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1

Alternative splicing of DNA damage response genes and gastrointestinal cancers  

PubMed Central

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

Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

2014-01-01

2

The evolutionary relationship between gene duplication and alternative splicing.  

PubMed

Gene duplication and alternative splicing (AS) are the two major evolutionary mechanisms that can bring the functional variation by increasing gene diversification. The purpose of this research is to understand the evolutionary relationship between these two different mechanisms, utilizing available data resources. We found the proportion of AS loci and the average number of AS isoforms per locus to be larger in duplicated genes compared to those in singleton genes. However we also found that small gene families have larger proportion of AS loci and larger average number of AS isoforms per locus than large gene families. These results suggest that gene duplication allows for more alternative splicing events to occur on newly duplicated copies than on singletons, probably due to the reduced functional constraint on the duplicates. Smaller average number of AS isoforms in the larger gene families can be explained by the decreased possibility for new useful function to be created via a new alternative splicing event. PMID:18835337

Jin, Lihua; Kryukov, Kirill; Clemente, Jose C; Komiyama, Tomoyoshi; Suzuki, Yoshiyuki; Imanishi, Tadashi; Ikeo, Kazuho; Gojobori, Takashi

2008-12-31

3

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

Microsoft Academic Search

Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alterna- tive splicing of

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

2002-01-01

4

Conserved Alternative Splicing Patterns and Splicing Signals in the Drosophila Sodium Channel Gene Para  

PubMed Central

We cloned genomic DNA corresponding to the Drosophila virilis homologue of para, a gene encoding a sodium channel ?-subunit, and obtained many partial cDNA clones from embryos and adults. Para protein has been well conserved, and the optional elements at six different sites of alternative splicing in D. melanogaster are present in D. virilis, in addition to one new optional exon. Among 31 different splice-types observed in D. virilis, the stage-specific pattern of alternative splicing seen in D. melanogaster is also conserved. Comparison of genomic DNA sequence revealed three aspects that vary between alternatively and constitutively used exon sequences. Sixteen short blocks (10-75 bp), the only recognizably conserved intron sequence, were disproportionately associated with alternatively used splice sites. Silent site substitutions were found much less frequently in alternative than constitutive exon elements, and the degree of match to the Drosophila splice site consensus tended to be lower at less frequently selected alternative splice junctions. This study shows that the developmentally regulated variability of para products is highly conserved and therefore likely to be of functional significance and suggests that a variety of different sequence-dependent mechanisms may regulate this pattern of alternative splicing. PMID:8536968

Thackeray, J. R.; Ganetzky, B.

1995-01-01

5

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

E-print Network

RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity ...

Horvitz, H Robert

6

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

Microsoft Academic Search

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

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

2012-01-01

7

Complex Alternative Splicing  

PubMed Central

Alternative splicing is a powerful means of controlling gene expression and increasing protein diversity. Most genes express a limited number of mRNA isoforms, but there are several examples of genes that use alternative splicing to generate hundreds, thousands, and even tens of thousands of isoforms. Collectively such genes are considered to undergo complex alternative splicing. The best example is the Drosophila Down syndrome cell adhesion molecule (Dscam) gene, which can generate 38,016 isoforms by the alternative splicing of 95 variable exons. In this review, we will describe several genes that use complex alternative splicing to generate large repertoires of mRNAs and what is known about the mechanisms by which they do so. PMID:18380340

Park, Jung Woo; Graveley, Brenton R.

2015-01-01

8

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

SciTech Connect

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

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

2008-11-07

9

Identication of a novel alternative splicing isoform of human amyloid precursor protein gene, APP639  

E-print Network

Identi®cation of a novel alternative splicing isoform of human amyloid precursor protein gene, APP, Meibergdreef 33, 1105 AZ Amsterdam ZO, the Netherlands Keywords: alternative splicing, Alzheimer's disease, APP derived from a large amyloid precursor protein (APP). To date, several alternatively spliced human APP

Tian, Weidong

10

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

PubMed Central

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

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

2011-01-01

11

The Caenorhabditis elegans gene mfap-1 encodes a nuclear protein that affects alternative splicing.  

PubMed

RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre-mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing. PMID:22829783

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

2012-01-01

12

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. PMID:24358217

Sallée, Marion; Fontès, Michel; Louis, Laurence; Cérini, Claire; Brunet, Philippe; Burtey, Stéphane

2013-01-01

13

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

SciTech Connect

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

Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A. [Universite de Paris (France)] [and others

1995-09-20

14

Gene Selection, Alternative Splicing, and Post-translational Processing Regulate Neuroligin Selectivity for -Neurexins  

E-print Network

Gene Selection, Alternative Splicing, and Post-translational Processing Regulate Neuroligin1/NX1 binding. Our data indicate that gene selection, mRNA splicing, and post. In the mammalian central nervous system, the development and maintenance of a functional neuronal network is based

Sandini, Giulio

15

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

E-print Network

factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). WeRNP complex and the SF1/U2AF65/U2AF35 protein complex recognize the 59 and 39 splice sites of an intronThe Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing

Horvitz, H. Robert

16

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

PubMed Central

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

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

2013-01-01

17

Regulation of Translation Factor EEF1D Gene Function by Alternative Splicing  

PubMed Central

Alternative splicing is an exquisite mechanism that allows one coding gene to have multiple functions. The alternative splicing machinery is necessary for proper development, differentiation and stress responses in a variety of organisms, and disruption of this machinery is often implicated in human diseases. Previously, we discovered a long form of eukaryotic elongation factor 1B? (eEF1B?; this long-form eEF1B? results from alternative splicing of EEF1D transcripts and regulates the cellular stress response by transcriptional activation, not translational enhancement, of heat-shock responsive genes. In this review, we discuss the molecular function of EEF1D alternative splicing products and the estimated implication of human diseases. PMID:25686034

Kaitsuka, Taku; Matsushita, Masayuki

2015-01-01

18

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

SciTech Connect

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

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)] [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)] [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

2011-10-14

19

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing  

E-print Network

experimental test of the effect of methylation on alternative slicing at the whole genome level has never beenRNA interference knockdown of DNA methyl- transferase 3 affects gene alternative splicing of Medicine, Houston, TX 77030; f Divisions of Animal and Plant Sciences, University of Missouri, Columbia, MO

Jacobsen, Steve

20

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. PMID:15805497

Kim, Namshin; Shin, Seokmin; Lee, Sanghyuk

2005-01-01

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

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

PubMed Central

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 an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome. PMID:22299024

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

2012-01-01

23

[Progress in the study on alternative splicing and functions of kininogen genes].  

PubMed

The kininogen gene and its coded proteins are slightly different in various species. In human beings, bovine and mouse, for example, there is an alternatively spliced kininogen gene K, encoding two kinds of proteins. By contrast, there is still another constituted spliced kininogen gene T which encodes only one kind of protein in the rat. The kininogen, belonging to the family 3 of cystatin superfamily, is a kind of multifunctional proteins with multiple domains, which maintains the normal physiological condition in human and some other organisms. The antagonism of hemoglutination and antihemoglutination of kininogen can not only recuperate the damaged blood vessels to prevent them from bleeding ceaselessly, but also restrain the formation of thrombus. In this review, we briefly discussed alternative splicing of kininogen gene and the multifunction of kininogen protein, as well as primarily hemoglutination and antihemoglutination, in human beings, bovine, mouse and rat which have been currently studied in detail. Our aims are to provide the beneficial references for further understanding the mechanism of evolution and alternative splicing of kininogen gene, and elucidating the multifunction roles of kininogen protein. Besides, it would be helpful for developing new medicines to regulate the vascular permeability and blood pressure and to restrain tumor. PMID:17138555

Zhou, Li-Wei; Ma, Fei; Li, Qing-Wei

2006-12-01

24

Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis  

PubMed Central

Isochorismate synthase (ICS) converts chorismate to isochorismate for the biosynthesis of phylloquinone, an essential cofactor for photosynthetic electron transport. ICS is also required for salicylic acid (SA) synthesis during Arabidopsis defense. In several other species, including Populus, SA is derived primarily from the phenylpropanoid pathway. We therefore sought to investigate ICS regulation in Populus to learn the extent of ICS involvement in SA synthesis and defense. Arabidopsis harbors duplicated AtICS genes that differ in their exon-intron structure, basal expression, and stress inducibility. In contrast, we found a single ICS gene in Populus and six other sequenced plant genomes, pointing to the AtICS duplication as a lineage-specific event. The Populus ICS encodes a functional plastidic enzyme, and was not responsive to stresses that stimulated phenylpropanoid accumulation. Populus ICS underwent extensive alternative splicing that was rare for the duplicated AtICSs. Sequencing of 184 RT-PCR Populus clones revealed 37 alternative splice variants, with normal transcripts representing ?50% of the population. When expressed in Arabidopsis, Populus ICS again underwent alternative splicing, but did not produce normal transcripts to complement AtICS1 function. The splice-site sequences of Populus ICS are unusual, suggesting a causal link between junction sequence, alternative splicing, and ICS function. We propose that gene duplication and alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. AtICS1 represents a divergent isoform for inducible SA synthesis during defense. Populus ICS primarily functions in phylloquinone biosynthesis, a process that can be sustained at low ICS transcript levels. PMID:19996170

Yuan, Yinan; Chung, Jeng-Der; Fu, Xueyan; Johnson, Virgil E.; Ranjan, Priya; Booth, Sarah L.; Harding, Scott A.; Tsai, Chung-Jui

2009-01-01

25

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

PubMed Central

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

2014-01-01

26

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

PubMed

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

Skandalis, Adonis; Uribe, Elke

2004-01-01

27

Diabetes-Induced Changes in the Alternative Splicing of the Slo Gene in Corporal Tissue  

PubMed Central

Objectives Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel ?-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Methods Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. Results A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Conclusions Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity. PMID:17150299

Davies, Kelvin P.; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C.; Desai, Pratik; Verselis, Vytas K.; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J.

2007-01-01

28

Leydig cells express the myelin proteolipid protein gene and incorporate a new alternatively spliced exon.  

PubMed

Although the myelin proteolipid protein gene (Plp1) is highly expressed in the central nervous system encoding the most abundant myelin protein in oligodendrocytes, it is also expressed in other tissues, including testis. Transgenic studies with mice that harbor Plp1-lacZ fusion genes suggest that Leydig cells are the source of Plp1 gene expression in testis. However, virtually nothing is known about Plp1 gene regulation in Leydig cells, which is the focus of this study. The first intron contains both positive and negative regulatory elements that are important in regulating Plp1 gene expression in oligodendrocytes. To test whether these elements are functional in Leydig cells, a battery of Plp1-lacZ fusion genes with partial deletion of Plp1 intron 1 sequence was transfected into the mouse Leydig cell line, TM3. Results presented here suggest that an enhancer, which is very potent in oligodendrocytes, is only nominally active in TM3 cells. The intron also contains several negative regulatory elements that are operative in TM3 cells. Moreover a new exon (exon 1.2) was identified within the first 'intron' resulting in novel splice variants in TM3 cells. Western blot analysis suggests that these splice variants, along with those containing another alternatively spliced exon (exon 1.1) derived from intron 1 sequence, give rise to multiple Plp1 gene products in the mouse testis. PMID:19232385

Li, Shenyang; Greuel, Brian T; Meng, Fanxue; Pereira, Glauber B; Pitts, Adria; Dobretsova, Anna; Wight, Patricia A

2009-05-01

29

Diverse modes of alternative splicing of human splicing factor SF1 deduced from the exon–intron structure of the gene  

Microsoft Academic Search

Several cDNAs encoding the essential human splicing facor (SF) 1 have been cloned. Comparison of the cDNA sequences suggested that the corresponding mRNAs are generated by alternative splicing from a common pre-mRNA. To confirm this assumption and to analyze possible modes used in the generation of these mRNAs, we have determined the structure of the gene encoding SF1. The gene

Angela Krämer; Mireille Quentin; Frank Mulhauser

1998-01-01

30

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

PubMed Central

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

2013-01-01

31

ZRANB2 localizes to supraspliceosomes and influences the alternative splicing of multiple genes in the transcriptome.  

PubMed

Alternative splicing is a major source of protein diversity in humans. The human splicing factor zinc finger, Ran-binding domain containing protein 2 (ZRANB2) is a splicing protein whose specific endogenous targets are unknown. Its upregulation in grade III ovarian serous papillary carcinoma could suggest a role in some cancers. To determine whether ZRANB2 is part of the supraspliceosome, nuclear supernatants from human embryonic kidney 293 cells were prepared and then fractioned on a glycerol gradient, followed by Western blotting. The same was done after treatment with a tyrosine kinase to induce phosphorylation. This showed for the first time that ZRANB2 is part of the supraspliceosome, and that phosphorylation affects its subcellular location. Studies were then performed to understand the splicing targets of ZRANB2 at the whole-transcriptome level. HeLa cells were transfected with a vector containing ZRANB2 or with a vector-only control. RNA was extracted, converted to cDNA and hybridized to Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays. At the FDR ?1.3 significance level we found that ZRANB2 influenced the alternative splicing of primary transcripts of CENTB1, WDR78, C10orf18, CABP4, SMARCC2, SPATA13, OR4C6, ZNF263, CAPN10, SALL1, ST18 and ZP2. Several of these have been implicated in tumor development. In conclusion ZRANB2 is part of the supraspliceosome and causes differential splicing of numerous primary transcripts, some of which might have a role in cancer. PMID:23666063

Yang, Yee Hwa J; Markus, M Andrea; Mangs, A Helena; Raitskin, Oleg; Sperling, Ruth; Morris, Brian J

2013-09-01

32

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

PubMed

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

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

2012-08-01

33

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

PubMed Central

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

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

2014-01-01

34

Splice variants of the condensin II gene Ncaph2 include alternative reading frame translations of exon 1.  

PubMed

Condensins I and II are five-protein complexes that are important for the condensation of chromatin. They are essential for mitosis and important for regulating gene expression during interphase. Here, we investigated the transcription and translation of the mouse Ncaph2 gene, which encodes a subunit of condensin II. We identified three splice variants within the first exon, a NAGNAG splice variant at the beginning of exon 16 and alternative 3'-UTRs. In total, Ncaph2 is potentially capable of generating 12 unique mRNA transcripts and six unique proteins. We confirm that Ncaph2 can generate three different N-termini, all encoded by exon 1, one of which is translated from an alternative reading frame. This alternative reading frame splice variant appears to be a novel outcome of splicing. If this is applicable to other genes, it would account for a previously unappreciated level of eukaryotic protein diversity. PMID:22333158

Theodoratos, Angelo; Wilson, Laurence O W; Gosling, Katharine M; Fahrer, Aude M

2012-04-01

35

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.  

PubMed

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton. PMID:20212022

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

36

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates  

PubMed Central

Pre-mRNA 5? spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ?250,000 high-quality reads corresponding to 8790 genes, ?58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of “infrequently trans-splicedgenes, accounting for ?28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing ?80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of ±3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca2+ homeostasis, and actin cytoskeleton. PMID:20212022

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B.; Macmil, Simone L.; Roe, Bruce A.; Zeller, Robert W.; Satou, Yutaka; Hastings, Kenneth E.M.

2010-01-01

37

ASD: a bioinformatics resource on alternative splicing  

Microsoft Academic Search

Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on alternative splicing. We present the continuation and upgrade of the ASD (T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) NucleicAcidsRes.32,D64-D69)thatconsistsofcom- putationally and manually generated data. Its largest parts

Stefan Stamm; Jean-jack M. Riethoven; Vincent Le Texier; Chellappa Gopalakrishnan; Vasudev Kumanduri; Yesheng Tang; Nuno L. Barbosa-morais; Thangavel Alphonse Thanaraj

2006-01-01

38

Characterization of a gene encoding a single-subunit bacteriophage-type RNA polymerase from maize which is alternatively spliced.  

PubMed

Single-subunit RNA polymerases belonging to the T3/T7 bacteriophage family are thought to be common throughout eukaryotes. We report the isolation and characterization of a nucleus-encoded single-subunit RNA polymerase gene from maize. This gene is highly homologous to other single-subunit RNA polymerase genes from Arabidopsis, Chenopodium. yeast and Neurospora crassa involved in organellar transcription. Genomic Southern analysis reveals 10 to 15 hybridising fragments, suggesting that maize contains a small gene family. The isolated gene contains 19 exons and its genomic structure is highly conserved when compared to the three Arabidopsis homologues. Unlike the case in Arabidopsis, intron-12 of the maize bacteriophage-type RNA polymerase gene is alternatively spliced. Quantitative RT-PCR revealed that the resultant alternatively spliced transcript represents approximately 21 to 26% of the total polymerase mRNA in maize coleoptiles. The orthologous wheat bacteriophage-type RNA polymerase is also alternatively spliced and the intron exhibits 78% identity to maize intron-12. The conservation in alternative splicing between wheat and maize and its absence from Arabidopsis suggest a functional requirement for the alternatively spliced product. PMID:9829825

Young, D A; Allen, R L; Harvey, A J; Lonsdale, D M

1998-10-01

39

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

PubMed Central

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

Kim, Namshin; Shin, Seokmin; Lee, Sanghyuk

2004-01-01

40

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

PubMed Central

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

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

1995-01-01

41

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development  

PubMed Central

During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion functions, and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization, These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is down-regulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics. PMID:24752171

Giudice, Jimena; Xia, Zheng; Wang, Eric T.; Scavuzzo, Marissa A.; Ward, Amanda J.; Kalsotra, Auinash; Wang, Wei; Wehrens, Xander H.T.; Burge, Christopher B.; Li, Wei; Cooper, Thomas A.

2014-01-01

42

Complex pattern of alternative splicing in the normal uroporphyrinogen decarboxylase gene: implications for diagnosis of familial porphyria cutanea tarda.  

PubMed

We describe multiple alternative transcripts of uroporphyrinogen decarboxylase mRNA in normal individuals and patients with familial porphyria cutanea tarda. mRNA was reverse-transcribed, subjected to the polymerase chain reaction, and analyzed for nucleotide sequence. Seven different transcripts were characterized, and a cryptic splice acceptor site was identified in intron 1. In all mRNAs the exons abutted at previously defined exon boundaries. Characterization of the splice junctions in the genomic DNA showed that splice donor and acceptor sequences complied with the consensus sequences for these sites except for the splice acceptor sequences of exons 3 and 10. THese deviations were present in two normal individuals and one patient with familial porphyria cutanea tarda and were thus unable to explain the multiple aberrant uroporphyrinogen decarboxylase transcripts. We conclude that apparent deletions observed in transcripts derived from the uroporphyrinogen decarboxylase gene in patients with familial porphyria cutanea tarda should be interpreted with caution. PMID:7923766

McManus, J F; Begley, C G; Ratnaike, S

1994-10-01

43

Subgroup Specific Alternative Splicing in Medulloblastoma  

PubMed Central

Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

2014-01-01

44

Splicing and alternative splicing in rice and humans.  

