3K27me3, defined the use of alternative promoters, and we identified a combinatorial role of H3K4me3 and H3K27me3 in regulating the expression of transcripts, including transcript variants of a gene during development. We observed a strong bias of both H3K4me3 and H3K27me3 for CpG-rich promoters and an exponential relationship between their enrichment and corresponding transcript expression. Furthermore, the majority of genes associated with neurological diseases expressed multiple transcripts through alternative promoters, and we demonstrated aberrant use of alternative promoters in medulloblastoma, cancer arising in the cerebellum. The transcriptomes of developing and adult cerebella presented in this study emphasize the importance of analyzing gene regulation and function at the isoform level.

Pal, Sharmistha; Gupta, Ravi; Kim, Hyunsoo; Wickramasinghe, Priyankara; Baubet, Valerie; Showe, Louise C.; Dahmane, Nadia; Davuluri, Ramana V.

2011-01-01

262

Alternative Splicing of Exon 14 Determines Nuclear or Cytoplasmic Localisation of FMR1 Protein Isoforms  

Microsoft Academic Search

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to sev- eral isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of

Annie Sittler; Didier Devys; Chantal Weber; Jean-Louis Mandel

1996-01-01

263

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

PubMed

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 were 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 functions. PMID:21925157

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

2011-11-15

264

The ASRG database: identification and survey of Arabidopsis thaliana genes involved in pre-mRNA splicing  

PubMed Central

A total of 74 small nuclear RNA (snRNA) genes and 395 genes encoding splicing-related proteins were identified in the Arabidopsis genome by sequence comparison and motif searches, including the previously elusive U4atac snRNA gene. Most of the genes have not been studied experimentally. Classification of these genes and detailed information on gene structure, alternative splicing, gene duplications and phylogenetic relationships are made accessible as a comprehensive database of Arabidopsis Splicing Related Genes (ASRG) on our website.

Wang, Bing-Bing; Brendel, Volker

2004-01-01

265

Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2  

PubMed Central

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or ?-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.

Ruepp, Marc-David; Ranzini, Francesco; Maffioletti, Andrea; Alvarez, Reinaldo; Muhlemann, Oliver; Barabino, Silvia M. L.

2013-01-01

266

Gene structure-based splice variant deconvolution using a microarry platform  

Microsoft Academic Search

Motivation: Alternative splicing allows a single gene to generate multiple mRNAs, which can be translated into functionally and structurally diverse proteins. One gene can have multiple variants coexisting at different concentrations. Estimating the relative abundance of each variant is important for the study of underlying biological function. Microarrays are standard tools that measure gene expression. But most design and analysis

Hui Wang; Earl Hubbell; Jing-shan Hu; Gangwu Mei; Melissa S. Cline; Gang Lu; Tyson Clark; Michael A. Siani-rose; Manuel Ares; David Kulp; David Haussler

2003-01-01

267

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

2013-06-01

268

Alternative splicing results in RET isoforms with distinct trafficking properties  

PubMed Central

RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling.

Richardson, Douglas S.; Rodrigues, David M.; Hyndman, Brandy D.; Crupi, Mathieu J. F.; Nicolescu, Adrian C.; Mulligan, Lois M.

2012-01-01

269

The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA.  

PubMed Central

Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle.

Hodges, D; Cripps, R M; O'Connor, M E; Bernstein, S I

1999-01-01

270

Statistical and Computational Methods for High-Throughput Sequencing Data Analysis of Alternative Splicing  

PubMed Central

The burgeoning field of high-throughput sequencing significantly improves our ability to understand the complexity of transcriptomes. Alternative splicing, as one of the most important driving forces for transcriptome diversity, can now be studied at an unprecedent resolution. Efficient and powerful computational and statistical methods are in urgent need to facilitate the characterization and quantification of alternative splicing events. Here we discuss methods in splice junction read mapping, and methods in exon-centric or isoform-centric quantification of alternative splicing. In addition, we discuss HITS-CLIP and splicing QTL analyses which are novel high-throughput sequencing based approaches in the dissection of splicing regulation.

2013-01-01

271

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

PubMed Central

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 and demonstrated editing at this site in human brain. We have shown that in cell culture, base substitutions to mimic editing at different combinations of the six sites profoundly affect relative splicing at the normal and the upstream alternative splice site, but splicing at the downstream alternative splice site was consistently rare. Editing combinations in different splice variants from human brain were determined and are consistent with the effects of editing on splicing observed in cell culture. As RNA editing usually occurs close to exon/intron boundaries, this is likely to be a general phenomenon and suggests an important novel role for RNA editing.

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

2004-01-01

272

HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements*  

PubMed Central

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.

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

2009-01-01

273

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia.  

PubMed

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-?E4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy. PMID:24668151

Rebhandl, Stefan; Huemer, Michael; Zaborsky, Nadja; Gassner, Franz Josef; Catakovic, Kemal; Felder, Thomas Klaus; Greil, Richard; Geisberger, Roland

2014-07-01

274

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

PubMed Central

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 comprehensive analysis of AS by investigating the expression of known and predicted exons. Identifying of AS events from exon array has raised much attention, however, new and powerful algorithms for exon array data analysis are still absent till now. Results Here, we considered identifying of AS events in the framework of variable selection and developed a regression method for AS detection (REMAS). Firstly, features of alternatively spliced exons were scaled by reasonably defined variables. Secondly, we designed a hierarchical model which can represent gene structure and transcriptional influence to exons, and the lasso type penalties were introduced in calculation because of huge variable size. Thirdly, an iterative two-step algorithm was developed to select alternatively spliced genes and exons. To avoid negative effects introduced by small sample size, we ranked genes as parameters indicating their AS capabilities in an iterative manner. After that, both simulation and real data evaluation showed that REMAS could efficiently identify potential AS events, some of which had been validated by RT-PCR or supported by literature evidence. Conclusion As a new lasso regression algorithm based on hierarchical model, REMAS has been demonstrated as a reliable and effective method to identify AS events from exon array data.

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

2009-01-01

275

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

276

Differential Requirements for Alternative Splicing and Nuclear Export Functions of Equine Infectious Anemia Virus Rev Protein  

Microsoft Academic Search

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized

MATTHEW E. HARRIS; RICHARD R. GONTAREK; DAVID DERSE; THOMAS J. HOPE

1998-01-01

277

Alternative Splicing in the Voltage-Gated Sodium Channel DmNav Regulates Activation, Inactivation, and Persistent Current  

PubMed Central

Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. In this study we report the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na+ gene (DmNav, paralytic), in late-stage embryos of Drosophila melanogaster. We identify 27 unique splice variants, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j, e, and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage (V1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V1/2 of steady-state inactivation. The magnitude of the persistent Na+ current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L. Transcripts containing k have significantly smaller persistent currents compared with those containing L. Finally, we show that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). Our results provide a functional basis for understanding how alternative splicing of a voltage-gated Na+ channel results in diversity in neuronal signaling.