PubMed

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

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-09-01

45

Splicing and alternative splicing in rice and humans  

PubMed Central

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

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-01-01

46

Homer1 Alternative Splicing Is Regulated by Gonadotropin-Releasing Hormone and Modulates Gonadotropin Gene Expression  

PubMed Central

Hypothalamic gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive physiology by regulating follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression in the pituitary. Analysis of gonadotrope deep-sequencing data identified a global regulation of pre-mRNA splicing by GnRH. Homer1, a gene encoding a postsynaptic density scaffolding protein, was selected for further study. Homer1 expresses a short splice form, Homer1a, and more-abundant long transcripts Homer1b/c. GnRH induced a modest increase in Homer1b/c expression and a dramatic increase in the Homer1a splice form. G protein knockdown studies suggested that the Homer1 induction, but not the regulated splicing, was G?q/11 dependent. Phosphorylation of the splicing regulator SRp20 was found to be induced by GnRH. SRp20 depletion attenuated the GnRH-induced increase in the Homer1a-to-Homer1b/c ratio and modulated the effects of GnRH on FSH? and LH? expression. Homer1 gene knockdown resulted in increased GnRH-induced FSH? and LH? transcript levels. Furthermore, splice-form-specific reduction of Homer1b/c increased both FSH? and LH? mRNA induction, whereas reduction of Homer1a had the opposite effect on FSH? induction. These results indicate that the regulation of Homer1 splicing by GnRH contributes to gonadotropin gene control. PMID:24591653

Wang, Qian; Chikina, Maria D.; Pincas, Hanna

2014-01-01

47

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. PMID:18315870

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

2008-01-01

48

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

PubMed Central

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.

2013-01-01

49

Alternative Splicing Programs in Prostate Cancer  

PubMed Central

Prostate cancer (PCa) remains one of the most frequent causes of death for cancer in the male population. Although the initial antiandrogenic therapies are efficacious, PCa often evolves into a hormone-resistant, incurable disease. The genetic and phenotypic heterogeneity of this type of cancer renders its diagnosis and cure particularly challenging. Mounting evidence indicates that alternative splicing, the process that allows production of multiple mRNA variants from each gene, contributes to the heterogeneity of the disease. Key genes for the biology of normal and neoplastic prostate cells, such as those encoding for the androgen receptor and cyclin D1, are alternatively spliced to yield protein isoforms with different or even opposing functions. This review illustrates some examples of genes whose alternative splicing regulation is relevant to PCa biology and discusses the possibility to exploit alternative splicing regulation as a novel tool for prognosis, diagnosis, and therapeutic approaches to PCa. PMID:23983695

Sette, Claudio

2013-01-01

50

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

PubMed

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

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

2015-02-01

51

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. 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

2009-01-01

52

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. PMID:22719917

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

2012-01-01

53

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. PMID:23766705

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

2013-01-01

54

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

55

Alternative Splicing in the Fly and the Worm: Splicing Databases for Drosophila melanogaster and Caenorhabditis elegans  

Microsoft Academic Search

Alternative splicing is a widespread cellular phenomenon, which regulates gene expression in eukaryotic genomes. Availability of accumulating transcript and genomic sequence data has lead to generation of a wide-range of alternative splicing databases. Generally the available databases focus on mammalian transcriptomes. Here, we present two new alternative splicing databases for the model organisms, Drosophila melanogaster and Caenorhabditis elengans. Databases presented

Bahar Taneri; Ben Snyder; Alexey Novoradovsky; Terry Gaasterland

2011-01-01

56

Evidence for alternative splicing mechanisms in meadow fescue (Festuca pratensis) and perennial ryegrass (Lolium perenne) Rubisco activase gene.  

PubMed

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression. PMID:25577732

Jurczyk, Barbara; Hura, Katarzyna; Trzemecka, Anna; Rapacz, Marcin

2015-03-15

57

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

PubMed

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

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

2009-09-01

58

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

PubMed Central

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

Lareau, Liana F.; Brenner, Steven E.

2015-01-01

59

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

Microsoft Academic Search

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

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

2003-01-01

60

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. PMID:24817852

Sheng, Juan-Juan; Jin, Jian-Ping

2014-01-01

61

Alternative splicing of the imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and DNA binding activities  

Microsoft Academic Search

ZAC encodes a zinc finger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZAC?2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal

Benoit Bilanges; Annie Varrault; Abhijit Mazumdar; Colette Pantaloni; Anke Hoffmann; Joël Bockaert; Dietmar Spengler; Laurent Journot

2001-01-01

62

Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes  

PubMed Central

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

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

2013-01-01

63

Gene structure, alternative splicing, and chromosomal localization of pro-apoptotic Bcl2 relative Bim  

Microsoft Academic Search

.   Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three\\u000a isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses\\u000a and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated\\u000a the organization

Philippe Bouillet; Li Chen Zhang; David C. S. Huang; Graham C. Webb; Cynthia D. K. Bottema; Paul Shore; Helen J. Eyre; Grant R. Sutherland; Jerry M. Adams

2001-01-01

64

Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11/hippostasin.  

PubMed

The human kallikrein (KLK) family consists of 15 genes located on human chromosome 19q13.4. KLK11/hippostasinis a member of the kallikrein family and is expressed in various tissues. Two types of KLK11 isoforms, isoform 1 and isoform 2, have been predicted from cDNA sequences. Isoform 1 has been isolated from human hippocampus, whereas isoform 2 has been isolated from prostate. However, the regulation and characteristics of these isoforms are unknown. We identified the first three exons (1a, 1b, and 1c) by determining their transcription initiation sites. Exon 1b contained the initiation codon of isoform 2, and noncoding exons 1a and 1c contributed to isoform 1 mRNA. The dual luciferase promoter assay revealed three promoter regions, corresponding to the first exon of each isoform. Reverse transcription and PCR showed that exon 1a was expressed in the hippocampus, thalamus, and non-central nervous system (CNS) tissues, whereas exon 1b was detected only in non-CNS tissues. Exon 1c was observed in both CNS and non-CNS tissues, except for salivary glands. In vitro mutagenesis revealed that the initiation codon for isoform 2 in exon 1b was functional. Isoform 2 had additional hydrophilic amino acids at the amino terminal and was secreted from the neuroblastoma cell line Neuro2a. Isoform 1 fused with green fluorescent protein (GFP) was distributed to cellular processes, whereas isoform 2-GFP was retained in the Golgi apparatus. We suggest that not only alternative splicing but also tissue-specific use of multiple promoters regulate the expression and intracellular trafficking of KLK11/hippostasin isoforms. PMID:16911518

Mitsui, Shinichi; Nakamura, Terukazu; Okui, Akira; Kominami, Katsuya; Uemura, Hidetoshi; Yamaguchi, Nozomi

2006-08-01

65

Mutational bias is the driving force for shaping the synonymous codon usage pattern of alternatively spliced genes in rice (Oryza sativa L.).  

PubMed

Alternative splicing plays important roles in diverse aspects of plant development, metabolism, and stress responses. However, the regulatory mechanisms of alternative splicing of genes still remain incompletely elucidated, especially in plants. In this study, the synonymous codon usage pattern of alternatively spliced (AS) genes in rice was firstly explored using the combination of correspondence analysis (CA), internal CA, correlation and ANOVA analyses. The results show that alternatively and non-alternatively spliced (non-AS) genes have similar tendency for overall codon usage, but exhibit significant difference in 58 out of 64 codons. AS and non-AS genes are both under strong purifying selection, but the former ones have significant lower mutation rate and are prone to be enriched towards the chromosomal ends. In the group of AS genes, the variability in synonymous codon usage between genes is mainly due to the variations in GC content, CDS length, as well as gene functions. Mutational bias that accounts for 25.85 % of the total codon usage variability plays a major role in shaping the codon usage pattern of AS genes. In contrast, no obvious evidence is found for the contributions of translational selection, AS types, the conservation of AS events, and numbers of AS variants to the codon usage divergence between AS genes. These findings may be useful for further understanding the mechanisms of origination, differentiation and regulation of alternatively spliced genes in plants. PMID:25407289

Liu, Qingpo; Hu, Haichao; Wang, Hong

2015-04-01

66

ECgene: genome annotation for alternative splicing  

PubMed Central

ECgene provides annotation for gene structure, function and expression, taking alternative splicing events into consideration. The gene-modeling algorithm combines the genome-based expressed sequence tag (EST) clustering and graph-theoretic transcript assembly procedures. The website provides several viewers and applications that have many unique features useful for the analysis of the transcript structure and gene expression. The summary viewer shows the gene summary and the essence of other annotation programs. The genome browser and the transcript viewer are available for comparing the gene structure of splice variants. Changes in the functional domains by alternative splicing can be seen at a glance in the transcript viewer. We also provide two unique ways of analyzing gene expression. The SAGE tags deduced from the assembled transcripts are used to delineate quantitative expression patterns from SAGE libraries available publically. Furthermore, the cDNA libraries of EST sequences in each cluster are used to infer qualitative expression patterns. It should be noted that the ECgene website provides annotation for the whole transcriptome, not just the alternatively spliced genes. Currently, ECgene supports the human, mouse and rat genomes. The ECgene suite of tools and programs is available at http://genome.ewha.ac.kr/ECgene/. PMID:15608289

Kim, Pora; Kim, Namshin; Lee, Younghee; Kim, Bumjin; Shin, Youngah; Lee, Sanghyuk

2005-01-01

67

Riboswitch Control of Gene Expression in Plants by Splicing and Alternative 3? End Processing of mRNAs[W][OA  

PubMed Central

The most widespread riboswitch class, found in organisms from all three domains of life, is responsive to the vitamin B1 derivative thiamin pyrophosphate (TPP). We have established that a TPP-sensing riboswitch is present in the 3? untranslated region (UTR) of the thiamin biosynthetic gene THIC of all plant species examined. The THIC TPP riboswitch controls the formation of transcripts with alternative 3? UTR lengths, which affect mRNA accumulation and protein production. We demonstrate that riboswitch-mediated regulation of alternative 3? end processing is critical for TPP-dependent feedback control of THIC expression. Our data reveal a mechanism whereby metabolite-dependent alteration of RNA folding controls splicing and alternative 3? end processing of mRNAs. These findings highlight the importance of metabolite sensing by riboswitches in plants and further reveal the significance of alternative 3? end processing as a mechanism of gene control in eukaryotes. PMID:17993623

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

2007-01-01

68

Vitamin D and alternative splicing of RNA.  

PubMed

The active form of vitamin D (1?,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. PMID:25447737

Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

2015-04-01

69

Using an exon microarray to identify a global profile of gene expression and alternative splicing in K562 cells exposed to sodium valproate.  

PubMed

To investigate the effect of valproate treatment on the K562 cell line, a model for chronic myelogenous leukaemia, the growth and survival of the K562 cell line were investigated using the Annexin-V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarrays. A significant increase in cell apoptosis was observed in valproate-exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of the genes observed to be differentially expressed that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed to be alternatively spliced by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line. PMID:22200904

Zhang, Xiang-Zhong; Yin, Ai-Hua; Zhu, Xiao-Yu; Ding, Qian; Wang, Chun-Huai; Chen, Yun-Xian

2012-04-01

70

SOX2 modulates alternative splicing in transitional cell carcinoma.  

PubMed

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor. PMID:20138825

Tung, Chun-Liang; Hou, Pei-Hsuan; Kao, Yung-Ling; Huang, Yu-Wen; Shen, Chiung-Chun; Cheng, Yi-Hsin; Wu, Shu-Fen; Lee, Moon-Sing; Li, Chin

2010-03-12

71

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

PubMed Central

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

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

2014-01-01

72

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

PubMed

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

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

2015-04-01

73

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

PubMed Central

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

2014-01-01

74

Tau exon 10 alternative splicing and tauopathies  

PubMed Central

Abnormalities of microtubule-associated protein tau play a central role in neurofibrillary degeneration in several neurodegenerative disorders that collectively called tauopathies. Six isoforms of tau are expressed in adult human brain, which result from alternative splicing of pre-mRNA generated from a single tau gene. Alternative splicing of tau exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively). Approximately equal levels of 3R-tau and 4R-tau are expressed in normal adult human brain, but the 3R-tau/4R-tau ratio is altered in the brains in several tauopathies. Discovery of silence mutations and intronic mutations of tau gene in some individuals with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), which only disrupt tau exon 10 splicing but do not alter tau's primary sequence, demonstrates that dysregulation of tau exon 10 alternative splicing and consequently of 3R-tau/4R-tau balance is sufficient to cause neurodegeneration and dementia. Here, we review the gene structure, transcripts and protein isoforms of tau, followed by the regulation of exon 10 splicing that determines the expression of 3R-tau or 4R-tau. Finally, dysregulation of exon 10 splicing of tau in several tauopathies is discussed. Understanding the molecular mechanisms by which tau exon 10 splicing is regulated and how it is disrupted in tauopathies will provide new insight into the mechanisms of these tauopathies and help identify new therapeutic targets to treat these disorders. PMID:18616804

Liu, Fei; Gong, Cheng-Xin

2008-01-01

75

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

PubMed Central

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (?18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 – now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation. PMID:18454200

Barberan-Soler, Sergio; Zahler, Alan M.

2008-01-01

76

The adaptive significance of unproductive alternative splicing in primates.  

PubMed

Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase ? as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase ? transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing. PMID:20719917

Skandalis, Adonis; Frampton, Mark; Seger, Jon; Richards, Miriam H

2010-10-01

77

Directing alternative splicing: cast and scenarios  

Microsoft Academic Search

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

Benoit Chabot

1996-01-01

78

Shotgun proteomics aids discovery of novel protein-coding genes, alternative splicing, and “resurrected” pseudogenes in the mouse genome  

PubMed Central

Recent advances in proteomic mass spectrometry (MS) offer the chance to marry high-throughput peptide sequencing to transcript models, allowing the validation, refinement, and identification of new protein-coding loci. We present a novel pipeline that integrates highly sensitive and statistically robust peptide spectrum matching with genome-wide protein-coding predictions to perform large-scale gene validation and discovery in the mouse genome for the first time. In searching an excess of 10 million spectra, we have been able to validate 32%, 17%, and 7% of all protein-coding genes, exons, and splice boundaries, respectively. Moreover, we present strong evidence for the identification of multiple alternatively spliced translations from 53 genes and have uncovered 10 entirely novel protein-coding genes, which are not covered in any mouse annotation data sources. One such novel protein-coding gene is a fusion protein that spans the Ins2 and Igf2 loci to produce a transcript encoding the insulin II and the insulin-like growth factor 2–derived peptides. We also report nine processed pseudogenes that have unique peptide hits, demonstrating, for the first time, that they are not just transcribed but are translated and are therefore resurrected into new coding loci. This work not only highlights an important utility for MS data in genome annotation but also provides unique insights into the gene structure and propagation in the mouse genome. All these data have been subsequently used to improve the publicly available mouse annotation available in both the Vega and Ensembl genome browsers (http://vega.sanger.ac.uk). PMID:21460061

Brosch, Markus; Saunders, Gary I.; Frankish, Adam; Collins, Mark O.; Yu, Lu; Wright, James; Verstraten, Ruth; Adams, David J.; Harrow, Jennifer; Choudhary, Jyoti S.; Hubbard, Tim

2011-01-01

79

Modulation of p53? and p53? expression by regulating the alternative splicing of TP53 gene modifies cellular response.  

PubMed

In addition to the tumor suppressor p53 protein, also termed p53?, the TP53 gene produces p53? and p53? through alternative splicing of exons 9? and 9? located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53? and p53? at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9?/9?. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and ? variant, supporting our experimental data. Using siRNA specifically targeting exons 9?/9?, we demonstrate that cell growth can be driven by modulating p53? and p53? expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53? and p53? promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53? enhanced p53? transcriptional activity on the p21 and Bax promoters, while p53? increased p53? transcriptional activity on the Bax promoter only. Moreover, p53? and p53? co-immunoprecipitate with p53? only in the presence of p53-responsive promoter. Interestingly, although p53? and p53? promote apoptosis in MCF7 cells, p53? and p53? maintain cell growth in response to TG003 in a p53?-dependent manner. The dual activities of p53? and p53? isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53? and p53? regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. PMID:24926616

Marcel, V; Fernandes, K; Terrier, O; Lane, D P; Bourdon, J-C

2014-09-01

80

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

PubMed Central

Background The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. Methodology/Principal findings We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5? UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. Conclusions/Significance The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat. PMID:21603624

Rodríguez-Suárez, Cristina; Atienza, Sergio G.; Pistón, Fernando

2011-01-01

81

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

82

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

PubMed Central

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 (p38MAPK)-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/p38MAPK 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/p38MAPK 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; Kuwano, Yuki; Nishida, Kensei; Kajita, Keisuke; Kurokawa, Ken; Satake, Yuzuru; Shoda, Katsutoshi; Imoto, Issei; Rokutan, Kazuhito

2014-01-01

83

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)

2001-08-07

84

Heritability of alternative splicing in the human genome  

PubMed Central

Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5? splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled. PMID:17671095

Kwan, Tony; Benovoy, David; Dias, Christel; Gurd, Scott; Serre, David; Zuzan, Harry; Clark, Tyson A.; Schweitzer, Anthony; Staples, Michelle K.; Wang, Hui; Blume, John E.; Hudson, Thomas J.; Sladek, Rob; Majewski, Jacek

2007-01-01

85

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

PubMed Central

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

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

2011-01-01

86

Ca 2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human ? 1C subunit gene  

Microsoft Academic Search

L-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of

R. D Zühlke; A Bouron; N. M Soldatov; H Reuter

1998-01-01

87

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii  

PubMed Central

Background Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes. PMID:20163725

2010-01-01

88

Should pharmacologists care about alternative splicing? IUPHAR Review 4  

PubMed Central

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

Bonner, T I

2014-01-01

89

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

PubMed

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

Yap, Karen; Makeyev, Eugene V

2013-09-01

90

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

PubMed Central

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

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

2014-01-01

91

Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis.  

PubMed

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes. PMID:25372122

Burkhardt, Alyssa; Buchanan, Alex; Cumbie, Jason S; Savory, Elizabeth A; Chang, Jeff H; Day, Brad

2015-03-01

92

Functional interactions between alternatively spliced forms of Pax6 in crystallin gene regulation and in haploinsufficiency  

Microsoft Academic Search

Pax6 is essential for development of the eye, olfac- tory system, brain and pancreas. Haploinsufficiency of Pax6 causes abnormal eye development. Two forms of Pax6 protein, PAX6 and PAX6(5a), differ in a 14 amino acid insertion encoded by an alterna- tively spliced exon 5a in the N-terminal DNA-binding paired domain (PD), and they are simultaneously expressed. Here, we show that

Bharesh K. Chauhan; Ying Yang; Kveta Cveklovaand AlesCvekl

2004-01-01

93

Alternative splicing generates secretory isoforms of human CD1.  

PubMed Central

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

Woolfson, A; Milstein, C

1994-01-01

94

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

95

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

PubMed Central

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

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

2015-01-01

96

Identification and expression pattern of two novel alternative splicing variants of EEF1D gene of dairy cattle.  

PubMed

Our recent genome-wide association study (GWAS) has identified 105 genome-wide significant SNPs for milk production traits. Of these, one SNP (rs109661298) for milk fat percentage is located within the first intron of the bovine EEF1D gene on BTA14, thus that the EEF1D gene was considered as a novel candidate in dairy cattle. Until now, however, its genomic organization remains undetermined yet and no studies of EEF1D in relation to milk production traits have been reported. To layout the groundwork for the validation of gene function in dairy cattle, we herein investigated its expression pattern in lactating dairy cows. With rapid amplification of 5' cDNA end (5' RACE), two novel alternatively spliced transcript variants of 1202bp and 2195bp were isolated in bovine mammary in lactation, named EEF1Da and EEF1Db (GenBank: KC190039 and KC190038KC190039KC190038). Such two variants contain the different first exon from each other (exon1a vs exon1b: 294bp vs 1287bp) with no overlap and the same remaining 7 exons. Coding sequence similarity between EEF1Da and EEF1Db and three of human EEF1D transcript variants were 85-88%. With semi-quantitative and quantitative real-time RT-PCR, we found that the mRNA level of EEF1Da was similar to the overall EEF1D mRNA and much higher than EEF1Db in the mammary of lactating cows, indicating EEF1Da functions as the dominant transcript variant to encode the EEF1D protein. Tissue expression pattern showed that the mRNA expression of EEF1D and EEF1Da in mammary gland was significantly higher compared with other 7 tissues (P<0.05, P<0.01) with the exception of EEF1D between mammary and lung. Together, our findings present the first report on the alternative splicing of the bovine EEF1D gene and provided basis for further investigation on function validation of EEF1D in dairy cattle. PMID:24239553

Xie, Yan; Yang, Shaohua; Cui, Xiaogang; Jiang, Li; Zhang, Shengli; Zhang, Qin; Zhang, Yuan; Sun, Dongxiao

2014-01-25

97

Analysis of smu-1, a Gene That Regulates the Alternative Splicing of unc-52 Pre-mRNA in Caenorhabditis elegans  

PubMed Central

Mutations in the smu-1 gene of Caenorhabditis elegans were previously shown to suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. We show that a presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. We have used reverse transcription-PCR and RNase protection to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. We have identified smu-1 molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. We propose that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes. PMID:11438655

Spike, Caroline A.; Shaw, Jocelyn E.; Herman, Robert K.

2001-01-01

98

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

PubMed

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

Philippou, Anastassios; Armakolas, Athanasios; Koutsilieris, Michael

2013-01-01

99

The landscape of alternative splicing in cervical squamous cell carcinoma  

PubMed Central

Background Alternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. In this study, we identified alternative splicing events in four pairs of cervical squamous cell carcinoma (CSCC) and adjacent nontumor tissues using RNA sequencing. Methods The transcripts of the four paired samples were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted to detect the alternative spliced genes-related signal pathways. The alternative spliced genes were validated by reverse transcription-polymerase chain reaction (RT-PCR). Results There were 35 common alternative spliced genes in the four CSCC samples; they were novel and CSCC specific. Sixteen pathways were significantly enriched (P<0.05). One novel 5?AS site in the KLHDC7B gene, encoding kelch domain-containing 7B, and an exon-skipping site in the SYCP2 gene, encoding synaptonemal complex 2, were validated by RT-PCR. The KLHDC7B gene with 5?AS was found in 67.5% (27/40) of CSCC samples and was significantly related with cellular differentiation and tumor size. The exon-skipping site of the SYCP2 gene was found in 35.0% (14/40) of CSCC samples and was significantly related with depth of cervical invasion. Conclusion The KLHDC7B and the SYCP2 genes with alternative spliced events might be involved in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC. PMID:25565867

Guo, Peng; Wang, Dan; Wu, Jun; Yang, Junjun; Ren, Tong; Zhu, Baoli; Xiang, Yang

2015-01-01

100

Characterization of Tissue-Specific and Developmentally Regulated Alternative Splicing of Exon 64 in the COL5A1 Gene  

PubMed Central

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

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

2014-01-01

101

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

102

Statistical and Computational Studies on Alternative Splicing  

Microsoft Academic Search

\\u000a The accumulating genome sequences and other high-throughput data have shed light on the extent and importance of alternative\\u000a splicing in functional regulation. Alternative splicing dramatically increases the transcriptome and proteome diversity of\\u000a higher organisms by producing multiple splice variants from different combinations of exons. It has an important role in many\\u000a biological processes including nervous system development and programmed cell

Liang Chen

103

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

PubMed Central

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

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

2013-01-01

104

Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human alpha1C subunit gene.  

PubMed

L-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of Ca2+ channel splice variants derived from the human alpha1C gene to the DHP isradipine. Among seven alpha1C channels we observed up to 10-fold differences in IC50 values for isradipine, as well as changes in the voltage dependence of DHP action. PMID:9607315

Zühlke, R D; Bouron, A; Soldatov, N M; Reuter, H

1998-05-01

105

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

PubMed Central

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

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

2014-01-01

106

Alternative splicing networks regulated by signaling in human T cells  

PubMed Central

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

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

2012-01-01

107

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

USGS Publications Warehouse

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

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

2009-01-01

108

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. PMID:24155671

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

2013-01-01

109

Cross-kingdom patterns of alternative splicing and splice recognition  

PubMed Central

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

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

2008-01-01

110

Autoregulation of transformer-2 Alternative Splicing Is Necessary for Normal Male Fertility in Drosophila  

Microsoft Academic Search

In the male germline of Drosophila the transformer-2 protein is required for differential splicing of pre- mRNAs from the exuperantia and att genes and autoregulates alternative splicing of its own pre-mRNA. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing\\/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron

M. Elaine McGuffin; Dawn Chandler; Darshna Somaiya; Brigitte Dauwalder; William Mattox

111

Detection and measurement of alternative splicing using splicing-sensitive microarrays  

Microsoft Academic Search

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting

Karpagam Srinivasan; Lily Shiue; Justin D. Hayes; Ross Centers; Sean Fitzwater; Rebecca Loewen; Lillian R. Edmondson; Jessica Bryant; Michael Smith; Claire Rommelfanger; Valerie Welch; Tyson A. Clark; Charles W. Sugnet; Kenneth J. Howe; Yael Mandel-Gutfreund; Manuel Ares

2005-01-01

112

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

113

Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts  

PubMed Central

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo. PMID:1918153

1991-01-01

114

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

115

Studies of exon scrambling and mutually exclusive alternative splicing  

E-print Network

The goals of this thesis work were to study two special alternative splicing events: exon scrambling at the RNA splicing level and mutually exclusive alternative splicing (MEAS) by computational and experimental methods. ...

Kong, Rong, 1979-

2005-01-01

116

The distribution pattern of genetic variation in the transcript isoforms of the alternatively spliced protein-coding genes in the human genome.  

PubMed

By enabling the transcription of multiple isoforms from the same gene locus, alternative-splicing mechanisms greatly expand the diversity of the human transcriptome and proteome. Currently, the alternatively spliced transcripts from each protein-coding gene locus in the human genome can be classified as either principal or non-principal isoforms, providing that they differ with respect to cross-species conservation or biological features. By mapping the variants from the 1000 Genomes Project onto the coding region of each isoform, an interesting pattern of the genetic variation distributions of the coding regions for these two types of transcript isoforms was revealed on a whole-genome scale: compared with the principal isoform-specific coding regions, the non-principal isoform-specific coding regions are significantly enriched in amino acid-changing variants, particularly those that have a strong impact on protein function and have higher derived allele frequencies, suggesting that non-principal isoform-specific substitutions are less likely to be related to phenotype changes or disease. The results herein can help us better understand the potential consequences of alternatively spliced products from a population perspective. PMID:25820936

Liu, Ting; Lin, Kui

2015-04-21

117

Conditional Control of Alternative Splicing through Light-Triggered Splice-Switching Oligonucleotides.  

PubMed

The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF ? ON and ON ? OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals. PMID:25734836

Hemphill, James; Liu, Qingyang; Uprety, Rajendra; Samanta, Subhas; Tsang, Michael; Juliano, Rudolph L; Deiters, Alexander

2015-03-18

118

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. PMID:22876330

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

2012-01-01

119

Probabilistic Inference of Alternative Splicing Events in Microarray Data  

Microsoft Academic Search

Alternative splicing (AS) is an important and frequent step in mammalian gene expression that allows a single gene to specify multiple products, and is crucial for the regulation of fundamental biological processes. The extent of AS regulation, and the mechanisms involved, are not well un- derstood. We have developed a custom DNA microarray platform for surveying AS levels on a

Ofer Shai; Brendan J. Frey; Quaid Morris; Qun Pan; Christine Misquitta; Benjamin J. Blencowe

2004-01-01

120

Redefining the structure of the mouse connexin43 gene: selective promoter usage and alternative splicing mechanisms yield transcripts with different translational efficiencies.  

PubMed

The connexin43 (cx43) gene was originally described as consisting of two exons, one coding for most of the 5'-untranslated region (5'-UTR), and the other for the protein sequence and 3'-UTR. We now report that in mouse four additional exons are expressed, all coding for novel 5'-UTRs. Altogether, we found nine different cx43 mRNA species (GenBank accession numbers NM010288, and AY427554 through AY427561) generated by differential promoter usage and alternative splicing mechanisms. The relative abundance of these different mRNAs varied with the tissue source. In addition, the different transcripts showed varying translational efficiencies in several cell lines, indicating the presence of cis-RNA elements that regulate cx43 translation. We propose that it is the promoter driving the expression of the cx43 gene that determines exon choice in the downstream splicing events in a cell-type-dependent fashion. This in turn will affect the translation efficiency of the transcript orchestrating the events that lead to the final expression profile of cx43. Since a similar organization of the cx43 gene was also observed in rat it is likely that the complex regulation of cx43 expression involving transcription, splicing and translation mechanisms is a common trait conserved during evolution. PMID:15328367

Pfeifer, Ingrid; Anderson, Curtis; Werner, Rudolf; Oltra, Elisa

2004-01-01

121

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica  

PubMed Central

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3?-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment. PMID:22368181

Kabran, Philomène; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuvéglise, Cécile

2012-01-01

122

Correspondence Alternative Splicing at NAGNAG  

E-print Network

splicing at NAGNAGs mainly results in the insertion/deletion of one amino acid. While such subtle events]: biases towards intron phase 1 and single amino acid insertions/deletions, correlation of amino acid. Meanwhile, Akerman and Mandel-Gutfreund found a high conservation of the intronic flanking regions [5

Will, Sebastian

123

AsMamDB: an alternative splice database of mammals  

PubMed Central

The objective of database AsMamDB is to facilitate the systematic study of alternatively spliced genes of mammals. Version 1.0 of AsMamDB contains 1563 alternatively spliced genes of human, mouse and rat, each associated with a cluster of nucleotide sequences. The main information provided by AsMamDB includes gene alternative splicing patterns, gene structures, locations in chromosomes, products of genes and tissues where they express. Alternative splicing patterns are represented by multiple alignments of various gene transcripts and by graphs of their topological structures. Gene structures are illustrated by exon, intron and various regulatory elements distributions. There are 4204 DNAs, 3977 mRNAs, 8989 CDSs and 126 931 ESTs in the current database. More than 130 000 GenBank entries are covered and 4443 MEDLINE records are linked. DNA, mRNA, exon, intron and relevant regulatory element sequences are provided in FASTA format. More information can be obtained by using the web-based multiple alignment tool Asalign and various category lists. AsMamDB can be accessed at http://166.111.30.6 5/ASMAM DB.html. PMID:11125106

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

2001-01-01

124

Alternative Splicing of Type II Procollagen: IIB or not IIB?  

PubMed Central

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

2015-01-01

125

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

PubMed Central

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 and complexity of alternative splicing regulation by an intricate protein–RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative pre-mRNA splicing. PMID:19773805

Chen, Mo; Manley, James L.

2010-01-01

126

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. PMID:19178699

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

2009-01-01

127

Global regulation of alternative splicing during myogenic differentiation  

E-print Network

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely ...

Bland, Christopher S.

128

PELP1 oncogenic functions involve alternative splicing via PRMT6  

PubMed Central

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a proto-oncogene that functions as coactivator of the estrogen receptor and is an independent prognostic predictor of shorter survival of breast cancer patients. The dysregulation of PELP1 in breast cancer has been implicated in oncogenesis, metastasis, and therapy resistance. Although several aspects of PELP1 have been studied, a complete list of PELP1 target genes remains unknown, and the molecular mechanisms of PELP1 mediated oncogenesis remain elusive. In this study, we have performed a whole genome analysis to profile the PELP1 transcriptome by RNA-sequencing and identified 318 genes as PELP1 regulated genes. Pathway analysis revealed that PELP1 modulates several pathways including the molecular mechanisms of cancer, estrogen signaling, and breast cancer progression. Interestingly, RNA-seq analysis also revealed that PELP1 regulates the expression of several genes involved in alternative splicing. Accordingly, the PELP1 regulated genome includes several uniquely spliced isoforms. Mechanistic studies show that PELP1 binds RNA with a preference to poly-C, co-localizes with the splicing factor SC35 at nuclear speckles, and participates in alternative splicing. Further, PELP1 interacts with the arginine methyltransferase PRMT6 and modifies PRMT6 functions. Inhibition of PRMT6 reduced PELP1-mediated estrogen receptor activation, cellular proliferation, and colony formation. PELP1 and PRMT6 are co-recruited to estrogen receptor target genes, PELP1 knockdown affects the enrichment of histone H3R2 di-methylation, and PELP1 and PRMT6 coordinate to regulate the alternative splicing of genes involved in cancer. Collectively, our data suggest that PELP1 oncogenic functions involve alternative splicing leading to the activation of unique pathways that support tumor progression and that the PELP1-PRMT6 axis may be a potential target for breast cancer therapy. PMID:24447537

Mann, Monica; Zou, Yi; Chen, Yidong; Brann, Darrell; Vadlamudi, Ratna

2014-01-01

129

Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer  

PubMed Central

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

Sette, Claudio

2013-01-01

130

Cross-kingdom patterns of alternative splicing and splice recognition  

E-print Network

Background: Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently ...

McGuire, Abigail Manson

131

Gene recognition via spliced sequence alignment.  

PubMed

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%. PMID:8799154

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

1996-08-20

132

Argonaute proteins couple chromatin silencing to alternative splicing.  

PubMed

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1?, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification. PMID:22961379

Ameyar-Zazoua, Maya; Rachez, Christophe; Souidi, Mouloud; Robin, Philippe; Fritsch, Lauriane; Young, Robert; Morozova, Nadya; Fenouil, Romain; Descostes, Nicolas; Andrau, Jean-Christophe; Mathieu, Jacques; Hamiche, Ali; Ait-Si-Ali, Slimane; Muchardt, Christian; Batsché, Eric; Harel-Bellan, Annick

2012-10-01

133

Genomewide comparative analysis of alternative splicing in plants  

E-print Network

Genomewide comparative analysis of alternative splicing in plants Bing-Bing Wang* and Volker in mamma- lian 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 c

Brendel, Volker

134

The functional modulation of epigenetic regulators by alternative splicing  

Microsoft Academic Search

BACKGROUND: Epigenetic regulators (histone acetyltransferases, methyltransferases, chromatin-remodelling enzymes, etc) play a fundamental role in the control of gene expression by modifying the local state of chromatin. However, due to their recent discovery, little is yet known about their own regulation. This paper addresses this point, focusing on alternative splicing regulation, a mechanism already known to play an important role in

Sergio Lois; Noemí Blanco; Marian Martínez-Balbás; Xavier de la Cruz

2007-01-01

135

A chloroplast retrograde signal regulates nuclear alternative splicing  

PubMed Central

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.

2015-01-01

136

Deep surveying of alternative splicing complexity in the  

E-print Network

system of Illumina to survey splicing complexity in diverse, normal human tissues using mRNA- SeqDeep surveying of alternative splicing complexity in the human transcriptome by high the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice

137

Alternative splicing of the androgen receptor in polycystic ovary syndrome  

PubMed Central

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

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

2015-01-01

138

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

Microsoft Academic Search

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

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

2010-01-01

139

Analysis of two alleles of the urease gene from potato: polymorphisms, expression, and extensive alternative splicing of the corresponding mRNA.  

PubMed

Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Desiree) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25-50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism. PMID:15533883

Witte, Claus-Peter; Tiller, Sarah; Isidore, Edwige; Davies, Howard V; Taylor, Mark A

2005-01-01

140

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 Central

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

141

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

PubMed Central

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

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

2014-01-01

142

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. PMID:23865674

2013-01-01

143

The splice of life: Alternative splicing and neurological disease  

Microsoft Academic Search

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

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

2001-01-01

144

WebScipio: Reconstructing alternative splice variants of eukaryotic proteins.  

PubMed

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. PMID:23677611

Hatje, Klas; Hammesfahr, Björn; Kollmar, Martin

2013-07-01

145

Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.  