Lin, Wei-Hsiang; Wright, Duncan E.; Muraro, Nara I.; Baines, Richard A.

2009-01-01

278

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.  

PubMed Central

In Caenorhabditis elegans, pre-mRNAs that are trans-spliced are distinguished by the presence of an 'outron', intron-like RNA at the 5' end followed by a splice acceptor. We report that trans-splicing of the rol-6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans-splice site, thereby converting rol-6 into a conventional gene with a spliced intron near its 5' end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis-splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans-splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans-splicing is the presence of an outron. Clearly, cis- and trans-splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing. Images

Conrad, R; Liou, R F; Blumenthal, T

1993-01-01

279

Dynamic integration of splicing within gene regulatory pathways  

PubMed Central

Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control.

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

2013-01-01

280

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

281

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations  

Microsoft Academic Search

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat

Thilo Dörk; Rainer Fislage; Thomas Neumann; Brigitte Wulf; Burkhard Tümmler

1994-01-01

282

Differential expression of alternative splice variants of CTLA4 in Kuwaiti autoimmune disease patients.  

PubMed

Cytotoxic T lymphocyte associated antigen4 (CTLA4) is a candidate susceptibility gene for the study of autoimmune diseases. The present study sought to explore the expression profile of the CTLA4 gene in autoimmune patients, such as rheumatoid arthritis (RA), systemic lupus erythematous (SLE) and Hashimoto's thyroiditis (HT), compared to healthy controls (HCs). A total of 88 (22 RA, 22 SLE 22 HT, 22 HCs) age-, gender- and ethnicity-matched individuals were recruited. The hypersensitive capillary electrophoresis method was employed to detect the CTLA4 splice variants. PCRs of the patient's cDNA using CTLA4-specific primers followed by cloning and sequencing were used to distinguish the various splice variants. The biochemical properties of all known CTLA4 variants were analysed using the ExPASy and ESEfinder programmes. Six alternatively spliced variants of the CTLA4 gene were detected in this study. These included mCTLA4-672, sCTLA4-562, N-CTLA4-292, L-CTLA4-277, ssCTLA4-214 and K-CTLA4-142 bp. K-CTLA4-142 bp and N-CTLA4-292 bp represented two novel splice variants of the CTLA4 gene. A reduction in the frequency of mCTLA4-672 bp and sCTLA4-562 bp was observed in SLE and RA patients compared to healthy controls. The shortest splice variant, K-CTLA4-142 bp, was predominantly detected in all of the tested cohorts,while the decreased expression of the N-CTLA4-292 bp variant was observed in the autoimmune subjects. The exonic splice enhancer motifs of the SRp40 protein were found exactly at the splice junction of wCTLA4 (-ACAGAGC-, 2.7) and K-CTLA4 (-TGAAAAG-, 3.37), and that of the SRp55 protein was found at the splice junction of L-CTLA4 (-TGTGTG-, 2.82). Our study highlights the discrepancies in the expression spectrum of the CTLA4 gene in autoimmune patients and healthy subjects. The abnormal expression pattern of the CTLA4 gene in autoimmune patients suggests that in addition to allelic variation, the expression pattern of CTLA4 could contribute to autoimmunity. PMID:24498648

AlFadhli, Suad; Nizam, Rasheeba

2014-01-25

283

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

284

ASH2L : alternative splicing and downregulation during induced megakaryocytic differentiation of multipotential leukemia cell lines  

Microsoft Academic Search

Drosophila ash2 is a member of the trxG gene super family, some human homologues of which are involved in hematopoiesis and leukemia. We report here the identification of the human homologue of Drosophila ash2 and its alternative splicing isoform, ASH2L1 and ASH2L2. ASH2L proteins are 60% homologous to Drosophila ash2. ASH2L also has a zinc finger motif (C2C2) although it

Junhua Wang; Yan Zhou; Bin Yin; Guangwei Du; Xiaowei Huang; Guangtao Li; Yan Shen; Jiangang Yuan; Boqin Qiang

2001-01-01

285

Alternative splicing in teleost fish genomes: same-species and cross-species analysis and comparisons  

Microsoft Academic Search

Alternative splicing (AS) is a mechanism by which the coding diversity of the genome can be greatly increased. Rates of AS\\u000a are known to vary according to the complexity of eukaryotic species potentially explaining the tremendous phenotypic diversity\\u000a among species with similar numbers of coding genes. Little is known, however, about the nature or rate of AS in teleost fish.

Jianguo Lu; Eric Peatman; Wenqi Wang; Qing Yang; Jason Abernathy; Shaolin Wang; Huseyin Kucuktas; Zhanjiang Liu

2010-01-01

286

The emerging role of pre-messenger RNA splicing in stress responses: sending alternative messages and silent messengers.  

PubMed

Alternative splicing (AS) of pre-messenger RNAs is a major process contributing to both transcriptome and proteome diversity in various physiological and pathological situations. There is also accumulating evidence that various stresses impact on AS. In particular, recent analyses of the transcriptome reveal large numbers of AS events that are regulated by genotoxic stress inducers like radiations and chemotherapeutic agents. Many AS events have the potential to affect the relative production of protein isoforms with different activities, as shown in the case of several genes involved in apoptosis. There is also increasing evidence that stresses induce "non-productive" splice variants, leading to a decrease in gene expression levels or preventing increases in protein levels despite transcriptional stimulation. This is typically achieved by the production of splice variants that are subject to nonsense-mediated decay. In addition, recent studies suggest that pre-mRNA splicing efficiency or fidelity may be altered by stresses. For example, various genotoxic agents induce multiple exon skipping in MDM2 transcripts, thereby preventing the production of the main p53-ubiquitin ligase and favoring p53 activity in response to genotoxic agents. In terms of mechanisms, stresses can impact on pre-mRNA splicing by inducing post-translational modifications and subcellular redistribution of splicing factors, or by targeting the communication between the splicing and transcription machineries. Altogether, these data suggest that splicing regulatory networks play a key role in the cellular responses triggered by stresses. PMID:21712650

Dutertre, Martin; Sanchez, Gabriel; Barbier, Jérôme; Corcos, Laurent; Auboeuf, Didier

2011-01-01

287

claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing.  