PubMed

To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies. PMID:20650682

Zhang, Huan; Dungan, Christopher F; Lin, Senjie

2011-01-01

146

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

SciTech Connect

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

Markus, M. Andrea [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Heinrich, Bettina [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Raitskin, Oleg [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Adams, David J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Mangs, Helena [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Goy, Christine [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Ladomery, Michael [Centre for Research in Biomedicine, Bristol Genomics Research Institute, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY (United Kingdom); Sperling, Ruth [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Stamm, Stefan [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Morris, Brian J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia)]. E-mail: brianm@medsci.usyd.edu.au

2006-10-15

147

Novel Development-Related Alternative Splices in Human Testis Identified by cDNA Microarrays  

Microsoft Academic Search

Alternative splicing of premessenger RNA is an im- portant regulatory mechanism that increases the diversity of proteins transcribed from a single gene. This is particularly important in the testis because germ cell expansion and differentiation require many cellular changes and regulatory steps. To investigate novel devel- opment-related alternative splicings in the human testis, comple- mentary DNA microarray studies were conducted

XIAOYAN HUANG; JIANMIN LI; LI LU; MIN XU; JUNHUA XIAO; LANLAN YIN; HU ZHU; ZUOMIN ZHOU; JIAHAO SHA

2005-01-01

148

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

EPA Science Inventory

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

149

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

SciTech Connect

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

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

1988-11-01

150

Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster  

PubMed Central

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster. PMID:21233220

Hartmann, Britta; Castelo, Robert; Miñana, Belén; Peden, Erin; Blanchette, Marco; Rio, Donald C.; Singh, Ravinder; Valcárcel, Juan

2011-01-01

151

Alternative Splicing of Exon 17 and a Missense Mutation in Exon 20 of the Insulin Receptor Gene in Two Brothers with a Novel Syndrome of Insulin Resistance (Congenital Fiber-Type Disproportion Myopathy)  

Microsoft Academic Search

The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to

Peter Vorwerk; Claus T. Christoffersen; Jørn Müller; Henrik Vestergaard; Oluf Pedersen; Pierre De Meyts

1999-01-01

152

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. PMID:24623796

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

153

Expansion of the eukaryotic proteome by alternative splicing  

PubMed Central

The collection of components required to carry out the intricate processes involved in generating and maintaining a living, breathing and, sometimes, thinking organism is staggeringly complex. Where do all of the parts come from? Early estimates stated that about 100,000 genes would be required to make up a mammal; however, the actual number is less than one-quarter of that, barely four times the number of genes in budding yeast. It is now clear that the ‘missing’ information is in large part provided by alternative splicing, the process by which multiple different functional messenger RNAs, and therefore proteins, can be synthesized from a single gene. PMID:20110989

Nilsen, Timothy W.; Graveley, Brenton R.

2012-01-01

154

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

PubMed Central

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

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

2013-01-01

155

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

PubMed Central

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

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

2014-01-01

156

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

PubMed

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of ?-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. PMID:25737553

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

2015-03-17

157

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

PubMed Central

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

Sheives, Paul; Lynch, Kristen W

2002-01-01

158

Alteration of conserved alternative splicing in AMELX causes enamel defects.  

PubMed

Tooth enamel is the most highly mineralized tissue in vertebrates. Enamel crystal formation and elongation should be well controlled to achieve an exceptional hardness and a compact microstructure. Enamel matrix calcification occurs with several matrix proteins, such as amelogenin, enamelin, and ameloblastin. Among them, amelogenin is the most abundant enamel matrix protein, and multiple isoforms resulting from extensive but well-conserved alternative splicing and postsecretional processing have been identified. In this report, we recruited a family with a unique enamel defect and identified a silent mutation in exon 4 of the AMELX gene. We show that the mutation caused the inclusion of exon 4, which is almost always skipped, in the mRNA transcript. We further show, by generating and characterizing a transgenic animal model, that the alteration of the ratio and quantity of the developmentally conserved alternative splicing repertoire of AMELX caused defects in enamel matrix mineralization. PMID:25117480

Cho, E S; Kim, K-J; Lee, K-E; Lee, E-J; Yun, C Y; Lee, M-J; Shin, T J; Hyun, H-K; Kim, Y-J; Lee, S-H; Jung, H-S; Lee, Z H; Kim, J-W

2014-10-01

159

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

Microsoft Academic Search

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

Irina M. Shapiro; Albert W. Cheng; Nicholas C. Flytzanis; Michele Balsamo; John S. Condeelis; Maja H. Oktay; Christopher B. Burge; Frank B. Gertler

2011-01-01

160

A Novel SLC12A3 Splicing Mutation Skipping of Two Exons and Preliminary Screening for Alternative Splice Variants in Human Kidney  

Microsoft Academic Search

Background: Gitelman’s syndrome is a mild autosomal recessive disorder caused by inactivating mutations of SLC12A3. However, severe phenotype may be associated with compound heterozygous nonfunctional variants such as frameshift and splicing mutations. Because most multi-exon genes are alternatively spliced as shown by recent studies, SLC12A3, with 26 exons, is likely to be alternatively spliced as well. Methods: A case of

Leping Shao; Liqiu Liu; Zhimin Miao; Hong Ren; Weiming Wang; Yanhua Lang; Shaoheng Yue; Nan Chen

2008-01-01

161

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

PubMed Central

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

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

1995-01-01

162

A house finch (Haemorhous mexicanus) spleen transcriptome reveals intra- and interspecific patterns of gene expression, alternative splicing and genetic diversity in passerines  

PubMed Central

Background With its plumage color dimorphism and unique history in North America, including a recent population expansion and an epizootic of Mycoplasma gallisepticum (MG), the house finch (Haemorhous mexicanus) is a model species for studying sexual selection, plumage coloration and host-parasite interactions. As part of our ongoing efforts to make available genomic resources for this species, here we report a transcriptome assembly derived from genes expressed in spleen. Results We characterize transcriptomes from two populations with different histories of demography and disease exposure: a recently founded population in the eastern US that has been exposed to MG for over a decade and a native population from the western range that has never been exposed to MG. We utilize this resource to quantify conservation in gene expression in passerine birds over approximately 50 MY by comparing splenic expression profiles for 9,646 house finch transcripts and those from zebra finch and find that less than half of all genes expressed in spleen in either species are expressed in both species. Comparative gene annotations from several vertebrate species suggest that the house finch transcriptomes contain ~15 genes not yet found in previously sequenced vertebrate genomes. The house finch transcriptomes harbour ~85,000 SNPs, ~20,000 of which are non-synonymous. Although not yet validated by biological or technical replication, we identify a set of genes exhibiting differences between populations in gene expression (n = 182; 2% of all transcripts), allele frequencies (76 FST ouliers) and alternative splicing as well as genes with several fixed non-synonymous substitutions; this set includes genes with functions related to double-strand break repair and immune response. Conclusions The two house finch spleen transcriptome profiles will add to the increasing data on genome and transcriptome sequence information from natural populations. Differences in splenic expression between house finch and zebra finch imply either significant evolutionary turnover of splenic expression patterns or different physiological states of the individuals examined. The transcriptome resource will enhance the potential to annotate an eventual house finch genome, and the set of gene-based high-quality SNPs will help clarify the genetic underpinnings of host-pathogen interactions and sexual selection. PMID:24758272

2014-01-01

163

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

PubMed Central

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

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

2013-01-01

164

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5? splice site selection  

PubMed Central

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5? splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5? splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5? splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5? splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5? splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5? splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator. PMID:25294838

Bielli, Pamela; Bordi, Matteo; Biasio, Valentina Di; Sette, Claudio

2014-01-01

165

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors  

Microsoft Academic Search

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction.

Johann Soret; Nadia Bakkour; Sophie Maire; Sébastien Durand; Latifa Zekri; Mathieu Gabut; Weronika Fic; Gilles Divita; Christian Rivalle; Daniel Dauzonne; Chi Hung Nguyen; Philippe Jeanteur; Jamal Tazi

2005-01-01

166

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

167

An alternative splicing product of the murine trpv1 gene dominant negatively modulates the activity of TRPV1 channels.  

PubMed

Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1alpha and TRPV1beta and contain 839 and 829 amino acids, respectively. TRPV1beta arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1alpha in many tissues and significant expression of TRPV1beta in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1alpha formed a Ca(2+)-permeable channel activated by ligands known to stimulate TRPV1. TRPV1beta was not functional by itself but its co-expression inhibited the function of TRPV1alpha. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1beta than TRPV1alpha protein was found in cells and on the cell surface, indicating that the beta isoform is highly unstable. Our data suggest that TRPV1beta is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli. PMID:15234965

Wang, Chunbo; Hu, Hong-Zhen; Colton, Craig K; Wood, Jackie D; Zhu, Michael X

2004-09-01

168

Differential characterization of three alternative spliced isoforms of DPPX.  

PubMed

Transient subthreshold-activating somato-dendritic A-type K(+) currents (I(SA)s) have fundamental roles in neuronal function. They cause delayed excitation, influence spike repolarization, modulate the frequency of repetitive firing, and have important roles in signal processing in dendrites. We previously reported that DPPX proteins are key components of the channels mediating these currents (Kv4 channels) (Nadal, M.S., Ozaita, A., Amarillo, Y., Vega-Saenz, E., Ma, Y., Mo, W., Goldberg, E.M., Misumi, Y., Ikehara, Y., Neubert, T.A., Rudy, B., 2003. The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461). The DPPX gene encodes alternatively spliced transcripts that generate single-spanning transmembrane proteins with a short, divergent intracellular domain and a large extracellular domain. We characterized the modulatory effects on Kv4.2-mediated currents and the rat brain distribution of three splice variants of the DPPX subfamily of proteins. These three splice isoforms--DPPX-S, DPPX-L, and DPPX-K--are expressed in adult rat brain and modify the voltage dependence and kinetic properties of Kv4.2 channels expressed in Xenopus oocytes. Analysis of a deletion mutant that lacks the variable N-terminus showed that the N-terminus is not necessary for the modulation of Kv4 channels. Using in situ hybridization analysis, we found that the three splice variants are prominently expressed in brain regions where Kv4 subunits are also expressed. DPPX-K and DPPX-S mRNAs have a widespread distribution, whereas DPPX-L transcripts are concentrated in few specific areas of the rat brain. The emerging diversity of DPPX splice variants, differing only in the N-terminus of the protein, opens up intriguing possibilities for the modulation of Kv4 channels. PMID:16764835

Nadal, Marcela S; Amarillo, Yimy; Vega-Saenz de Miera, Eleazar; Rudy, Bernardo

2006-06-13

169

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

PubMed Central

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

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

1993-01-01

170

SOX2 modulates alternative splicing in transitional cell carcinoma  

Microsoft Academic Search

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration

Chun-Liang Tung; Pei-Hsuan Hou; Yung-Ling Kao; Yu-Wen Huang; Chiung-Chun Shen; Yi-Hsin Cheng; Shu-Fen Wu; Moon-Sing Lee; Chin Li

2010-01-01

171

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

PubMed

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

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

2013-01-01

172

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. PMID:23569438

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

2013-01-01

173

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. PMID:24740254

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

2014-01-01

174

RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing  

PubMed Central

Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure. PMID:22466703

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

2013-01-01

175

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

176

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. PMID:25018755

Sprink, Thorben; Hartung, Frank

2014-01-01

177

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

PubMed Central

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

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

2012-01-01

178

The ASAP II database: analysis and comparative genomics of alternative splicing in 15 animal species  

Microsoft Academic Search

We have greatly expanded the Alternative Splicing Annotation Project (ASAP) database: (i) its human alternative splicing data are expanded ? 3-fold over the previous ASAP database, to nearly 90000 distinct alternative splicing events; (ii) it now provides genome-wide alternative splicing analyses for 15 vertebrate, insect and other animal species; (iii) it provides comprehensive comparative genomics information for comparing alternative splicing

Namshin Kim; Alexander V. Alekseyenko; Meenakshi Roy; Christopher Lee

2007-01-01

179

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. PMID:22235189

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

2012-01-01

180

Unusual Alternative Splicing within the Human Kallikrein Genes KLK2 and KLK3 Gives Rise to Novel Prostate-specific Proteins  

Microsoft Academic Search

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and hu- man kallikrein 2 are produced and secreted in the pros- tate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3

Anat David; Nicola Mabjeesh; Idit Azar; Sharon Biton; Sharon Engel; Jeanne Bernstein; Jacob Romano; Yoav Avidor; Tova Waks; Zelig Eshhar; Salomon Z. Langer; Beatriz Lifschitz-Mercer; Haim Matzkin; Galit Rotman; Amir Toporik; Kinneret Savitsky; Liat Mintz

2002-01-01

181

Alternative Splicing of PTC7 in Saccharomyces cerevisiae Determines Protein Localization  

PubMed Central

It is well established that higher eukaryotes use alternative splicing to increase proteome complexity. In contrast, Saccharomyces cerevisiae, a single-cell eukaryote, conducts predominantly regulated splicing through retention of nonfunctional introns. In this article we describe our discovery of a functional intron in the PTC7 (YHR076W) gene that can be alternatively spliced to create two mRNAs that code for distinct proteins. These two proteins localize to different cellular compartments and have distinct cellular roles. The protein translated from the spliced mRNA localizes to the mitochondria and its expression is carbon-source dependent. In comparison, the protein translated from the unspliced mRNA contains a transmembrane domain, localizes to the nuclear envelope, and mediates the toxic effects of Latrunculin A exposure. In conclusion, we identified a definitive example of functional alternative splicing in S. cerevisiae that confers a measurable fitness benefit. PMID:19564484

Juneau, Kara; Nislow, Corey; Davis, Ronald W.

2009-01-01

182

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

Microsoft Academic Search

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

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

2007-01-01

183

Sequences of Exon 6 and the Adjacent Intron Boundaries of the Rat Platelet-Derived Growth Factor A-Chain Gene: Implications for Alternative Splicing  

Microsoft Academic Search

Platelet-derived growth factor (PDGF) is a potent stimulator of vascular smooth muscle cell growth. Two isoforms of PDGF A-chain mRNA that either include (long form) or exclude (short form) exon 6 are produced as a result of alternative splicing in mouse, rabbit, and human. The short form of PDGF A-chain is expressed in both resting and activated cells, while the

Wen-Yang Hu; Tomohiro Nakayama; Noboru Fukuda; Hirobumi Kishioka; Masayoshi Soma; Yoichi Izumi; Katsuo Kanmatsuse

1997-01-01

184

Alternative splicing of delta-like 1 homolog ( DLK1) in the pig and human  

Microsoft Academic Search

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be

Jeffrey A. Deiuliis; Bing Li; Pasha A. Lyvers-Peffer; Steven J. Moeller; Kichoon Lee

2006-01-01

185

Detection of alternative splicing during epithelial-mesenchymal transition.  

PubMed

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

Huang, Huilin; Xu, Yilin; Cheng, Chonghui

2014-01-01

186

Tissue-specific classification of alternatively spliced human exons  

E-print Network

Alternative splicing is involved in numerous cellular functions and is often disrupted and involved in disease. Previous research has identified methods to distinguish alternative conserved exons (ACEs) in human and mouse. ...

Rothman, Craig Jeremy

2007-01-01

187

Genome Wide Identification of Aberrant Alternative Splicing Events in Myotonic Dystrophy Type 2  

PubMed Central

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis. PMID:24722564

Fasanaro, Pasquale; Bugiardini, Enrico; Cardani, Rosanna; Manteiga, Jose M. Garcia.; Riba, Michela; Cittaro, Davide; Stupka, Elia; Meola, Giovanni; Martelli, Fabio

2014-01-01

188

Physiological significance of alternatively spliced exon combinations of the single-copy gene class A chitin synthase in the insect Ostrinia furnacalis (Lepidoptera).  

PubMed

Insect chitin synthase is an essential enzyme involved in chitin biosynthesis in insects. Chitin synthase A (CHSA) is expressed in different insect tissues during different developmental stages. CHSA contains alternative-splicing exons that allow tissue- and development-specific chitin synthesis. Here, we report that OfCHSA from the lepidopteran Ostrinia furnacalis contains two alternative-splicing exons, exons 2a and 2b and exons 19a and 19b. Although four combinations of these exons are theoretically possible, we found that transcripts containing exon 2a were dominant during most developmental stages, including embryonic development, larval-larval moulting, the larval-pupal transition and pupal-adult metamorphosis. Unexpectedly, 2b-containing transcripts were much more responsive to 20-hydroxyecdysone regulation than 2a-containing ones, suggesting that although OfCHSA isoforms encoded by 2b-containing transcripts are normally expressed at very low levels, they play unique roles. Spliced exons 2a and 2b have also been observed in Bombyx mori; therefore, this work provides new insights into the regulation of insect chitin synthase, particularly in lepidopteran insects. PMID:22607200

Qu, M; Yang, Q

2012-08-01

189

RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing.  

PubMed

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5' and 3' untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

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

2013-06-01

190

Multiple Alternative Splicing and Differential Expression Pattern of the Glycogen Synthase Kinase-3? (GSK3?) Gene in Goat (Capra hircus)  

PubMed Central

Glycogen synthase kinase-3? (GSK3?) has been identified as a key protein kinase involved in several signaling pathways, such as Wnt, IGF-? and Hedgehog. However, knowledge regarding GSK3? in the goat is limited. In this study, we cloned and characterized the goat GSK3? gene. Six novel GSK3? transcripts were identified in different tissues and designated as GSK3?1, 2, 3, 4, 5 and 6. RT-PCR was used to further determine whether the six GSK3? transcripts existed in different goat tissues. Bioinformatics analysis revealed that the catalytic domain (S_TKc domain) is missing from GSK3?2 and GSK3?4. GSK3?3 and GSK3?6 do not contain the negative regulatory sites that are controlled by p38 MAPK. Furthermore, qRT-PCR and western blot analysis revealed that all the GSK3? transcripts were expressed at the highest level in the heart, whereas their expression levels in the liver, spleen, kidney, brain, longissimus dorsi muscle and uterus were different. These studies provide useful information for further research on the functions of GSK3? isoforms. PMID:25334049

Hou, Yuguo; Wang, Yilin; Wang, Yan; Zhong, Tao; Li, Li; Zhang, Hongping; Wang, Linjie

2014-01-01

191

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

192

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing  

PubMed Central

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.

2014-01-01

193

Female-specific insect lethality engineered using alternative splicing.  

PubMed

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

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

2007-03-01

194

A survey of computational methods in transcriptome-wide alternative splicing analysis.  

PubMed

Abstract Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. Consequently the identification and quantification of differentially spliced transcripts is pivotal for transcriptome analysis. Here, we review the currently available computational approaches for the analysis of RNA-sequencing data with a focus on exon-skipping events of alternative splicing and discuss the novelties as well as challenges faced to perform differential splicing analyses. In accordance with operational needs we have classified the software tools, which may be instrumental for a specific analysis based on the experimental objectives and expected outcomes. In addition, we also propose a framework for future directions by pinpointing more extensive experimental validation to assess the accuracy of the software predictions and improvements that would facilitate visualizations, data processing, and downstream analyses along with their associated software implementations. PMID:25719337

Wang, Jianbo; Ye, Zhenqing; Huang, Tim H-M; Shi, Huidong; Jin, Victor

2015-03-01

195

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

PubMed

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

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

2014-01-01

196

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. PMID:23022282

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

2012-01-01

197

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. PMID:24739459

2014-01-01

198

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. PMID:22978521

2012-01-01

199

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

PubMed Central

RNA splicing is required to remove introns from pre-mRNA and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating SR splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here we tested the impact of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike to other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine, and are not simply due to reduced Top1 activity as TOP1 downregulation by siRNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by DRB blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc. PMID:20817775

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

2010-01-01

200

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

PubMed

RNA splicing is required to remove introns from pre-mRNA, and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating serine/arginine-rich splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here, we tested the effect of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine and are not simply due to reduced Top1 activity, as Top1 downregulation by short interfering RNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by 5,6-dichloro-1-?-d-ribofuranosylbenzimidazole (DRB) blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc. PMID:20817775

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

2010-10-15

201

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

202

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. PMID:24069034

Barberán-Soler, Sergio; Ragle, James Matthew

2013-01-01

203

Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy  

PubMed Central

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

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

2013-01-01

204

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

Microsoft Academic Search

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

J H Marden

2008-01-01

205

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

PubMed Central

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

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

2014-01-01

206

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

Microsoft Academic Search

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

Amir Dori; Jonathan Cohen; William F. Silverman

2005-01-01

207

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

PubMed

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

Ohe, Kenji; Hagiwara, Masatoshi

2015-04-17

208

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

PubMed Central

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

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

2014-01-01

209

The fibronectin gene as a model for splicing and transcription studies  

Microsoft Academic Search

The fibronectm (FN) gene has become paradigmatic to illustrate genome evolution by exon shuffling, generation of protein diversity by alterna- tive mRNA splicing, and topological coordination between transcription and splicing. Alternative splic- ing in three sites of the primary transcript gives rise to multiple FN polypeptides. This process is cell type-, development- and age-regulated. The differ- ent FN variants seem

A. R. KORNBUHrF; C. C. PESCE; C. R. ALONSO; P. CRAMER; A. SREBROW; S. WERBA; A. F. MURO; Ingenieria Gen

210

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

PubMed

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

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

2012-08-01

211

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. PMID:22210893

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

212

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress  

Microsoft Academic Search

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and\\/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines.

Ravi K. Singh; Aixa Tapia-Santos; Thomas W. Bebee; Dawn S. Chandler

2009-01-01

213

Splicing Programs and Cancer  

PubMed Central

Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets. PMID:22132318

Germann, Sophie; Gratadou, Lise; Dutertre, Martin; Auboeuf, Didier

2012-01-01

214

Tissue-specific alternative splicing analysis reveals the diversity of chromosome 18 transcriptome.  

PubMed

The Chromosome-centric Human Proteome Project (C-HPP) is aimed to identify the variety of protein products and transcripts of the number of chromosomes. The Russian part of C-HPP is devoted to the study of the human chromosome 18. Using widely accepted Tophat and SpliceGrapher, a tool for accurate splice sites and alternative mRNA isoforms prediction, we performed the extensive mining of the splice variants of chromosome 18 transcripts and encoded protein products in liver, brain, lung, kidney, blood, testis, derma, and skeletal muscles. About 6.1 billion of the reads represented by 450 billion of the bases have been analyzed. The relative frequencies of splice events as well as gene expression profiles in normal tissues are evaluated. Using ExPASy PROSITE, the novel features and possible functional sites of previously unknown splice variants were highlighted. A set of unique proteotypic peptides enabling the identification of novel alternative protein species using mass-spectrometry is constructed. The revealed data will be integrated into the gene-centric knowledgebase of the Russian part of C-HPP available at http://kb18.ru and http://www.splicing.zz.mu/. PMID:24320163

Shargunov, Alexander V; Krasnov, George S; Ponomarenko, Elena A; Lisitsa, Andrey V; Shurdov, Mikhail A; Zverev, Vitaliy V; Archakov, Alexander I; Blinov, Vladimir M

2014-01-01

215

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

216

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

PubMed Central

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo. PMID:24155930

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

2013-01-01

217

Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein  

PubMed Central

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (?9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that ?9-CETP protein is expressed in the liver but fails to circulate in the blood. PMID:24393849

Suhy, Adam; Hartmann, Katherine; Newman, Leslie; Papp, Audrey; Toneff, Thomas; Hook, Vivian; Sadee, Wolfgang

2014-01-01

218

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. PMID:24575124

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

2014-01-01

219

Bax?2 Family Alternative Splicing Salvages Bax Microsatellite-Frameshift Mutations  

PubMed Central

Mutation or aberrant splicing can interrupt gene expression. Tumor suppressor Bax is one of the susceptible genes prone to microsatellite frameshifting mutations in coding regions. As a result, tumors exhibiting microsatellite instability (MSI) often present a “Bax-negative” phenotype. We previously reported that some Bax-negative cells in fact contain a functional Bax isoform (Bax?2), generated when unique alternative splicing “salvages” the shifted reading frame introduced by a microsatellite mutation. Here we compared Bax alternative splicing profiles in a range of cell lines and primary tumors with and without Bax microsatellite mutations. We found that MSI tumors exhibit a high Bax alternative splicing frequency, especially in exon 2, and produce a family of alternatively spliced isoforms that retain many important Bax functional domains. Surprisingly, these Bax?2 family isoforms can rescue Bax from all common microsatellite frameshift mutations. Production of Bax?2 requires specific cis mutations, while trans components are not cell-type specific. Furthermore, all Bax?2 family isoforms are more potent cell death inducers than the parental Bax without directly targeting mitochondria. These results indicate that the Bax?2 family can potentially salvage Bax tumor suppressor expression otherwise lost to mutation. PMID:24386510

Haferkamp, Bonnie; Zhang, Honghong; Kissinger, Samuel; Wang, Xin; Lin, Yuting; Schultz, Megan

2013-01-01

220

Dual-specificity splice sites function alternatively as 5 and 3 splice sites  

E-print Network

-scale sequencing projects and recent splicing- microarray studies, estimates of mammalian genes expressing multiple genome- wide, high-quality alignment of mRNA/EST and genome sequences and experimentally verified by RT of genome sequences and a large amount of mRNA/EST data, especially in human and mouse, genome

221

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

222

Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation  

PubMed Central

Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions. PMID:24442672

Zhou, Xuexia; Wu, Wenwu; Li, Huang; Cheng, Yuanming; Wei, Ning; Zong, Jie; Feng, Xiaoyan; Xie, Zhiqin; Chen, Dai; Manley, James L.; Wang, Hui; Feng, Ying

2014-01-01

223

Increased Dosage of Dyrk1A Alters Alternative Splicing Factor (ASF)-regulated Alternative Splicing of Tau in Down Syndrome*S??  

PubMed Central

Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS. PMID:18658135

Shi, Jianhua; Zhang, Tianyi; Zhou, Chunlei; Chohan, Muhammad Omar; Gu, Xiaosong; Wegiel, Jerzy; Zhou, Jianhua; Hwang, Yu-Wen; Iqbal, Khalid; Grundke-Iqbal, Inge; Gong, Cheng-Xin; Liu, Fei

2008-01-01

224

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

PubMed Central

Alternative splicing is a fundamental step in regulation of gene expression of many tumor suppressors and oncogenes in cancer. Signalling through the Ras-MAPK and PI3K-mTOR pathways is misregulated and hyperactivated in most types of cancer. However, the regulation of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing is less well established. Recent studies have shown the contribution of alternative splicing regulation of these signalling pathways which can lead to cellular transformation, cancer development, and tumor maintenance. This review will discuss findings in the literature which describe new modes of regulation of components of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing. We will also describe the mechanisms by which signals from extracellular stimuli can be communicated to the splicing machinery and to specific RNA-binding proteins that ultimately control exon definition events. PMID:24078813

Bonomi, Serena

2013-01-01

225

Tracking the evolution of alternatively spliced exons within the Dscam family  

Microsoft Academic Search

BACKGROUND: The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17), potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within

Mack E Crayton; Bradford C Powell; Todd J Vision; Morgan C Giddings

2006-01-01

226

ALTERNATE PATCHED SPLICE FORMS ARE EXPRESSED IN A TISSUE SPECIFIC MANNER DURING EARLY EMBRYONIC DEVELOPMENT  

Technology Transfer Automated Retrieval System (TEKTRAN)

BACKGROUND: The Hedgehog (Hh) pathway is critical for embryonic patterning of nearly every organ system in the developing fetus and is highly conserved across phylogeny. We have previously characterized three alternate splice forms of the Ptc gene, including a novel Exon 1C isoform in the mouse, but...

227

Functional coordination of alternative splicing in the mammalian central nervous system  

Microsoft Academic Search

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

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

2007-01-01

228

QUANTIFYING ALTERNATIVE SPLICING FROM PAIRED-END RNA-SEQUENCING DATA.  

PubMed

RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence sub-optimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a non-parametric, highly flexible manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream analyses. We found a several fold improvement in estimation mean square error compared popular approaches in simulations, and substantially higher consistency between replicates in experimental data. Our findings indicate the need for adjusting the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper. PMID:24795787

Rossell, David; Stephan-Otto Attolini, Camille; Kroiss, Manuel; Stöcker, Almond

2014-03-01

229

QUANTIFYING ALTERNATIVE SPLICING FROM PAIRED-END RNA-SEQUENCING DATA  

PubMed Central

RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence sub-optimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a non-parametric, highly flexible manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream analyses. We found a several fold improvement in estimation mean square error compared popular approaches in simulations, and substantially higher consistency between replicates in experimental data. Our findings indicate the need for adjusting the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper* PMID:24795787

Rossell, David; Stephan-Otto Attolini, Camille; Kroiss, Manuel; Stöcker, Almond

2014-01-01

230

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

231

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

PubMed

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

Dehghanian, Fariba; Hojati, Zohreh; Kay, Maryam

2014-10-01

232

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

PubMed Central

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

Dehghanian, Fariba; Hojati, Zohreh; Kay, Maryam

2014-01-01

233

Duplicated gephyrin genes showing distinct tissue distribution and alternative splicing patterns mediate molybdenum cofactor biosynthesis, glycine receptor clustering, and escape behavior in zebrafish.  

PubMed

Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABA(A) receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish. PMID:20843816

Ogino, Kazutoyo; Ramsden, Sarah L; Keib, Natalie; Schwarz, Günter; Harvey, Robert J; Hirata, Hiromi

2011-01-01

234

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses[OPEN  

PubMed Central

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely. PMID:24179132

Staiger, Dorothee; Brown, John W.S.

2013-01-01

235

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. PMID:15575968

Wang, Bing-Bing; Brendel, Volker

2004-01-01

236

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

PubMed Central

Fine mapping of calcineurin (PPP3CA) gene identified single nucleotide polymorphisms (SNPs) and simple sequence repeat polymorphisms that are associated with addiction vulnerability. A trinucleotide repeat marker, located in the 5? untranslated region (5?UTR) of the PPP3CA mRNA, exhibited significantly different genotype and allele frequencies between abusers and controls in the NIDA African–American sample. The polymorphism showed allelic-specific expression in mRNA extracted from postmortem brain specimens. Novel alternatively spliced isoforms of PPP3CA were identified and their expressions were found altered in brain regions of postmortem Alzheimer's disease patients. These data underscore the importance of calcineurin gene in the molecular mechanism of addiction and Alzheimer's diseases. PMID:20590401

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

2010-01-01

237

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges  

PubMed Central

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

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

2014-01-01

238

ORIGINAL ARTICLE Tau Alternative Splicing and Frontotemporal Dementia  

E-print Network

ORIGINAL ARTICLE Tau Alternative Splicing and Frontotemporal Dementia Amar Kar, MS,* David Kuo, BS with frontotemporal lobe dementia. These mutations affect either biochemical/biophysical pro- perties or the delicate of genetics and molecular pathogenesis of tauopathies with the focus on fronto- temporal lobe dementia. We

Wu, Jane Y.

239

An Alternative Splicing Switch Regulates Embryonic Stem Cell  

E-print Network

An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming Mathieu for Systems Biology, Samuel Lunenfeld Research Institute 4Center for Stem Cells and Tissue Engineering, Samuel expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)- specific AS event

Zandstra, Peter W.

240

Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN  

PubMed Central

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation. PMID:24179125

Reddy, Anireddy S.N.; Marquez, Yamile; Kalyna, Maria; Barta, Andrea

2013-01-01

241

Intron retention: a human DKC1 gene common splicing event.  

PubMed

Identification of alternatively spliced transcripts produced by a gene is a crucial step in deciphering the bulk of its biological roles and the overall processes that regulate its activity. By using a combination of bioinformatic and molecular approaches we identified, cloned, and characterized 3 novel alternative splice isoforms derived from human dyskeratosis congenita 1 (hDKC1), an essential human gene causative of the X-linked dyskeratosis congenita disease and involved in multiple functions related to cell growth, proliferation, and telomere maintenance. Expression of the new isoforms, all characterized by intron retention, was confirmed by RT-PCR in a panel of diverse cell lines and normal human tissues, and despite the presence of premature termination codons, was not down-regulated by the mechanism of nonsense-mediated decay. Accumulation of these transcripts fluctuated distinctly in the diverse tissues and during in vitro differentiation of Caco2 cells, suggesting that their ratio may contribute to the gene functional diversity across different cell types. Intriguingly, the structure of one isoform leads to exonize an intronically encoded small nucleolar RNA (snoRNA), highlighting an additional layer of complexity that can contribute to overall gene regulation. PMID:24219293

Turano, Mimmo; Angrisani, Alberto; Di Maio, Nunzia; Furia, Maria

2013-12-01

242

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.  

PubMed

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

243

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

PubMed Central

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

2012-01-01

244

Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs  

SciTech Connect

A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.

O'Malley, K.L.; Anhalt, M.J.; Martin, B.M.; Kelsoe, J.R.; Winfield, S.L.; Ginns, E.I.

1987-11-03

245

Alternative splicing of the mouse amelogenin primary RNA transcript  

Microsoft Academic Search

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively

J. P. Simmer; C. C. Hu; E. C. Lau; P. Sarte; H. C. Slavkin; A. G. Fincham

1994-01-01

246

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

PubMed Central

Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays (HJAY). We analyzed these data using a computational pipeline specifically designed to detect AS with a low false positive rate. This identified 181 splice events representing 156 genes as candidates for AS. RT-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype-specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. siRNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype specific AS. The subtype specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues. PMID:20605923

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

2010-01-01

247

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

PubMed Central

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5? and 3? untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

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

2013-01-01

248

Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis.  

PubMed

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition. PMID:25087874

Singh, Ravi K; Xia, Zheng; Bland, Christopher S; Kalsotra, Auinash; Scavuzzo, Marissa A; Curk, Tomaz; Ule, Jernej; Li, Wei; Cooper, Thomas A

2014-08-21

249

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

PubMed Central

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 TCL1tg 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

2014-01-01

250

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

251

Alternative splicing of EKLF/KLF1 in murine primary erythroid tissues.  

PubMed

Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non-erythroid Krüppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krüppel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation. PMID:25283745

Yien, Yvette Y; Gnanapragasam, Merlin Nithya; Gupta, Ritama; Rivella, Stefano; Bieker, James J

2015-01-01

252

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

Microsoft Academic Search

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA pre- cursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for

ERIC ALLEMAND; RENATA GATTONI; HENRI-MARC BOURBON; JAMES STEVENIN; JAVIER F. CACERES; JOHANN SORET; JAMAL TAZI

2001-01-01

253

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

Microsoft Academic Search

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

Joachim Krebs; Jody Groenendyk; Marek Michalak

2011-01-01

254

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 Central

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, Tomoaki; Nagashima, Kunio; Ward, Jerrold M.; Minoo, Parviz; Zimonjic, Drazen B.; Popescu, Nicholas C.; Kimura, Shioko

2001-01-01

255

Differing patterns of selection in alternative and constitutive splice sites  

PubMed Central

In addition to allowing identification of putative functional elements as regions having reduced substitution rates, comparison of genome sequences can also provide insights into these elements at the nucleotide level, by indicating the pattern of tolerated substitutions. We created data sets of orthologous alternative and constitutive splice sites in mouse, rat, and human and analyzed the substitutions occurring within them. Our results illuminate differences between alternative and constitutive sites and, in particular, strongly support the idea that alternative sites are under selection to be weak. PMID:17556528

Garg, Kavita; Green, Phil

2007-01-01

256

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier  

PubMed Central

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies. PMID:20949073

Hinzpeter, Alexandre; Aissat, Abdel; Sondo, Elvira; Costa, Catherine; Arous, Nicole; Gameiro, Christine; Martin, Natacha; Tarze, Agathe; Weiss, Laurence; de Becdelièvre, Alix; Costes, Bruno; Goossens, Michel; Galietta, Luis J.; Girodon, Emmanuelle; Fanen, Pascale

2010-01-01

257

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

258

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

259

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

Microsoft Academic Search

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

German Gaston Leparc; Robi David Mitra

2007-01-01

260

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. PMID:23299472

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

2013-01-01

261

Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts  

PubMed Central

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour. PMID:20126463

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

2010-01-01

262

Using single-strand conformational polymorphism gel electrophoresis to analyze mutually exclusive alternative splicing.  

PubMed

Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful. PMID:14769996

Celotto, Alicia M; Graveley, Brenton R

2004-01-01

263

Alternative splicing of human T-cell-specific MAL mRNA and its correlation with the exon/intron organization of the gene.  

PubMed

Sequence analysis of the T-cell-specific MAL gene revealed four exons, each encoding a hydrophobic, presumably membrane-associated, segment and its adjacent hydrophilic sequence. Amplification by the polymerase chain reaction of cDNA from different T-cell samples indicated the existence of four different forms of MAL mRNA, termed MAL-a, -b, -c, and -d, that arise from differential usage of exons II and/or III. As the three introns were located between complete codons, the reading frame was maintained in all the transcripts. A model resembling the structures postulated for different proteolipid proteins is proposed for the protein encoded by each alternative mRNA species. PMID:8088843

Rancaño, C; Rubio, T; Alonso, M A

1994-05-15

264

Modeling alternative splicing variants from RNA-Seq data with isoform graphs.  