PubMed

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach. PMID:11585919

Niimi, T; Nagashima, K; Ward, J M; Minoo, P; Zimonjic, D B; Popescu, N C; Kimura, S

2001-11-01

288

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

PubMed

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

McNeil, Bonnie A; Zimmerly, Steven

2014-06-01

289

Stepwise assembly of the Nova-regulated alternative splicing network in the vertebrate brain  

PubMed Central

Novel organismal structures in metazoans are often undergirded by complex gene regulatory networks; as such, understanding the emergence of new structures through evolution requires reconstructing the series of evolutionary steps leading to these underlying networks. Here, we reconstruct the step-by-step assembly of the vertebrate splicing network regulated by Nova, a splicing factor that modulates alternative splicing in the vertebrate central nervous system by binding to clusters of YCAY motifs on pre-RNA transcripts. Transfection of human HEK293T cells with Nova orthologs indicated vertebrate-like splicing regulatory activity in bilaterian invertebrates, thus Nova acquired the ability to bind YCAY clusters and perform vertebrate-like splicing modulation at least before the last common ancestor of bilaterians. In situ hybridization studies in several species showed that Nova expression became restricted to CNS later on, during chordate evolution. Finally, comparative genomics studies revealed a diverse history for Nova-regulated exons, with target exons arising through both de novo exon creation and acquisition of YCAY motifs by preexisting exons throughout chordate and vertebrate history. In addition, we find that tissue-specific Nova expression patterns emerged independently in other lineages, suggesting independent assembly of tissue-specific regulatory networks.

Irimia, Manuel; Denuc, Amanda; Burguera, Demian; Somorjai, Ildiko; Martin-Duran, Jose M.; Genikhovich, Grigory; Jimenez-Delgado, Senda; Technau, Ulrich; Roy, Scott W.; Marfany, Gemma; Garcia-Fernandez, Jordi

2011-01-01

290

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

PubMed

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

Provost-Javier, K N; Rasgon, J L

2014-08-01

291

Alternative splicing by participation of the group II intron ORF in extremely halotolerant and alkaliphilic Oceanobacillus iheyensis.  

PubMed

Group II introns inserted into genes often undergo splicing at unexpected sites, and participate in the transcription of host genes. We identified five copies of a group II intron, designated Oi.Int, in the genome of an extremely halotolerant and alkaliphilic bacillus, Oceanobacillus iheyensis. The Oi.Int4 differs from the Oi.Int3 at four bases. The ligated exons of the Oi.Int4 could not be detected by RT-PCR assays in vivo or in vitro although group II introns can generally self-splice in vitro without the involvement of an intron-encoded open reading frame (ORF). In the Oi.Int4 mutants with base substitutions within the ORF, ligated exons were detected by in vitro self-splicing. It was clear that the ligation of exons during splicing is affected by the sequence of the intron-encoded ORF since the splice sites corresponded to the joining sites of the intron. In addition, the mutant introns showed unexpected multiple products with alternative 5' splice sites. These findings imply that alternative 5' splicing which causes a functional change of ligated exons presumably has influenced past adaptations of O. iheyensis to various environmental changes. PMID:21487203

Chee, Gab-Joo; Takami, Hideto

2011-01-01

292

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

293

Novel alternative splicing isoform biomarkers identification from high-throughput plasma proteomics profiling of breast cancer  

PubMed Central

Background In the biopharmaceutical industry, biomarkers define molecular taxonomies of patients and diseases and serve as surrogate endpoints in early-phase drug trials. Molecular biomarkers can be much more sensitive than traditional lab tests. Discriminating disease biomarkers by traditional method such as DNA microarray has proved challenging. Alternative splicing isoform represents a new class of diagnostic biomarkers. Recent scientific evidence is demonstrating that the differentiation and quantification of individual alternative splicing isoforms could improve insights into disease diagnosis and management. Identifying and characterizing alternative splicing isoforms are essential to the study of molecular mechanisms and early detection of complex diseases such as breast cancer. However, there are limitations with traditional methods used for alternative splicing isoform determination such as transcriptome-level, low level of coverage and poor focus on alternative splicing. Results Therefore, we presented a peptidomics approach to searching novel alternative splicing isoforms in clinical proteomics. Our results showed that the approach has significant potential in enabling discovery of new types of high-quality alternative splicing isoform biomarkers. Conclusions We developed a peptidomics approach for the proteomics community to analyze, identify, and characterize alternative splicing isoforms from MS-based proteomics experiments with more coverage and exclusive focus on alternative splicing. The approach can help generate novel hypotheses on molecular risk factors and molecular mechanisms of cancer in early stage, leading to identification of potentially highly specific alternative splicing isoform biomarkers for early detection of cancer.

2013-01-01

294

Genome-wide prediction of cis-acting RNA elements regulating tissue-specific pre-mRNA alternative splicing  

PubMed Central

Background Human genes undergo various patterns of pre-mRNA splicing across different tissues. Such variation is primarily regulated by trans-acting factors that bind on exonic and intronic cis-acting RNA elements (CAEs). Here we report a computational method to mechanistically identify cis-acting RNA elements that contribute to the tissue-specific alternative splicing pattern. This method is an extension of our previous model, SplicingModeler, which predicts the significant CAEs that contribute to the splicing differences between two tissues. In this study, we introduce tissue-specific functional levels estimation step, which allows evaluating regulatory functions of predicted CAEs that are involved in more than two tissues. Results Using a publicly available Affymetrix Genechip® Human Exon Array dataset, our method identifies 652 cis-acting RNA elements (CAEs) across 11 human tissues. About one third of predicted CAEs can be mapped to the known RBP (RNA binding protein) binding sites or match with other predicted exonic splicing regulator databases. Interestingly, the vast majority of predicted CAEs are in intronic regulatory regions. A noticeable exception is that many exonic elements are found to regulate the alternative splicing between cerebellum and testes. Most identified elements are found to contribute to the alternative splicing between two tissues, while some are important in multiple tissues. This suggests that genome-wide alternative splicing patterns are regulated by a combination of tissue-specific cis-acting elements and "general elements" whose functional activities are important but differ across multiple tissues. Conclusion In this study, we present a model-based computational approach to identify potential cis-acting RNA elements by considering the exon splicing variation as the combinatorial effects of multiple cis-acting regulators. This methodology provides a novel evaluation on the functional levels of cis-acting RNA elements by estimating their tissue-specific functions on various tissues.

Wang, Xin; Wang, Kejun; Radovich, Milan; Wang, Yue; Wang, Guohua; Feng, Weixing; Sanford, Jeremy R; Liu, Yunlong

2009-01-01

295

Characterization of the 13q14 tumor suppressor locus in CLL: identification of ALT1, an alternative splice variant of the LEU2 gene.  