PubMed

Next-generation sequencing (NGS) technologies need new methodologies for alternative splicing (AS) analysis. Current computational methods for AS analysis from NGS data are mainly based on aligning short reads against a reference genome, while methods that do not need a reference genome are mostly underdeveloped. In this context, the main developed tools for NGS data focus on de novo transcriptome assembly (Grabherr et al., 2011 ; Schulz et al., 2012). While these tools are extensively applied for biological investigations and often show intrinsic shortcomings from the obtained results, a theoretical investigation of the inherent computational limits of transcriptome analysis from NGS data, when a reference genome is unknown or highly unreliable, is still missing. On the other hand, we still lack methods for computing the gene structures due to AS events under the above assumptions--a problem that we start to tackle with this article. More precisely, based on the notion of isoform graph (Lacroix et al., 2008), we define a compact representation of gene structures--called splicing graph--and investigate the computational problem of building a splicing graph that is (i) compatible with NGS data and (ii) isomorphic to the isoform graph. We characterize when there is only one representative splicing graph compatible with input data, and we propose an efficient algorithmic approach to compute this graph. PMID:24200390

Beretta, Stefano; Bonizzoni, Paola; Vedova, Gianluca Della; Pirola, Yuri; Rizzi, Raffaella

2014-01-01

265

Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis.  

PubMed

Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing. PMID:25482508

Tejedor, J Ramón; Papasaikas, Panagiotis; Valcárcel, Juan

2015-01-01

266

Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle.  

PubMed

Nitric oxide (NO) functions as a molecular mediator in numerous processes in cellular development and physiology. Differential expression and regulation of a family of three NO synthase (NOS) gene products help achieve this diversity of action. Previous studies identify post-translational modification and interaction of NOS with specific protein targets as tissue-specific modes of regulation. Here, we show that alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle as its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family. PMID:8626668

Silvagno, F; Xia, H; Bredt, D S

1996-05-10

267

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.  

PubMed

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. PMID:25151644

Hulse, R P; Beazley-Long, N; Hua, J; Kennedy, H; Prager, J; Bevan, H; Qiu, Y; Fernandes, E S; Gammons, M V; Ballmer-Hofer, K; Gittenberger de Groot, A C; Churchill, A J; Harper, S J; Brain, S D; Bates, D O; Donaldson, L F

2014-11-01

268

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

PubMed Central

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance. PMID:20221253

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

2010-01-01

269

Whole Transcriptome Sequencing Reveals Gene Expression and Splicing Differences in Brain Regions Affected by Alzheimer's Disease  

PubMed Central

Recent studies strongly indicate that aberrations in the control of gene expression might contribute to the initiation and progression of Alzheimer's disease (AD). In particular, alternative splicing has been suggested to play a role in spontaneous cases of AD. Previous transcriptome profiling of AD models and patient samples using microarrays delivered conflicting results. This study provides, for the first time, transcriptomic analysis for distinct regions of the AD brain using RNA-Seq next-generation sequencing technology. Illumina RNA-Seq analysis was used to survey transcriptome profiles from total brain, frontal and temporal lobe of healthy and AD post-mortem tissue. We quantified gene expression levels, splicing isoforms and alternative transcript start sites. Gene Ontology term enrichment analysis revealed an overrepresentation of genes associated with a neuron's cytological structure and synapse function in AD brain samples. Analysis of the temporal lobe with the Cufflinks tool revealed that transcriptional isoforms of the apolipoprotein E gene, APOE-001, -002 and -005, are under the control of different promoters in normal and AD brain tissue. We also observed differing expression levels of APOE-001 and -002 splice variants in the AD temporal lobe. Our results indicate that alternative splicing and promoter usage of the APOE gene in AD brain tissue might reflect the progression of neurodegeneration. PMID:21283692

Twine, Natalie A.; Janitz, Karolina; Wilkins, Marc R.; Janitz, Michal

2011-01-01

270

Heteroduplex formation and S1 digestion for mapping alternative splicing sites.  

PubMed

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies. PMID:18949713

Ferreira, E N; Rangel, M C R; Pineda, P B; Vidal, D O; Camargo, A A; Souza, S J; Carraro, D M

2008-01-01

271

Leucine-rich repeat kinase 2 and alternative splicing in Parkinson's disease.  

PubMed

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD) and are associated with pleiomorphic neuropathology. We hypothesize that LRRK2 mediates its pathogenic effect through alternative splicing of neurodegeneration genes. Methods used in this study included western blotting analysis of subcellular protein fractions, exon-array analysis of RNA from cultured neuroblastoma cells transfected with LRRK2 expression vectors, and reverse-transcription polymerase chain reaction (RT-PCR) of RNA from cultured cells and postmortem tissue. Overexpression of the LRRK2 G2019S mutant resulted in a significant (2.6-fold; P = 0.020) decrease in nuclear transactive response DNA-binding protein 43 levels. Exon-array analyses revealed that wild-type LRRK2 had a significant effect on the expression of genes with nuclear (P < 10(-22) ) and cell-cycle functions (P < 10(-15) ). We replicated changes in gene expression in 30% of selected genes by quantitative RT-PCR. Overexpression of LRRK2 resulted in the altered splicing of two genes associated with PD, with an increased inclusion of exon 10 of microtubule-associated protein tau (1.7-fold; P = 0.001) and exon 5 of the alpha-synuclein (SNCA) gene (1.6-fold; P =0.005). Moreover, overexpression of LRRK2 (G2019S) and two mutant genes associated with neurodegeneration, TARDBP (M337V) and FUS (R521H), were associated with decreased inclusion out of the dystonin (DST) 1e precursor exons in SK-N-MC cells. Altered splicing of SNCA (1.9-fold; P < 0.001) and DST genes (log(2) 2.3-fold; P = 0.005) was observed in a cohort of PD, compared with neurologically healthy, brains. This suggests that aberrant RNA metabolism is an important contributor to idiopathic PD. PMID:22528366

Elliott, David A; Kim, Woojin S; Gorissen, Sarsha; Halliday, Glenda M; Kwok, John B J

2012-07-01

272

Whole Transcriptome Sequencing Reveals Gene Expression and Splicing Differences in Brain Regions Affected by Alzheimer's Disease  

Microsoft Academic Search

Recent studies strongly indicate that aberrations in the control of gene expression might contribute to the initiation and progression of Alzheimer's disease (AD). In particular, alternative splicing has been suggested to play a role in spontaneous cases of AD. Previous transcriptome profiling of AD models and patient samples using microarrays delivered conflicting results. This study provides, for the first time,

Natalie A. Twine; Karolina Janitz; Marc R. Wilkins; Michal Janitz

2011-01-01

273

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. PMID:23707073

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

274

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

275

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. PMID:24844180

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

2014-01-01

276

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

PubMed

Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms. (1) The presence of mutations, particularly in exon 10 and intron 10-11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data. PMID:25826665

Lisowiec, Jolanta; Magner, Dorota; Kierzek, Elzbieta; Lenartowicz, Elzbieta; Kierzek, Ryszard

2015-03-01

277

Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition  

PubMed Central

Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons (ASEs), and that inhibition of DNA methylation results in aberrant splicing of ASEs. The methyl-CpG-binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased histone acetylation and aberrant ASE-skipping events. We further show that inhibition of histone deacetylase (HDAC) activity leads to exon skipping that shows a highly significant degree of overlap with that caused by MeCP2 knockdown. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative RNA splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through the subsequent recruitment of HDACs. PMID:23938295

Maunakea, Alika K; Chepelev, Iouri; Cui, Kairong; Zhao, Keji

2013-01-01

278

HNRNPA1 regulates HMGCR alternative splicing and modulates cellular cholesterol metabolism  

PubMed Central

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

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

2014-01-01

279

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. PMID:20109226

2010-01-01

280

Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens.  

PubMed

Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes. PMID:24777346

Chang, Chiung-Yun; Lin, Wen-Dar; Tu, Shih-Long

2014-04-28

281

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. PMID:24472559

2014-01-01

282

The thyroperoxidase doublet is not produced by alternative splicing.  

PubMed

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that the TPO doublet is not the consequence of translation of TPO2. PMID:8824887

Cetani, F; Costagliola, S; Tonacchera, M; Panneels, V; Vassart, G; Ludgate, M

1995-12-29

283

Analysis and prediction of gene splice sites in four Aspergillus genomes.  

PubMed

Several Aspergillus fungal genomic sequences have been published, with many more in progress. Obviously, it is essential to have high-quality, consistently annotated sets of proteins from each of the genomes, in order to make meaningful comparisons. We have developed a dedicated, publicly available, splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window size on single local networks for training, to cover both donor and acceptor site information. We have applied NetAspGene to other Aspergilli, including Aspergillus nidulans, Aspergillus oryzae, and Aspergillus niger. Evaluation with independent data sets reveal that NetAspGene performs substantially better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene. PMID:18948220

Wang, Kai; Ussery, David Wayne; Brunak, Søren

2009-03-01

284

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. PMID:24287856

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

2013-01-01

285

Retrotransposons of the Tnt1B family are mobile in Nicotiana plumbaginifolia and can induce alternative splicing of the host gene upon insertion  

Microsoft Academic Search

Active retrotransposons have been identified in Nicotiana plumbaginifolia by their ability to disrupt the nitrate reductase gene in chlorate–resistant mutants selected from protoplast–derived cultures. In mutants E23 and F97, two independent insertions of Tnp2, a new retrotransposon closely related to the tobacco Tnt1 elements, were detected in the nitrate reductase gene. These two Tnp2 elements are members of the Tnt1B

Christian Meyer

2001-01-01

286

Molecular Heterogeneity and Alternative Splicing of Human Lactoperoxidase  

PubMed Central

Human lactoperoxidase (LPO) exists as two distinct molecules independent of glycosylation. The N-terminus of one form is blocked and has not been identified while the other is proteolytically processed at the N-terminus similar to myeloperoxidase. Our analysis identified alternatively spliced human LPO mRNAs that may explain the observed molecular heterogeneity of LPO. Two mRNAs omit propeptide encoding exons while retaining the 5? exon encoding the secretion signal, consistent with the heterogeneity and suggesting a possible functional role for the propeptide. Two LPO forms were expressed using baculovirus and both showed similar enzyme activity. LC/MS/MS analysis of trypsin digested, partially purified, salivary LPO confirmed the larger unprocessed LPO is present in saliva. To compare variant expression patterns, antisera were raised against purified recombinant (rhLPO) as well as against an antigenic peptide sequence within the exons encoding the propeptide region. Immunohistochemistry demonstrated proLPO was differently localized within gland cells compared to other forms of LPO. The data suggested splice variants may contribute to LPO molecular heterogeneity and its regulation by intracellular compartmental localization. PMID:19059195

Fragoso, Miryam A.; Torbati, Aliza; Fregien, Nevis; Conner, Gregory E.

2009-01-01

287

Profilin II Is Alternatively Spliced, Resulting in Profilin Isoforms That Are Differentially Expressed and Have Distinct Biochemical Properties  

Microsoft Academic Search

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all

ANJA LAMBRECHTS; ATTILA BRAUN; VERONIQUE JONCKHEERE; ATTILA ASZODI; LORENE M. LANIER; JOHAN ROBBENS; INGE VAN COLEN; JOEL VANDEKERCKHOVE; REINHARD FASSLER; CHRISTOPHE AMPE

2000-01-01

288

Alternative splicing in concert with protein intrinsic disorder enables increased functional diversity  

E-print Network

protein isoforms from a single gene, thereby contributing to protein diversity. Despite intensive efforts analyzed a set of 46 differentially spliced genes encoding experimentally characterized human proteins unstructured protein structure The splicing of pre-mRNA (1) was first described in 1977. Soon thereafter

Obradovic, Zoran

289

Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells  

PubMed Central

The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene. PMID:25313066

Alló, Mariano; Agirre, Eneritz; Bessonov, Sergey; Bertucci, Paola; Gómez Acuña, Luciana; Buggiano, Valeria; Bellora, Nicolás; Singh, Babita; Petrillo, Ezequiel; Blaustein, Matías; Miñana, Belén; Dujardin, Gwendal; Pozzi, Berta; Pelisch, Federico; Bechara, Elías; Agafonov, Dmitry E.; Srebrow, Anabella; Lührmann, Reinhard; Valcárcel, Juan; Eyras, Eduardo; Kornblihtt, Alberto R.

2014-01-01

290

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. PMID:18638396

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

2008-01-01

291

SMAR1, a Novel, Alternatively Spliced Gene Product, Binds the Scaffold\\/Matrix-Associated Region at the T Cell Receptor ? Locus  

Microsoft Academic Search

Rearrangement and expression of the T cell receptor ? gene are critical events for early T lymphocyte development. To characterize cis-regulatory elements and their associated trans-factors that mediate these events, we have previously identified a nuclear matrix\\/scaffold-associated region, referred to as MAR?, 400 bp upstream of the E? enhancer. Electrophoretic mobility shift assay showed that two known MAR-binding proteins, SATB1

Samit Chattopadhyay; Ruchika Kaul; Alan Charest; David Housman; Jianzhu Chen

2000-01-01

292

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization  

Microsoft Academic Search

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the

Gary D. Paterno; Zhihu Ding; Yuan-Y. Lew; Gord W. Nash; F. Corinne Mercer; Laura L. Gillespie

2002-01-01

293

Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments.  

PubMed Central

Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms. Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment. Recently a gene for Wilson disease (WD)(also named the ATP7B gene) was cloned. This gene encodes a copper transporter of the P-type ATPase. We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain. We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol. These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper. PMID:9307043

Yang, X L; Miura, N; Kawarada, Y; Terada, K; Petrukhin, K; Gilliam, T; Sugiyama, T

1997-01-01

294

The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform.  

PubMed

Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism. PMID:25620705

Dichmann, Darwin S; Walentek, Peter; Harland, Richard M

2015-02-01

295

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 PMID:8139926

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

1994-01-01

296

Alternate Splicing of mRNAs Encoding Human Mast Cell Growth Factor and Localization of the Gene to Chromosome 12q22-q24  

Microsoft Academic Search

Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides correspondingto munineand human MGF sequences. Sequencingof the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form correspondingin size to the munine cDNA, and an alternately splicedclone with a deletion of the sixth

Dirk M. Anderson; Douglas E. Williams; Robert Tushinski; Steve Gimpel; Linda A. Cannizzaro; Margaret Aronson; Carlo M. Croce; Kay Huebner; David Cosman; Stewart D. Lyman

1991-01-01

297

Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.  

PubMed

Alternative splicing (AS) of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs) at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs) as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes). AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms. PMID:21935421

Richardson, Dale N; Rogers, Mark F; Labadorf, Adam; Ben-Hur, Asa; Guo, Hui; Paterson, Andrew H; Reddy, Anireddy S N

2011-01-01

298

PTBP-dependent PSD-95 and CamKII? alternative splicing in the lens  

PubMed Central

Purpose Parallels described between neurons and lens fiber cells include detailed similarities in sub-cellular structures that increasingly show shared expression of genes involved in the construction and function of these structures in neurons. Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well. Both elongated cell types form membrane protrusions with similar size, shape, and spacing that exclude microtubules, contain F-actin, and are coated with the clathrin/AP-2 adaptor. Lenses express glutamate and gamma-aminobutyric acid (GABA) receptors with signaling and channel proteins shown to act together at neuronal membranes. Postsynaptic density protein 95 (PSD-95) and Ca2+/calmodulin-dependent protein kinase (CaMKII?) expression and functions illustrate the integration of aspects of neuronal molecular and cell biology and were investigated here in the lens. Methods Immunofluorescence, immunoblot, and RT–PCR methods were used to assess protein expression and alternative transcript splicing. Results We showed the essential dendritic spine scaffold protein PSD-95 is expressed in lenses and demonstrated lens PSD-95 transcripts undergo polypyrimidine tract binding protein (PTBP)-dependent alternative splicing of its pivotal exon 18 required to avoid nonsense-mediated decay, and showed PTBP-dependent alternative splicing of CaMKII? transcripts in the lens. The PSD-95 protein was observed at fiber cell membranes overlapping with N-methyl-D-aspartate (NMDA) and ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate and GABA receptor proteins, tyrosine phosphatase STEP, CaMKII?, the Ca(V)1.3 calcium channel, and clathrin, which were previously identified at lens fiber cell membranes. During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses. Conclusions Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens. PMID:25540577

Nandanoor, Anoop; Kasinathan, Chinnaswamy

2014-01-01

299

Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions  

PubMed Central

Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study. PMID:25605796

Mauger, Oriane; Klinck, Roscoe; Chabot, Benoit; Muchardt, Christian; Allemand, Eric; Batsché, Eric

2015-01-01

300

Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression  

PubMed Central

Background CD44 is considered as ‘a’ metastasis associated gene, despite the fact that it is an umbrella term for a group of molecules produced from a single gene by alternative splicing. However, little consideration is given to the above in the literature of colorectal carcinomas as well as other tumour types, leading to confusion and contradictory results about its possible role in tumour progression. Methods We compared the CD44 alternative splice pattern (ASP) of three genetically different human colorectal cancer cell lines (HT25, HT29, HCT116) using a series of PCR reactions and next- generation sequencing method, as well as identified a colorectal adenocarcinoma specific CD44 ASP. This ASP was further investigated in terms of its qualitative and quantitative stability in our experimental iso- and xenograft mouse models for colorectal cancer progression. A complex preclinical experimental set-up was established to separately test the different steps of tumour progression and the role of tumour microenvironment, respectively, focusing on the role of ‘CD44’ in this process. Results We managed to present a colorectal cancer-specific CD44 ASP, which remained unchanged from cell lines throughout primary tumour formation and metastatic progression. Furthermore, we report a unique roster of all expressed CD44 variant isoforms characteristic to colorectal cancer. Finally, on quantitative assessment of the variable exons v3 and v6, higher co-expression levels were found to be characteristic to metastatically potent tumour cells. Conclusion Particular CD44 variant isoforms seem to act as “metastasis genes” via tumour microenvironment-driven shifts in v3 and v6 expressions. However, this function may just affect a minority of tumour subclones. This fact and the huge potential number of different CD44 splice variants that can contain v3 and v6 domains can explain incoherence of clinical studies regarding functional asessment of CD44 variants, as well as diminish the chances of using CD44 variants for predictive purpose. PMID:23151220

2012-01-01

301

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

PubMed Central

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. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1–3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins. PMID:22295091

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

2012-01-01

302

Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo.  