PubMed

Chromosome 13q14 deletions constitute the most common genetic abnormality in chronic lymphocytic leukemia (CLL). To identify the putative tumor suppressor gene targeted by 13q14 genomic loss, we completely sequenced and characterized a segment of 790 kb at 13q14 spanning the minimal region of loss in CLL. Transcribed sequences in the region were identified through database homology searches and exon-prediction analysis. Two-hundred kb at the centromeric end of the sequence contain five CpG islands, three previously identified genes LEU5/RFP2, LEU2, and LEU1, seven of seven EST clusters composed of >10 ESTs, and a large number of predicted exons. Homology searches against the mouse EST database have allowed us to identify a highly conserved alternative first exon of the LEU2 gene, giving rise to a novel transcript, ALT1 (GenBank accession no. AF380424), which originates within a G+C region in the vicinity of the D13S272 marker. Two novel 3' exons of LEU2 were also identified and are present in both LEU2 and ALT1 transcripts. However, we have not identified any mutations in leukemia cases, or alterations in expression of mRNAs in the region, that might directly implicate these mRNAs in the pathology of CLL. The centromeric end of the sequence, where all reported genes are located, contains twice the expected amount of ALU repeats, whereas the telomeric end is LINE1 rich and contains four LINE1 elements longer than 4 kb, including two full-length LINE1 sequences. This feature of the sequence may favor the occurrence of chromosomal rearrangements and may confer instability to the region, resulting in deletions that may inactivate an as yet unidentified tumor suppressor. PMID:11559527

Bullrich, F; Fujii, H; Calin, G; Mabuchi, H; Negrini, M; Pekarsky, Y; Rassenti, L; Alder, H; Reed, J C; Keating, M J; Kipps, T J; Croce, C M

2001-09-15

296

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

297

Mnk2 Alternative Splicing Modulates the p38-MAPK Pathway and Impacts Ras-Induced Transformation.  

PubMed

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

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

2014-04-24

298

Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.  

PubMed

This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:24211518

Omenn, Gilbert S

2014-05-01

299

A High Throughput Assay to Identify Small Molecule Modulators of Alternative pre-mRNA Splicing  

PubMed Central

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-mRNA splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB) is overexpressed in ovarian tumors, compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high throughput screening HTS assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high throughput assay could be used to identify small molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.

Arslan, Ahmet Dirim; He, Xiaolong; Wang, Minxiu; Rumschlag-Booms, Emily; Rong, Lijun; Beck, William T.

2012-01-01

300

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

301

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

302

Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling  

PubMed Central

SUMMARY Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development, and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-L-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCFCOI1) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and 7 C-terminal (X5PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X5PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone, (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between alternative splicing of JAZ pre-mRNA and dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Chung, Hoo Sun; Cooke, Thomas F.; DePew, Cody L.; Patel, Lalita C.; Ogawa, Narihito; Kobayashi, Yuichi; Howe, Gregg A.

2010-01-01

303

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.  

PubMed Central

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones. Images

DeNoto, F M; Moore, D D; Goodman, H M

1981-01-01

304

Phosphorylation stabilizes alternatively spliced CD44 mRNA transcripts in breast cancer cells: Inhibition by antisense complementary to casein kinase II mRNA  

Microsoft Academic Search

CD44, the predominant vertebrate cell surface receptor for hyaluronan, exists in a variety of isoforms resulting from alternative splicing of a single gene. Particular spliced variants of CD44 correlate with increased cell motility, and with poor clinical prognosis in several kinds of carcinomas. Combinations of 9 variant exons that confer this enhanced motility on tumor cells are inserted into a

Bent Formby; Robert Stern

1998-01-01

305

Introduction to Cotranscriptional RNA Splicing  

PubMed Central

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.

Merkhofer, Evan C.; Hu, Peter; Johnson, Tracy L.

2014-01-01

306

Impact of Alternative Initiation, Splicing, and Termination on the Diversity of the mRNA Transcripts Encoded by the Mouse Transcriptome  

PubMed Central

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleotide distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Zavolan, Mihaela; Kondo, Shinji; Schonbach, Christian; Adachi, Jun; Hume, David A.; Hayashizaki, Yoshihide; Gaasterland, Terry

2003-01-01

307

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer  

PubMed Central

SUMMARY 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.

Babic, Ivan; Anderson, Erik S.; Tanaka, Kazuhiro; Guo, Deliang; Masui, Kenta; Li, Bing; Zhu, Shaojun; Gu, Yuchao; Villa, Genaro; Akhavan, David; Nathanson, David; Gini, Beatrice; Mareninov, Sergey; Li, Rui; Espindola C., Carolina; 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.

2013-01-01

308

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

2013-06-01

309

Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera)  

PubMed Central

In eusocial insects the production of daughters is generally restricted to mated queens, and unmated workers are functionally sterile. The evolution of this worker sterility has been plausibly explained by kin selection theory [Hamilton W (1964) J Theor Biol 7:1–52], and many traits have evolved to prevent conflict over reproduction among the females in an insect colony. In honeybees (Apis mellifera), worker reproduction is regulated by the queen, brood pheromones, and worker policing. However, workers of the Cape honeybee, Apis mellifera capensis, can evade this control and establish themselves as social parasites by activating their ovaries, parthenogenetically producing diploid female offspring (thelytoky) and producing queen-like amounts of queen pheromones. All these traits have been shown to be strongly influenced by a single locus on chromosome 13 [Lattorff HMG, et al. (2007) Biol Lett 3:292–295]. We screened this region for candidate genes and found that alternative splicing of a gene homologous to the gemini transcription factor of Drosophila controls worker sterility. Knocking out the critical exon in a series of RNAi experiments resulted in rapid worker ovary activation—one of the traits characteristic of the social parasites. This genetic switch may be controlled by a short intronic splice enhancer motif of nine nucleotides attached to the alternative splice site. The lack of this motif in parasitic Cape honeybee clones suggests that the removal of nine nucleotides from the altruistic worker genome may be sufficient to turn a honeybee from an altruistic worker into a parasite.

Jarosch, Antje; Stolle, Eckart; Crewe, Robin M.; Moritz, Robin F. A.

2011-01-01

310

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb  

PubMed Central

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4–6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24 hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4–6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4–5–6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

Ganassi, M.; Badodi, S.; Polacchini, A.; Baruffaldi, F.; Battini, R.; Hughes, S.M.; Hinits, Y.; Molinari, S.

2014-01-01

311

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.  

PubMed

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity. PMID:24844180

Ganassi, M; Badodi, S; Polacchini, A; Baruffaldi, F; Battini, R; Hughes, S M; Hinits, Y; Molinari, S

2014-07-01

312

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

PubMed Central

Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the host’s immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites.

2014-01-01

313

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

PubMed

Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the host's immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites. PMID:24472559

Hull, Rodney; Dlamini, Zodwa

2014-01-01

314

Cartography of neurexins: More than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons  

Microsoft Academic Search

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three ?- and three ?-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that a-neurexins are alternatively spliced at five canonical positions, and ?-neurexins at two. Characterization of many independent

Beate Ullrich; Yuri A Ushkaryov; Thomas C Südhof

1995-01-01

315

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

316

Alternative splicing of mRNA of mouse interleukin-4 and interleukin-6  

Microsoft Academic Search

Interleukin-4 and interleukin-6 are multifunctional regulatory proteins, which participate both in haemopoiesis and in immunopoiesis. The alternative splicing of these interleukins in humans is known to proceed in a tissue-specific manner. Additionally, changes in splicing can also be dependent on tissue pathology.In this work, we report on the presence of alternatively spliced mRNA (IL-4?2mRNA), lacking exon 2, in mouse bone

Olga P. Yatsenko; Maxim L. Filipenko; Eugene A. Khrapov; Elena N. Voronina; Vladimir A. Kozlov; Sergey V. Sennikov

2004-01-01

317

Molecular characterization of the mouse ribosomal protein S24 multigene family: a uniquely expressed intron-containing gene with cell-specific expression of three alternatively spliced mRNAs.  

PubMed Central

A family of 16 genes encoding the mouse ribosomal protein S24 was identified, and four members from this family were cloned. A single expressed intron-containing S24 gene (termed mrpS24) and one pseudogene (mrpS24p) were completely sequenced and characterized. The mrpS24 gene has seven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt). The cap site of S24 was mapped to a G residue four nt upstream of a polypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element. The 5' region (-325 to +33) of the mrpS24 gene has a functional promoter that was able to express the fused chloramphenicol acetyltransferase (CAT) reporter gene. Two different forms of mouse S24 cDNA clones were previously isolated. Sequence analysis showed that one of these cDNA clones (termed S24a) lacks the entire exon V sequence (18 nt), and the deduced amino acid sequence is missing a C-terminal lysine residue encoded by the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bp direct repeat, and its sequence is almost identical to the S24 cDNA sequence, but it lacks two mini-exons, V and VI (20 nt), as in the cases of the human and rat S24 cDNAs. RT-PCR experiments demonstrated the existence of a third form (S24c) that similarly lacks both of the mini-exons, and suggested that different species of S24 mRNA might arise from alternative splicing of the mini-exons V and VI. Northern blot analysis showed that S24 expression is down- and up-regulated during adipocyte differentiation and in cellular transformation, respectively. RNase protection assays and RT-PCR experiments suggested that these cell-specific changes of S24 mRNA levels are mainly due to fluctuations in S24c mRNA level. Our results provide the first indication that a ribosomal protein gene is regulated by alternative usage of two mini-exons in a cell-specific manner. Images

Xu, L; He, G P; Li, A; Ro, H S

1994-01-01

318

Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain.  

PubMed

We cloned a novel ankyrin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins. PMID:7615634

Peters, L L; John, K M; Lu, F M; Eicher, E M; Higgins, A; Yialamas, M; Turtzo, L C; Otsuka, A J; Lux, S E

1995-07-01

319

A correlation with exon expression approach to identify cis-regulatory elements for tissue-specific alternative splicing  

PubMed Central

Correlation of motif occurrences with gene expression intensity is an effective strategy for elucidating transcriptional cis-regulatory logic. Here we demonstrate that this approach can also identify cis-regulatory elements for alternative pre-mRNA splicing. Using data from a human exon microarray, we identified 56 cassette exons that exhibited higher transcript-normalized expression in muscle than in other normal adult tissues. Intron sequences flanking these exons were then analyzed to identify candidate regulatory motifs for muscle-specific alternative splicing. Correlation of motif parameters with gene-normalized exon expression levels was examined using linear regression and linear splines on RNA words and degenerate weight matrices, respectively. Our unbiased analysis uncovered multiple candidate regulatory motifs for muscle-specific splicing, many of which are phylogenetically conserved among vertebrate genomes. The most prominent downstream motifs were binding sites for Fox1- and CELF-related splicing factors, and a branchpoint-like element acuaac; pyrimidine-rich elements resembling PTB-binding sites were most significant in upstream introns. Intriguingly, our systematic study indicates a paucity of novel muscle-specific elements that are dominant in short proximal intronic regions. We propose that Fox and CELF proteins play major roles in enforcing the muscle-specific alternative splicing program, facilitating expression of unique isoforms of cytoskeletal proteins critical to muscle cell function.

Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Yamamoto, Miki; Marr, Henry; Arribere, Josh; Minovitsky, Simon; Poliakov, Alexander; Dubchak, Inna; Blume, John E.; Conboy, John G.

2007-01-01

320

Alternative splicing of BRAF transcripts and characterization of C-terminally truncated B-Raf isoforms in colorectal cancer.  

PubMed

The BRAF proto-oncogene is mutated in a subset of human tumors, including colorectal cancer. A splicing variant lacking exons 14 and 15 (BRAF del E14/15) has been described recently. However, the frequency of the variant, the kinase activity of the protein isoform, its biological function, and which allele it is derived from remains unknown. BRAF mRNA from colorectal cancer cell lines and colonic epithelium was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants and allelic origin of alternatively spliced transcripts were analyzed by DNA sequencing. Kinase activity of the B-Raf isoforms was determined by Western blotting after transfections with expression constructs of the different BRAF variants. Four additional BRAF transcript variants resulting in C-terminal truncation of the gene product were found. Alternative splicing was found at frequencies from 4.7 to 16.7% in normal and neoplastic colorectal cells. Alternative transcripts were shown to be derived from both wild-type and V600E alleles. All nonconsensus B-Raf protein variants were found to be kinase-dead and failed to coactivate full-length B-Raf. In conclusion, we present a highly sensitive method for the detection of aberrantly spliced transcripts. Alternative splicing of exons 14, 15, 15b, 16b and 16c occurs in a considerable fraction of BRAF mRNA in normal colon and colorectal cancer cells and is independent of the V600E mutational status of the parental allele. Splicing of nonfunctional transcripts affects overall cellular B-Raf activity and might represent a mechanism to decrease sensitivity to growth signals. PMID:23354951