PubMed

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5' splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3' splice site. The 5' and 3' splice site complexes are thought to be joined together by protein-protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF(65). Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5' and 3' splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival. PMID:25605964

Becerra, Soraya; Montes, Marta; Hernández-Munain, Cristina; Suñé, Carlos

2015-03-01

303

Regulation of human papillomavirus gene expression by splicing and polyadenylation.  

PubMed

Human papillomaviruses (HPVs) are small DNA tumour viruses that are present in more than 99% of all cervical cancers. The ability of these viruses to cause disease is partly attributed to the strict coordination of viral gene expression with the differentiation stage of the infected cell. HPV gene expression is regulated temporally at the level of RNA splicing and polyadenylation, and a dysregulated gene expression programme allows some HPV types to establish long-term persistence, which is a risk factor for cancer. In this Review, we summarize the role of splicing and polyadenylation in the regulation of HPV gene expression and discuss the viral and cellular factors that control these processes. PMID:23474685

Johansson, Cecilia; Schwartz, Stefan

2013-04-01

304

Species-specific alternative splicing leads to unique expression of sno-lncRNAs  

PubMed Central

Background Intron-derived long noncoding RNAs with snoRNA ends (sno-lncRNAs) are highly expressed from the imprinted Prader-Willi syndrome (PWS) region on human chromosome 15. However, sno-lncRNAs from other regions of the human genome or from other genomes have not yet been documented. Results By exploring non-polyadenylated transcriptomes from human, rhesus and mouse, we have systematically annotated sno-lncRNAs expressed in all three species. In total, using available data from a limited set of cell lines, 19 sno-lncRNAs have been identified with tissue- and species-specific expression patterns. Although primary sequence analysis revealed that snoRNAs themselves are conserved from human to mouse, sno-lncRNAs are not. PWS region sno-lncRNAs are highly expressed in human and rhesus monkey, but are undetectable in mouse. Importantly, the absence of PWS region sno-lncRNAs in mouse suggested a possible reason why current mouse models fail to fully recapitulate pathological features of human PWS. In addition, a RPL13A region sno-lncRNA was specifically revealed in mouse embryonic stem cells, and its snoRNA ends were reported to influence lipid metabolism. Interestingly, the RPL13A region sno-lncRNA is barely detectable in human. We further demonstrated that the formation of sno-lncRNAs is often associated with alternative splicing of exons within their parent genes, and species-specific alternative splicing leads to unique expression pattern of sno-lncRNAs in different animals. Conclusions Comparative transcriptomes of non-polyadenylated RNAs among human, rhesus and mouse revealed that the expression of sno-lncRNAs is species-specific and that their processing is closely linked to alternative splicing of their parent genes. This study thus further demonstrates a complex regulatory network of coding and noncoding parts of the mammalian genome. PMID:24734784

2014-01-01

305

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons  

PubMed Central

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2?) and Tra2? have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2? and Tra2? induces substantial shifts in splicing of endogenous Tra2? target exons, and that both constitutive and alternative target exons are under dual Tra2?–Tra2? control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker ?H2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability. PMID:25208576

Best, Andrew; James, Katherine; Dalgliesh, Caroline; Hong, Elaine; Kheirolahi-Kouhestani, Mahsa; Curk, Tomaz; Xu, Yaobo; Danilenko, Marina; Hussain, Rafiq; Keavney, Bernard; Wipat, Anil; Klinck, Roscoe; Cowell, Ian G.; Cheong Lee, Ka; Austin, Caroline A.; Venables, Julian P.; Chabot, Benoit; Santibanez Koref, Mauro; Tyson-Capper, Alison; Elliott, David J.

2014-01-01

306

From single-cell to cell-pool transcriptomes: Stochasticity in gene expression and RNA splicing  

PubMed Central

Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30–100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

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

2014-01-01

307

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

PubMed

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

Mandadi, Kranthi K; Scholthof, Karen-Beth G

2015-01-01

308

Gene Recognition Via Spliced Sequence Alignment  

Microsoft Academic Search

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

Mikhail S. Gelfand; Andrey A. Mironov; Pavel A. Pevzner

1996-01-01

309

Tra2? Protein Is Required for Tissue-specific Splicing of a Smooth Muscle Myosin Phosphatase Targeting Subunit Alternative Exon*  

PubMed Central

Alternative splicing of the smooth muscle myosin phosphatase targeting subunit (Mypt1) exon 23 (E23) is tissue-specific and developmentally regulated and, thus, an attractive model for the study of smooth muscle phenotypic specification. We have proposed that Tra2? functions as a tissue-specific activator of Mypt1 E23 splicing on the basis of concordant expression patterns and Tra2? activation of Mypt1 E23 mini-gene splicing in vitro. In this study we examined the relationship between Tra2? and Mypt1 E23 splicing in vivo in the mouse. Tra2? was 2- to 5-fold more abundant in phasic smooth muscle tissues, such as the portal vein, small intestine, and small mesenteric artery, in which Mypt1 E23 is predominately included as compared with the tonic smooth muscle tissues, such as the aorta and inferior vena cava, in which Mypt1 E23 is predominately skipped. Tra2? was up-regulated in the small intestine postnatally, concordant with a switch to Mypt1 E23 splicing. Targeting of Tra2? in smooth muscle cells using SM22?-Cre caused a substantial reduction in Mypt1 E23 inclusion specifically in the intestinal smooth muscle of heterozygotes, indicating sensitivity to Tra2? gene dosage. The switch to the Mypt1 E23 skipped isoform coding for the C-terminal leucine zipper motif caused increased sensitivity of the muscle to the relaxant effects of 8-Br-cyclic guanosine monophosphate (cGMP). We conclude that Tra2? is necessary for the tissue-specific splicing of Mypt1 E23 in the phasic intestinal smooth muscle. Tra2?, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation. PMID:22437831

Fu, Kang; Mende, Ylva; Bhetwal, Bhupal P.; Baker, Salah; Perrino, Brian A.; Wirth, Brunhilde; Fisher, Steven A.

2012-01-01

310

Alternative Cyclin D1 Splice Forms Differentially Regulate the DNA Damage Response  

PubMed Central

The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA-damage induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of ?H2AX phosphorylation, the assembly of DNA repair foci, and specific recruitment of DNA repair factors to chromatin, and G2/M arrest. Cyclin D1 deletion in fibroblasts or siRNA mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-FU-mediated DDR. Mechanistic studies demonstrated cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin. PMID:20940395

Li, Zhiping; Jiao, Xuanmao; Wang, Chenguang; Shirley, L. Andrew; Elsaleh, Hany; Dahl, Olav; Wang, Min; Soutoglou, Evi; Knudsen, Erik S.; Pestell, Richard G.

2010-01-01

311

Alternative splicing of Alu exons--two arms are better than one  

Microsoft Academic Search

Alus, primate-specific retroelements, are the most abundant repetitive elements in the human genome. They are composed of two related but distinct monomers, left and right arms. Intronic Alu ele- ments may acquire mutations that generate func- tional splice sites, a process called exonization. Most exonizations occur in right arms of antisense Alu elements, and are alternatively spliced. Here we show

Nurit Gal-Mark; Schraga Schwartz; Gil Ast

2008-01-01

312

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

PubMed Central

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

2013-01-01

313

Cytokines Interleukin-1? and Tumor Necrosis Factor-? Regulate Different Transcriptional and Alternative Splicing Networks in Primary ?-Cells  

PubMed Central

OBJECTIVE Cytokines contribute to pancreatic ?-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1? + interferon (IFN)-? and tumor necrosis factor (TNF)-? + IFN-? in primary rat ?-cells. RESEARCH DESIGN AND METHODS Fluorescence-activated cell sorter–purified rat ?-cells were exposed to IL-1? + IFN-? or TNF-? + IFN-? for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in ?-cells, with temporal differences in the number of genes modulated by IL-1? + IFN? or TNF-? + IFN-?. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of ?-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-?, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes. CONCLUSIONS The present study doubles the number of known genes expressed in primary ?-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in ?-cells. It also shows that cytokines modify alternative splicing in ?-cells, opening a new avenue of research for the field. PMID:19934004

Ortis, Fernanda; Naamane, Najib; Flamez, Daisy; Ladrière, Laurence; Moore, Fabrice; Cunha, Daniel A.; Colli, Maikel L.; Thykjaer, Thomas; Thorsen, Kasper; Ørntoft, Torben F.; Eizirik, Decio L.

2010-01-01

314

Alternative splicing and genetic diversity: silencers are more frequently modified by SNVs associated with alternative exon/intron borders  

PubMed Central

With the availability of a large amount of genomic data it is expected that the influence of single nucleotide variations (SNVs) in many biological phenomena will be elucidated. Here, we approached the problem of how SNVs affect alternative splicing. First, we observed that SNVs and exonic splicing regulators (ESRs) independently show a biased distribution in alternative exons. More importantly, SNVs map more frequently in ESRs located in alternative exons than in ESRs located in constitutive exons. By looking at SNVs associated with alternative exon/intron borders (by their common presence in the same cDNA molecule), we observed that a specific type of ESR, the exonic splicing silencers (ESSs), are more frequently modified by SNVs. Our results establish a clear association between genetic diversity and alternative splicing involving ESSs. PMID:21398627

de Souza, Jorge E. S.; Ramalho, Rodrigo F.; Galante, Pedro A. F.; Meyer, Diogo; de Souza, Sandro J.

2011-01-01

315

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

E-print Network

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 ...

Bradley, Robert K.

316

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

PubMed

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

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

2014-10-13

317

Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels  

PubMed Central

Synopsis The proposed Ca2+-activated Cl? channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1?ex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1?ex2 in HEK293 cells, and compared its properties to Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1?ex2 successfully formed Ca2+-activated Cl? channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2 expressing cells had no detectable Ca2+-activated Cl? currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1?ex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modeling of the Best1 protein structure. PMID:24341532

Kuo, Yu-Hung; Abdullaev, Iskandar F.; Hyzinski-García, María C.; Mongin, Alexander A.

2014-01-01

318

Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons  

PubMed Central

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence. PMID:25030907

Szafranski, Karol; Fritsch, Claudia; Schumann, Frank; Siebel, Lisa; Sinha, Rileen; Hampe, Jochen; Hiller, Michael; Englert, Christoph; Huse, Klaus; Platzer, Matthias

2014-01-01

319

Identification of differential splicing genes in gliomas using exon expression profiling  

PubMed Central

Diffuse gliomas are the most common type of malignant primary brain tumor, and their initiation and/or progression are often associated with alternative splicing. They produce an enormous economic burden on society and greatly impair the quality of life of those affected. The aim of the current study was to explore the differentially expressed genes (DEGs) observed in glioblastoma (GBM) and oligodendroglioma (OD) at the splicing level, and to analyze their functions in order to identify the underlying molecular mechanisms of gliomas. The exon-level expression profile data GSE9385 was downloaded from the Gene Expression Omnibus database, and included 26 GBM samples, 22 OD samples and 6 control brain samples. The differentially expressed exon-level probes were analyzed using the microarray detection of alternative splicing algorithm combined with the splicing index method, and the corresponding DEGs were identified. Next, a Gene Ontology enrichment analysis of the DEGs was performed. Additionally, the protein-protein interaction (PPI) networks were constructed based on the depth-first search algorithm. A total of 300 DEGs were identified to be shared by GBM and OD, including 97 upregulated and 203 downregulated DEGs. Furthermore, screening with a defined threshold identified 6 genes that were highly expressed in GBM, including AFF2, CACNA2D3 and ARPP21, while the 6 highly expressed genes in OD notably included CNTN2. The TP53 and HIST1H3A genes were the hub nodes in the PPI network of DEGs from GBM, while CNTN2 was linked to the highest degree in the OD PPI network. The present study provides a comprehensive bioinformatics analysis of DEGs in GBM and OD, which may provide a basis for understanding the initiation and/or progression of glioma development. PMID:25351872

YU, FENG; FU, WEI-MING

2015-01-01

320

Co-option of the piRNA pathway for germline-specific alternative splicing of C. elegans TOR.  

PubMed

Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3' splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3' splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a "nonself" intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations. PMID:25220461

Barberán-Soler, Sergio; Fontrodona, Laura; Ribó, Anna; Lamm, Ayelet T; Iannone, Camilla; Cerón, Julián; Lehner, Ben; Valcárcel, Juan

2014-09-25

321

SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.  

PubMed

Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, SpliceVista enables visualization, detection, and differential quantification of protein splice variants that are often missed in current proteomics pipelines. PMID:24692640

Zhu, Yafeng; Hultin-Rosenberg, Lina; Forshed, Jenny; Branca, Rui M M; Orre, Lukas M; Lehtiö, Janne

2014-06-01

322

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes  

PubMed Central

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution. PMID:23874930

Pope, Welkin H.; Jacobs-Sera, Deborah; Best, Aaron A.; Broussard, Gregory W.; Connerly, Pamela L.; Dedrick, Rebekah M.; Kremer, Timothy A.; Offner, Susan; Ogiefo, Amenawon H.; Pizzorno, Marie C.; Rockenbach, Kate; Russell, Daniel A.; Stowe, Emily L.; Stukey, Joseph; Thibault, Sarah A.; Conway, James F.; Hendrix, Roger W.; Hatfull, Graham F.

2013-01-01

323

Isolating and characterizing three alternatively spliced mu opioid receptor variants: mMOR-1A, mMOR-1O and mMOR-1P  

PubMed Central

Extensive alternative pre-mRNA splicing of the mu opioid receptor gene, OPRM1, has demonstrated an array of splice variants in mouse, rat and human. Three classes of splice variants have been identified: full length 7 transmembrane (TM) domain variants with C-terminal splicing, truncated 6TM variants and single TM variants. The current studies isolates and characterizes an additional three full length C-terminal splice variants generated from the mouse OPRM1 gene: mMOR-1A, mMOR-1O and mMOR-1P. Using RT-qPCR, we demonstrated differential expression of these variants' mRNAs among selected brain regions, supporting region-specific alternative splicing. When expressed in Chinese Hamster Ovary cells, all the variants displayed high mu binding affinity and selectivity with subtle differences in the affinities toward some agonists. [35S]?GTP binding assays revealed marked differences in agonist-induced G protein activation in both potency and efficacy among the variants. Together with the previous studies of mu agonist-induced phosphorylation and internalization in several carboxyl terminal splice variants, the current studies further suggest the existence of biased signaling of various agonists within each individual variant and/or among different variants. PMID:24375714

Xu, Jin; Xu, Mingming; Bolan, Elizabeth; Gilbert, Annie-Kim; Pasternak, Gavril W.; Pan, Ying-Xian

2014-01-01

324

Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria  

PubMed Central

Mitochondrial succinate dehydrogenase (SDH) is localized to the inner mitochondrial membrane and is responsible for the redox of succinic acid. SDH is a tetrameric iron-sulfur flavoprotein of the tricarboxylic acid cycle and respiratory chain. The SDH complex, subunit C (SDHC) transcript has deletion-type alternative splicing sites. Generally, alternative splicing produces variant proteins and expression patterns, as products of different genes. In certain cases, specific alternative splicing variants (ASVs) have been associated with human disease. Due to a frameshift mutation causing loss of the heme binding region, the SDHC ?5 isoform (lacking exon 5) exhibits no SDHC activity. To investigate whether the SDHC splicing variants can function as dominant-negative inhibitors, SDHC ASVs were overexpressed in HCT-15 human colorectal cancer cells. Using real-time reverse transcription-polymerase chain reaction, a dominant-negative effect of the ?5 isoform on SDHC mRNA was shown. In addition, ?5 overexpression increased the levels of reactive oxygen species. Furthermore, in the ?5 isoform-overexpressing cells, SDH activity was reduced. SDHC activation is a significant event during the electron transport chain, and the function of the SDHC ?5 variant may be significant for the differentiation of tumor cells. PMID:25435987

SATOH, NANA; YOKOYAMA, CHIKAKO; ITAMURA, NORIAKI; MIYAJIMA-NAKANO, YOSHIHARU; HISATOMI, HISASHI

2015-01-01

325

Transformation of eEF1B? into heat-shock response transcription factor by alternative splicing  

Microsoft Academic Search

Protein translation factors have crucial roles in a variety of stress responses. Here, we show that eukaryotic elongation factor 1B? (eEF1B?) changes its structure and function from a translation factor into a heat-shock response transcription factor by alternative splicing. The long isoform of eEF1B? (eEF1B?L) is localized in the nucleus and induces heat-shock element (HSE)-containing genes in cooperation with heat-shock

Taku Kaitsuka; Kazuhito Tomizawa; Masayuki Matsushita

2011-01-01

326

Identification of rare alternative splicing events in MS/MS data reveals a significant fraction of alternative translation initiation sites  

PubMed Central

Integration of transcriptome data is a crucial step for the identification of rare protein variants in mass-spectrometry (MS) data with important consequences for all branches of biotechnology research. Here, we used Splooce, a database of splicing variants recently developed by us, to search MS data derived from a variety of human tumor cell lines. More than 800 new protein variants were identified whose corresponding MS spectra were specific to protein entries from Splooce. Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events. Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites. Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences. To make the dataset generated here available to the community in a more effective form, the Splooce portal (http://www.bioinformatics-brazil.org/splooce) was modified to report the alternative splicing events supported by MS data. PMID:25405079

Kroll, José E.; de Souza, Sandro J.

2014-01-01

327

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs  

Microsoft Academic Search

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat\\/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were

MICHAEL BELSHAN; GREGORY S. PARK; PATRICIA BILODEAU; C. MARTIN STOLTZFUS; S. Carpenter

2000-01-01

328

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.  

PubMed

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases. PMID:24988226

Mora, Mireia; Hanzu, Felicia A; Pradas-Juni, Marta; Aranda, Gloria B; Halperin, Irene; Puig-Domingo, Manuel; Aguiló, Sira; Fernández-Rebollo, Eduardo

2014-01-01

329

New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1  

PubMed Central

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases. PMID:24988226

Mora, Mireia; Hanzu, Felicia A.; Pradas-Juni, Marta; Aranda, Gloria B.; Halperin, Irene; Puig-Domingo, Manuel; Aguiló, Sira; Fernández-Rebollo, Eduardo

2014-01-01

330

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression  

PubMed Central

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

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

2014-01-01

331

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators.  