Hirschi, Benjamin; Kolligs, Frank T

2013-08-01

321

Coding region intron\\/exon organization, alternative splicing, and X-chromosome inactivation of the KRAB\\/FPB-domain-containing human zinc finger gene ZNF41  

Microsoft Academic Search

ZNF41 belongs to a cluster of human zinc finger genes residing within a gene-rich region at Xp11.23. ZNF41 encodes a KRAB\\/FPB (Krüppel-associated\\/finger preceding box) domain, a potent transcription repression motif present in hundreds of vertebrate zinc finger protein genes, composed of two protein modules, A and B. Three introns, placed at identical positions in paralogous genes, interrupt four exons encoding

M. Rosati; A. Franzé; M. R. Matarazzo; G. Grimaldi

1999-01-01

322

Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse  

Microsoft Academic Search

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets:

Rotem Sorek; Gil Ast

2003-01-01

323

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts  

PubMed Central

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2/ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.

Skordis, Leigh A.; Dunckley, Matthew G.; Yue, Baigong; Eperon, Ian C.; Muntoni, Francesco

2003-01-01

324

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster.  

PubMed

Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3' RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics. PMID:24637386

Kim, Young Ho; Kwon, Deok Ho; Ahn, Hyo Min; Koh, Young Ho; Lee, Si Hyeock

2014-05-01

325

Quantification of type II procollagen splice forms using Alternative Transcript-qPCR (AT-qPCR)  

PubMed Central

During skeletal development, the onset of chondrogenic differentiation is marked by expression of the ?1(II) procollagen Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5? splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilage, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.

McAlinden, Audrey; Shim, Kyu-Hwan; Wirthlin, Louisa; Ravindran, Soumya; Hering, Thomas M.

2012-01-01

326

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2  

PubMed Central

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase–dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.

Saitoh, Noriko; Sakamoto, Chiyomi; Hagiwara, Masatoshi; Agredano-Moreno, Lourdes T.; Jimenez-Garcia, Luis Felipe; Nakao, Mitsuyoshi

2012-01-01

327

Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2?*  

PubMed Central

Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2? protein. The interaction between Tra2? and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2? protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2?, suggesting that Tra2? may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies.

Jiang, Zhihong; Tang, Hao; Havlioglu, Necat; Zhang, Xiaochun; Stamm, Stefan; Yan, Riqiang; Wu, Jane Y.

2007-01-01

328

Novel Fatty Acid Desaturase 3 (FADS3) Transcripts Generated By Alternative Splicing  

PubMed Central

Fatty acid desaturase 1 and 2 (FADS1 and FADS2) code for the key desaturase enzymes involved in the biosynthesis of long chain polyunsaturated fatty acids in mammals. FADS3 shares close sequence homology to FADS1 and FADS2 but the function of its gene product remains unknown. Alternative transcripts (AT) generated by alternative splicing (AS) are increasingly recognized as an important mechanism enabling a single gene to code for multiple gene products. We report the first AT of a FADS gene, FADS3, generated by AS. Aided by ORF Finder, we identified putative coding regions of eight AT for FADS3 with 1.34 kb (classical splicing), 1.14 (AT1), 0.77 (AT2), 1.25 (AT3), 0.51 (AT4), 0.74 (AT6), and 1.11 (AT7). In addition we identified a 0.51 kb length transcript (AT5) that has a termination codon within intron 8–9. The expression of each AT was analyzed in baboon neonate tissues and in differentiated and undifferentiated human SK-N-SH neuroblastoma cells. FADS3 AT are expressed in 12 neonate baboon tissues and showed reciprocal increases and decreases in expression changes in response to human neuronal cell differentiation. FADS3 AT, conserved in primates and under metabolic control in human cells, are a putative mediator of LCPUFA biosynthesis and/or regulation.

Park, Woo Jung; Kothapalli, Kumar SD; Reardon, Holly T; Kim, Luke Y.; Brenna, J. Thomas

2009-01-01

329

Alternative splicing of the guanine nucleotide-binding regulatory protein Go alpha generates four distinct mRNAs.  

PubMed Central

Go alpha a guanine nucleotide-binding (G) protein abundant in brain and other neural tissues, has been implicated in ion channel regulation. Concerted efforts in several laboratories have revealed multiple Go alpha mRNAs and protein isoforms in different contexts. Go alpha is a single copy gene in mammalian species, although the structure, number and tissue localization of Go alpha mRNAs reported by investigators are inconsistent. To define the cell-specific expression of alternatively spliced variants of Go alpha mRNA, we employed several strategies, including Northern hybridizations with sequences-specific oligonucleotides, selective digestions of Go alpha mRNA using RNase H, and adaptations of the polymerase chain reaction. Four distinct alternatively spliced variants were identified, a 5.7-kb Go alpha 2 mRNA and three Go alpha 1 mRNAs with different 3' UTRs. The UTRs of the three Go alpha 1s are composed of different combinations of what have been referred to as UTR-A and UTR-B. The sequences of the spliced segments are well conserved among mammalian species, suggesting a functional role for these alternatively spliced 3' UTRs in post-transcriptional and/or tissue-specific regulation of Go alpha expression. The position of the intron-exon splice boundary at nucleotide 31 following T of the TGA stop codon is conserved in the Gi alpha 2 and Gi alpha 3 genes, consistent with the notion that similar alternative splicing of 3' UTRs occurs in products of these related genes. Images

Murtagh, J J; Moss, J; Vaughan, M

1994-01-01

330

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

SciTech Connect

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

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

2003-03-25

331

Novel alternative splice variants of chicken NPAS3 are expressed in the developing central nervous system.  

PubMed

We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process. PMID:23962688

Shin, Jiheon; Kim, Jaesang

2013-11-10

332

Detecting Splicing Variants in Idiopathic Pulmonary Fibrosis from Non-Differentially Expressed Genes  

PubMed Central

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown cause that lacks a proven therapy for altering its high mortality rate. Microarrays have been employed to investigate the pathogenesis of IPF, but are presented mostly at the gene-expression level due to technologic limitations. In as much as, alternative RNA splicing isoforms are increasingly identified as potential regulators of human diseases, including IPF, we propose a new approach with the capacity to detect splicing variants using RNA-seq data. We conducted a joint analysis of differential expression and differential splicing on annotated human genes and isoforms, and identified 122 non-differentially expressed genes with a high degree of “switch” between major and minor isoforms. Three cases with variant mechanisms for alternative splicing were validated using qRT-PCR, among the group of genes in which expression was not significantly changed at the gene level. We also identified 35 novel transcripts that were unique to the fibrotic lungs using exon-exon junction evidence, and selected a representative for qRT-PCR validation. The results of our study are likely to provide new insight into the pathogenesis of pulmonary fibrosis and may eventuate in new treatment targets.

Deng, Nan; Sanchez, Cecilia G.; Lasky, Joseph A.; Zhu, Dongxiao

2013-01-01

333

SpliceCenter: A suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi, microarray, and peptide-based studies  

PubMed Central

Background Over 60% of protein-coding genes in vertebrates express mRNAs that undergo alternative splicing. The resulting collection of transcript isoforms poses significant challenges for contemporary biological assays. For example, RT-PCR validation of gene expression microarray results may be unsuccessful if the two technologies target different splice variants. Effective use of sequence-based technologies requires knowledge of the specific splice variant(s) that are targeted. In addition, the critical roles of alternative splice forms in biological function and in disease suggest that assay results may be more informative if analyzed in the context of the targeted splice variant. Results A number of contemporary technologies are used for analyzing transcripts or proteins. To enable investigation of the impact of splice variation on the interpretation of data derived from those technologies, we have developed SpliceCenter. SpliceCenter is a suite of user-friendly, web-based applications that includes programs for analysis of RT-PCR primer/probe sets, effectors of RNAi, microarrays, and protein-targeting technologies. Both interactive and high-throughput implementations of the tools are provided. The interactive versions of SpliceCenter tools provide visualizations of a gene's alternative transcripts and probe target positions, enabling the user to identify which splice variants are or are not targeted. The high-throughput batch versions accept user query files and provide results in tabular form. When, for example, we used SpliceCenter's batch siRNA-Check to process the Cancer Genome Anatomy Project's large-scale shRNA library, we found that only 59% of the 50,766 shRNAs in the library target all known splice variants of the target gene, 32% target some but not all, and 9% do not target any currently annotated transcript. Conclusion SpliceCenter provides unique, user-friendly applications for assessing the impact of transcript variation on the design and interpretation of RT-PCR, RNAi, gene expression microarrays, antibody-based detection, and mass spectrometry proteomics. The tools are intended for use by bench biologists as well as bioinformaticists.

Ryan, Michael C; Zeeberg, Barry R; Caplen, Natasha J; Cleland, James A; Kahn, Ari B; Liu, Hongfang; Weinstein, John N

2008-01-01

334

Mouse Models of Mutations and Variations in Autism Spectrum Disorder-Associated Genes: Mice Expressing Caps2/Cadps2 Copy Number and Alternative Splicing Variants  

PubMed Central

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by disturbances in interpersonal relationships and behavior. Although the prevalence of autism is high, effective treatments have not yet been identified. Recently, genome-wide association studies have identified many mutations or variations associated with ASD risk on many chromosome loci and genes. Identification of the biological roles of these mutations or variations is necessary to identify the mechanisms underlying ASD pathogenesis and to develop clinical treatments. At present, mice harboring genetic modifications of ASD-associated gene candidates are the best animal models to analyze hereditary factors involved in autism. In this report, the biological significance of ASD-associated genes is discussed by examining the phenotypes of mouse models with ASD-associated mutations or variations in mouse homologs, with a focus on mice harboring genetic modifications of the Caps2/Cadps2 (Ca2+-dependent activator protein for secretion 2) gene.

Sadakata, Tetsushi; Shinoda, Yo; Sato, Akira; Iguchi, Hirotoshi; Ishii, Chiaki; Matsuo, Makoto; Yamaga, Ryosuke; Furuichi, Teiichi

2013-01-01

335

Intron-independent Association of Splicing Factors with Active Genes  

Microsoft Academic Search

The cell nucleus is organized as discrete do- mains, often associated with specific events involved in chromosome organization, replication, and gene ex- pression. We have examined the spatial and functional relationship between the sites of heat shock gene tran- scription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluo- rescence and fluorescence in situ hybridization

Caroline Jolly; Claire Vourc; Michel Robert-Nicoud; Richard I. Morimoto

1999-01-01

336

Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTP?, ?, ? and PCP-2)  

PubMed Central

Background Four genes designated as PTPRK (PTP?), PTPRL/U (PCP-2), PTPRM (PTP?) and PTPRT (PTP?) code for a subfamily (type R2B) of receptor protein tyrosine phosphatases (RPTPs) uniquely characterized by the presence of an N-terminal MAM domain. These transmembrane molecules have been implicated in homophilic cell adhesion. In the human, the PTPRK gene is located on chromosome 6, PTPRL/U on 1, PTPRM on 18 and PTPRT on 20. In the mouse, the four genes ptprk, ptprl, ptprm and ptprt are located in syntenic regions of chromosomes 10, 4, 17 and 2, respectively. Results The genomic organization of murine R2B RPTP genes is described. The four genes varied greatly in size ranging from ~64 kb to ~1 Mb, primarily due to proportional differences in intron lengths. Although there were also minor variations in exon length, the number of exons and the phases of exon/intron junctions were highly conserved. In situ hybridization with digoxigenin-labeled cRNA probes was used to localize each of the four R2B transcripts to specific cell types within the murine central nervous system. Phylogenetic analysis of complete sequences indicated that PTP? and PTP? were most closely related, followed by PTP?. The most distant family member was PCP-2. Alignment of RPTP polypeptide sequences predicted putative alternatively spliced exons. PCR experiments revealed that five of these exons were alternatively spliced, and that each of the four phosphatases incorporated them differently. The greatest variability in genomic organization and the majority of alternatively spliced exons were observed in the juxtamembrane domain, a region critical for the regulation of signal transduction. Conclusions Comparison of the four R2B RPTP genes revealed virtually identical principles of genomic organization, despite great disparities in gene size due to variations in intron length. Although subtle differences in exon length were also observed, it is likely that functional differences among these genes arise from the specific combinations of exons generated by alternative splicing.

Besco, Julie; Popesco, Magdalena C; Davuluri, Ramana V; Frostholm, Adrienne; Rotter, Andrej

2004-01-01

337

A novel A + G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency  

Microsoft Academic Search

We have identified the underlying molecular de- fect in a patient with hepatic lipase (HL) deficiency present- ing with hypertriglyceridemia and premature cardiovascular disease. DNA sequencing of polymerase chain reaction (PCR) amplified DNA and digestion with BsrI established homozy- gosity for an A + G mutation in intron I of the patient's hepatic lipase gene. This mutation introduces an additional

Korbinian Brand; Klaus A. Dugi; John D. Brunzell; David N. Nevin; Silvia Santamarina-Fojo

338

Differential splicing of exon 5 of the Wilms tumour (WTI) gene.  

PubMed

The WTI gene encodes a developmentally regulated transcription factor whose function is altered by alternative splicing at two sites: the 17 amino acids of exon 5, whose functional effects are ill-defined, and the 3 amino acids (KTS) between exons 9 and 10, which determine sequence-specific DNA binding and nuclear localisation. Germline mutations, which prevent normal KTS splicing, can underlie the Denys-Drash syndrome, and disruptions of splicing of exon 5 may occur in Wilms tumours. We analysed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification the relative ratios of the four splice variants of WTI mRNA in normal and tumour tissues and found tissue-specific, developmental stage-specific, and species-specific differences in the splicing of exon 5 but not of KTS. We found no evidence for disrupted splicing in acute leukaemias or gonadal tumours. The significance of these findings is discussed, and the possibility is raised that WTI may orchestrate the appropriate response to growth and differentiation factor signalling, mediated by alterations in the relative levels of exon 5 containing WTI isoforms. PMID:9258661

Renshaw, J; King-Underwood, L; Pritchard-Jones, K

1997-08-01

339

NCAM1 association study of bipolar disorder and schizophrenia: polymorphisms and alternatively spliced isoforms lead to similarities and differences  

PubMed Central

Objective The neural cell adhesion molecule (NCAM1) is a multifunction transmembrane protein involved in synaptic plasticity, neurodevelopment, and neurogenesis. Multiple NCAM1 proteins were differentially altered in bipolar disorder and schizophrenia. Single nucleotide polymorphisms (SNPs) in the NCAM1 gene were significantly associated with bipolar disorder in the Japanese population. Bipolar disorder and schizophrenia may share common vulnerability or susceptibility risk factors for shared features in each disorder. Methods Both SNPs and splice variants in the NCAM1 gene were analysed in bipolar disorder and schizophrenia. A case-control study design for association of SNPs and differential exon expression in the NCAM1 gene was used. Results A genotypic association between bipolar disorder and SNP b (rs2303377 near mini-exon b) and a suggestive association between schizophrenia and SNP 9 (rs646558) were found. Three of the two marker haplotypes for SNP 9 and SNP b showed varying frequencies between bipolar and controls (P < 0.0001) as well as between schizophrenia and controls (P < 0.0001). There were nine NCAM1 transcripts present in postmortem brain samples that involve alternative splicing of NCAM1 mini-exons (a, b, c) and the secreted (SEC) exon. Significant differences in the amounts of four alternatively spliced isoforms were found between NCAM1 SNP genotypes. In exploratory analysis, the c—SEC alternative spliced isoform was significantly decreased in bipolar disorder compared to controls for NCAM1 SNP b heterozygotes (P = 0.013). Conclusions Diverse NCAM1 transcripts were found with possibly different functions. The results suggest that SNPs within NCAM1 contribute differential risk for both bipolar disorder and schizophrenia possibly by alternative splicing of the gene. Psychiatr Genet 17:55?67 © 2007 Lippincott Williams & Wilkins.

Atz, Mary E.; Rollins, Brandi; Vawter, Marquis P.

2007-01-01

340

Structural organization of mouse peroxisome proliferator-activated receptor {gamma} (mPPAR{gamma}) gene: Alternative promoter use and different splicing yield two mPPAR{gamma} isoforms  

SciTech Connect

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR {gamma} (mPPAR{gamma}) gene. This gene extends >105 kb and gives rise to two mRNAs (mPPAR{gamma}1 and mPPAR{gamma}2) that differ at their 5{prime} ends. The mPPAR{gamma}2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPAR{gamma}1 and reveals a different 5{prime} untranslated sequence. We show that mPPAR{gamma}1 mRNA is encoded by eight exons, whereas the mPPAR{gamma}2 mRNA is encoded by seven exons. Most of the 5{prime} untranslated sequence of mPPAR{gamma}1 mRNA is encoded by two exons, whereas the 5{prime} untranslated sequence of mPPAR{gamma}1 mRNA is encoded by two exons, whereas the 5{prime} untranslated sequence and the extra 30 N-terminal amino acids of mPPAR{gamma}2 are encoded by one exon, which is located between the second and third exons coding for mPPAR{gamma}1. The last six exons of mPPAR{gamma} gene code for identical sequences in mPPAR{gamma}1 and mPPAR{gamma}2 isoforms. The mPPAR{gamma}1 and mPPAR{gamma}2 isoforms are transcribed from different promoters. The mPPAR{gamma} gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase in the diversity of ligand and tissue-specific transcriptional responses. 22 refs., 5 figs.

Zhu, Y.; Qi, C.; Rao, M.S. [Northwestern Univ. Medical School, Chicago, IL (United States)] [and others

1995-08-15

341

Isolation, Characterization, and Mapping of a Zinc Finger Gene, ZFP95, Containing Both a SCAN Box and an Alternatively Spliced KRAB A Domain  

Microsoft Academic Search

A new zinc finger gene of the Krüppel family was identified by screening a human fetal cartilage cDNA library with degenerate oligonucleotides. Sequence analysis indicates that ZFP95 contains 12 highly conserved zinc finger motifs at the C-terminus and a SCAN box as well as a KRAB A domain at the N-terminus of the protein. ZFP95 represents a member of a

Sandra D. Dreyer; Qiping Zheng; Bernhard Zabel; Andreas Winterpacht; Brendan Lee

1999-01-01

342

A new AOX homologous gene OsIM1 from rice ( Oryza sativa L.) with an alternative splicing mechanism under salt stress  

Microsoft Academic Search

A differentially expressed OsIM1 gene was isolated from rice salt-tolerant mutant M-20 by differential display. Sequence analysis revealed that the amino-acid sequence of OsIM1 showed 66% and 62% identity with PTOX from tomato ( Capsicum annuum) and AtIM from Arabidopsis, both of which encoded chloroplast-orientated terminal oxidase. Comparison of the nucleotide sequence of the OsIM1 cDNA with its genomic sequence

Jin Kong; Ji-Ming Gong; Zhi-Gang Zhang; Jin-Song Zhang; Shou-Yi Chen

2003-01-01

343

Novel mutations in the GH gene (GH1) uncover putative splicing regulatory elements.  

PubMed

Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing. PMID:24635352

Babu, Deepak; Mellone, Simona; Fusco, Ileana; Petri, Antonella; Walker, Gillian E; Bellone, Simonetta; Prodam, Flavia; Momigliano-Richiardi, Patricia; Bona, Gianni; Giordano, Mara

2014-05-01