PubMed

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level. PMID:22203037

Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Hervé; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis; Chabot, Benoit

2012-03-01

332

Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators  

PubMed Central

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-xS splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level. PMID:22203037

Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Hervé; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis

2012-01-01

333

Regulation of alternative splicing of tau exon 10 by 9G8 and Dyrk1A  

PubMed Central

Adult human brain expresses six isoforms of tau protein as a result of alternative splicing. Alternative splicing of exon 10 (E10) leads to tau isoforms containing either three (3R) or four (4R) microtubule-binding repeats. Imbalance in the 3R-tau/4R-tau ratio causes neurofibrillary degeneration and dementia. Here, we demonstrated that the dual-specificity tyrosine phosphorylation–regulated kinase 1A (Dyrk1A) interacted with the splicing factor 9G8 and phosphorylated it at several serine residues. Dyrk1A itself promoted tau E10 inclusion, whereas 9G8 inhibited E10 inclusion, and these actions were variable depending on the cell types. Co-expression of Dyrk1A and 9G8 led to their translocation from the nucleus to the cytoplasm and suppressed their ability to regulate tau exon 10 splicing. This action is probably due to their interaction-induced translocation from the nucleus, where the regulation of tau E10 splicing occurs, to the cytoplasm. These findings provide novel insights into the molecular mechanism of the regulation of tau E10 splicing and further our understanding of the neurodegeneration caused by dysregulation of tau E10 splicing. PMID:21215488

Ding, Shaohong; Shi, Jianhua; Qian, Wei; Iqbal, Khalid; Grundke-Iqbal, Inge; Gong, Cheng-Xin; Liu, Fei

2010-01-01

334

Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.  

PubMed

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins. PMID:25636034

Biasiotto, Roberta; Akusjärvi, Göran

2015-01-01

335

Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene.  

PubMed

Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype. PMID:15968391

Ding, Qiulan; Wu, Wenman; Fu, Qihua; Wang, Xuefeng; Hu, Yiqun; Wang, Hongli; Wang, Zhenyi

2005-06-01

336

A homolog of splicing factor SF1 is essential for development and is involved in the alternative splicing of pre-mRNA in Arabidopsis thaliana.  

PubMed

During initial spliceosome assembly, SF1 binds to intron branch points and interacts with U2 snRNP auxiliary factor 65 (U2AF65). Here, we present evidence indicating that AtSF1, the Arabidopsis SF1 homolog, interacts with AtU2AF65a and AtU2AF65b, the Arabidopsis U2AF65 homologs. A mutant allele of AtSF1 (At5g51300) that contains a T-DNA insertion conferred pleiotropic developmental defects, including early flowering and abnormal sensitivity to abscisic acid. An AtSF1 promoter-driven GUS reporter assay showed that AtSF1 promoter activity was temporally and spatially altered, and that full AtSF1 promoter activity required a significant proportion of the coding region. DNA chip analyses showed that only a small proportion of the transcriptome was altered by more than twofold in either direction in the AtSF1 mutant. Expression of the mRNAs of many heat shock proteins was more than fourfold higher in the mutant strain; these mRNAs were among those whose expression was increased most in the mutant strain. An RT-PCR assay revealed an altered alternative splicing pattern for heat shock transcription factor HsfA2 (At2g26150) in the mutant; this altered splicing is probably responsible for the increased expression of the target genes induced by HsfA2. Altered alternative splicing patterns were also detected for the transcripts of other genes in the mutant strain. These results suggest that AtSF1 has functional similarities to its yeast and metazoan counterparts. PMID:24580679

Jang, Yun Hee; Park, Hyo-Young; Lee, Keh Chien; Thu, May Phyo; Kim, Soon-Kap; Suh, Mi Chung; Kang, Hunseung; Kim, Jeong-Kook

2014-05-01

337

Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A  

PubMed Central

Abnormal alternative splicing of tau exon 10 results in imbalance of 3R-tau and 4R-tau expression, which is sufficient to cause neurofibrillary degeneration. Splicing factor SC35, a member of the superfamily of the serine/arginine-rich (SR) proteins, promotes tau exon 10 inclusion. The molecular mechanism by which SC35 participates in tau exon 10 splicing remains elusive. In the present study, we found that tau pre-mRNA was coprecipitated by SC35 tagged with HA. Mutation of the SC35-like exonic splicing enhancer located at exon 10 of tau affected both the binding of SC35 to tau pre-mRNA and promotion of tau exon 10 inclusion, suggesting that SC35 acts on the SC35-like exonic splicing enhancer to promote tau exon 10 inclusion. Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) phosphorylated SC35 in vitro and interacted with it in cultured cells. Overexpression of Dyrk1A suppressed SC35?s ability to promote tau exon 10 inclusion. Downregulation of Dyrk1A promoted 4R-tau expression. Therefore, upregulation of Dyrk1A in Down syndrome brain or Alzheimer’s brain may cause dysregulation of tau exon 10 splicing through SC35, and probably together with other splicing factors, leading to the imbalance in 3R-tau and 4R-tau expression, which may initiate or accelerate tau pathology and cause neurofibrillary degeneration in the diseases. PMID:21470964

Qian, Wei; Liang, Hongwei; Shi, Jianhua; Jin, Nana; Grundke-Iqbal, Inge; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

2011-01-01

338

Tissue-specific effects of genetic and epigenetic variation on gene regulation and splicing.  

PubMed

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole. PMID:25634236

Gutierrez-Arcelus, Maria; Ongen, Halit; Lappalainen, Tuuli; Montgomery, Stephen B; Buil, Alfonso; Yurovsky, Alisa; Bryois, Julien; Padioleau, Ismael; Romano, Luciana; Planchon, Alexandra; Falconnet, Emilie; Bielser, Deborah; Gagnebin, Maryline; Giger, Thomas; Borel, Christelle; Letourneau, Audrey; Makrythanasis, Periklis; Guipponi, Michel; Gehrig, Corinne; Antonarakis, Stylianos E; Dermitzakis, Emmanouil T

2015-02-01

339

Tissue-Specific Effects of Genetic and Epigenetic Variation on Gene Regulation and Splicing  

PubMed Central

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole. PMID:25634236

Buil, Alfonso; Yurovsky, Alisa; Bryois, Julien; Padioleau, Ismael; Romano, Luciana; Planchon, Alexandra; Falconnet, Emilie; Bielser, Deborah; Gagnebin, Maryline; Giger, Thomas; Borel, Christelle; Letourneau, Audrey; Makrythanasis, Periklis; Guipponi, Michel; Gehrig, Corinne; Antonarakis, Stylianos E.; Dermitzakis, Emmanouil T.

2015-01-01

340

Splicing in disease: disruption of the splicing code and the decoding machinery  

Microsoft Academic Search

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases,

Guey-Shin Wang; Thomas A. Cooper

2007-01-01

341

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing*  

PubMed Central

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5? splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection. PMID:21622561

Guan, Wuxiang; Huang, Qinfeng; Cheng, Fang; Qiu, Jianming

2011-01-01

342

Intron 7 conserved sequence elements regulate the splicing of the SMN genes  

PubMed Central

Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. In humans there are two nearly identical SMN genes, SMN1 and SMN2. The SMN2 gene generates a truncated protein, due to a C to T nucleotide alteration in exon 7, which leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and the trans-factors that bind them. Here we identify conserved intronic sequences in the SMN genes. Five regions were examined due to conservation and their proximity to exons 6 through 8. Using mutagenesis two conserved elements located in intron 7 of the SMN genes that affect exon 7 splicing have been identified. Additional analysis of one of these regions showed decreased inclusion of exon 7 in SMN transcripts when deletions or mutations were introduced. Furthermore, multimerization of this conserved region was capable of restoring correct SMN splicing. Together these results describe a novel intronic splicing enhancer sequence located in the final intron of the SMN genes. This discovery provides insight into the splicing of the SMN genes using conserved intonic sequence as a tool to uncover regions of importance in pre-messenger RNA splicing. A better understanding of the way SMN pre-mRNA is spliced can lead to the development of new therapies. PMID:19701774

Gladman, Jordan T.; Chandler, Dawn S.

2009-01-01

343

Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.  

PubMed

Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs) have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA), the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4), which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%). Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD), in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms are missing the catalytic triad that is crucial for protease activity. PMID:23874499

Koumandou, Vassiliki Lila; Scorilas, Andreas

2013-01-01

344

Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tRNA synthetase includes alternative splicing.  

PubMed

We have investigated the transcriptional control elements of the human interferon (IFN)-gamma-induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met48 in exon III. The predicted 48-kDa protein corresponds exactly to an IFN-gamma-inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a gamma-activated sequence (GAS) element. IFN-gamma inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements. PMID:7814400

Tolstrup, A B; Bejder, A; Fleckner, J; Justesen, J

1995-01-01

345

Regulation of neurexin 1? tertiary structure and ligand binding through alternative splicing  

PubMed Central

Summary Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a “splice insert signaling code”. In particular, neurexin 1? carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1?+SS#4 reveals dramatic rearrangements to the “hyper-variable surface”, the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop ?10-?11 into a helix rearranging the binding site for neuroligins, and 3) rearranges the Ca2+-binding site required for ligand binding increasing its affinity. Our structures reveal the mechanism by which neurexin 1? isoforms acquire neuroligin splice isoform selectivity. PMID:18334217

Shen, Kaiser C.; Kuczynska, Dorota A.; Wu, Irene J.; Murray, Beverly H.; Sheckler, Lauren R.; Rudenko, Gabby

2008-01-01

346

Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB.  

PubMed

Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher Wj

2015-03-01

347

Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing  

PubMed Central

Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3? splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events. PMID:17804646

Alberstein, Moti; Amit, Maayan; Vaknin, Keren; O'Donnell, Amanda; Farhy, Chen; Lerenthal, Yaniv; Shomron, Noam; Shaham, Ohad; Sharrocks, Andrew D.; Ashery-Padan, Ruth; Ast, Gil

2007-01-01

348

Intron Retention: A Common Splicing Event within the Human Kallikrein Gene Family  

Microsoft Academic Search

Background: All human kallikrein (KLK) genes have at least one splice variant, some of which possess clinical utility in cancer diagnostics\\/prognostics. Given that in- trons <100 bp in length are retained in 95% of human genes and that splice variants of KLK3 and KLK4 retain intron III, we hypothesized that other proteins in this family, with a small intron III,

Iacovos P. Michael; Lisa Kurlender; Nader Memari; George M. Yousef; Daisy Du; Linda Grass; Carsten Stephan; Klaus Jung; Eleftherios P. Diamandis

349

TILLING mutants of durum wheat result in a high amylose phenotype and provide information on alternative splicing mechanisms.  

PubMed

The amylose/amylopectin ratio has a major influence over the properties of starch and determines its optimal end use. Here, high amylose durum wheat has been bred by combining knock down alleles at the two homoelogous genes encoding starch branching enzyme IIa (SBEIIa-A and SBEIIa-B). The complete silencing of these genes had a number of pleiotropic effects on starch synthesis: it affected the transcriptional activity of SBEIIb, ISA1 (starch debranching enzyme) and all of the genes encoding starch synthases (SSI, SSIIa, SSIII and GBSSI). The starch produced by grain of the double SBEIIa mutants was high in amylose (up to ?1.95 fold that of the wild type) and contained up to about eight fold more resistant starch. A single nucleotide polymorphism adjacent to the splice site at the end of exon 10 of the G364E mutant copies of both SBEIIa-A and SBEIIa-B resulted in the loss of a conserved exonic splicing silencer element. Its starch was similar to that of the SBEIIa double mutant. G364E SBEIIa pre-mRNA was incorrectly processed, resulting in the formation of alternative, but non-functional splicing products. PMID:25711820

Sestili, Francesco; Palombieri, Samuela; Botticella, Ermelinda; Mantovani, Paola; Bovina, Riccardo; Lafiandra, Domenico

2015-04-01

350

Differential expression of 24,426 human alternative splicing events and predicted cis-regulation in 48 tissues and cell lines  

PubMed Central

Alternative pre–messenger RNA splicing impacts development, physiology, and disease, but its regulation in humans is not well understood, partially due to the limited scale to which the expression of specific splicing events has been measured. We generated the first genome-scale expression compendium of human alternative splicing events using custom whole-transcript microarrays monitoring expression of 24,426 alternative splicing events in 48 diverse human samples. Over 11,700 genes and 9,500 splicing events were differentially expressed, providing a rich resource for studying splicing regulation. An unbiased, systematic screen of 21,760 4-mer to 7-mer words for cis-regulatory motifs identified 143 RNA 'words' enriched near regulated cassette exons, including six clusters of motifs represented by UCUCU, UGCAUG, UGCU, UGUGU, UUUU, and AGGG, which map to trans-acting regulators PTB, Fox, Muscleblind, CELF/CUG-BP, TIA-1, and hnRNP F/H, respectively. Each cluster showed a distinct pattern of genomic location and tissue specificity. For example, UCUCU occurs 110 to 35 nucleotides preceding cassette exons upregulated in brain and striated muscle but depleted in other tissues. UCUCU and UGCAUG appear to have similar function but independent action, occurring 5' and 3', respectively, of 33% of the cassette exons upregulated in skeletal muscle but co-occurring for only 2%. PMID:18978788

Castle, John C.; Zhang, Chaolin; Shah, Jyoti K.; Kulkarni, Amit V.; Cooper, Thomas A.; Johnson, Jason M.

2011-01-01

351

Alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via phosphorylation of SRp30a  

PubMed Central

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a. PMID:21045158

Shultz, Jacqueline C.; Goehe, Rachel W.; Wijesinghe, D. Shanaka; Murudkar, Charuta; Hawkins, Amy J.; Shay, Jerry W.; Minna, John D.; Chalfant, Charles E.

2010-01-01

352

Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees  

PubMed Central

Background Previous whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing. Results We confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites. Conclusions Cytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns. PMID:24079845

2013-01-01

353

Colony Forming Unit Endothelial Cells Do not Exhibit Telomerase Alternative Splicing Variants and Activity  

PubMed Central

Introduction: Endothelial progenitor colony forming unit-endothelial cells (CFU-EC) were first believed to be the progenitors of endothelial cells, named endothelial progenitor cells. Further studies revealed that they are monocytes regulating vasculogenesis. The main hindrance of these cells for therapeutic purposes is their low frequency and limited replicative potentials. This study was undertaken to determine telomerase activity and alternative splicing variants in CFU-EC as a potential cause of limited replicative capacity in these cells. Methods: CFU-EC were isolated from peripheral blood using a standard cell culture assay. Colonies were detached mechanically and alternative splicing variant mRNA were evaluated using real-time PCR. Telomerase enzyme activity was assessed using telomerase repeat amplification protocol. The same procedures were done on the cancer cell line Calu6 as the positive control. Results: The cultured peripheral blood mononuclear cells formed colonies with spindle-shaped monocytic cells sprouted from the clusters. These morphological characteristics fulfill the definition of CFU-EC. Telomere length amplification protocol assay revealed no telomerase activity and real-time PCR showed no expression of telomerase enzyme mRNA in CFU-EC. Both parameters were significantly higher in the cancer cell line Calu6 taken as the positive control. Conclusion: The absence of telomerase activity in the CFU-EC is a result of pre-transcriptional regulation of gene expression rather than other mechanisms for controlling telomerase activity such as post-transcriptional modifications. This finding can explain the limited proliferative activity of CFU-EC cells. We propose that absence of telomerase activity in CFU-EC can be attributable to their more mature monocytic nature that needs further investigations. PMID:23748893

Attar, Armin; Khosravi Maharlooi, Mohsen; Khoshkhou, Sara; Hosseini, Ahmad; Jaberipour, Mansoureh; Dehghan, Arman; Monabati, Ahmad

2013-01-01

354

An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.  

PubMed Central

We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances. PMID:10493923

Lin, Z; Wang, G; Demello, D E; Floros, J

1999-01-01

355

Alternative splicing contributes to the coordinated regulation of ferritin subunit levels in Bactrocera dorsalis (Hendel).  

PubMed

A constant ratio of ferritin heavy chain homolog (HCH) and light chain homolog (LCH) subunits seems to be required to compose the ferritin heteropolymer protein in insects. However, the mechanism by which insect LCH genes regulate protein levels remains unclear. We report that alternative promoters and alternative splicing contribute to maintaining a constant ratio of the two subunits, BdFer1HCH and BdFer2LCH (ferritin 1 HCH and ferritin 2 LCH), in Bactrocera dorsalis, a notorious quarantine pest. The genes BdFer1HCH and BdFer2LCH were identified with a series of potential transcription factor binding sites and were shown to be clustered within the genome in a "head to head" fashion. Thus, we unearthed a potential post-transcriptional mechanism to regulate the levels of LCH subunits, and confirmed that the expressions of BdFer1HCH and BdFer2LCH were induced by 20-hydroecdysone, iron overload, and immune challenge. PMID:24763285

Jiang, Xuan-Zhao; Cong, Lin; Niu, Jin-Zhi; Dou, Wei; Wang, Jin-Jun

2014-01-01

356

Alternative splicing contributes to the coordinated regulation of ferritin subunit levels in Bactrocera dorsalis (Hendel)  

PubMed Central

A constant ratio of ferritin heavy chain homolog (HCH) and light chain homolog (LCH) subunits seems to be required to compose the ferritin heteropolymer protein in insects. However, the mechanism by which insect LCH genes regulate protein levels remains unclear. We report that alternative promoters and alternative splicing contribute to maintaining a constant ratio of the two subunits, BdFer1HCH and BdFer2LCH (ferritin 1 HCH and ferritin 2 LCH), in Bactrocera dorsalis, a notorious quarantine pest. The genes BdFer1HCH and BdFer2LCH were identified with a series of potential transcription factor binding sites and were shown to be clustered within the genome in a “head to head” fashion. Thus, we unearthed a potential post-transcriptional mechanism to regulate the levels of LCH subunits, and confirmed that the expressions of BdFer1HCH and BdFer2LCH were induced by 20-hydroecdysone, iron overload, and immune challenge. PMID:24763285

Jiang, Xuan-Zhao; Cong, Lin; Niu, Jin-Zhi; Dou, Wei; Wang, Jin-Jun

2014-01-01

357

A novel extracellular domain variant of the human integrin alpha 7 subunit generated by alternative intron splicing.  

PubMed

The integrin alpha 7 beta 1 laminin receptor, which is expressed on replicating myoblasts, and upregulated during myogenic differentiation, is involved in cell adhesion and communication between muscle cells and the extracellular matrix. It is a major cell-surface substrate in skeletal muscle cells for the cell-surface, argininespecific, ADP-ribosyltransferase. Both the extracellular and cytoplasmic domains of the mouse alpha 7 subunit undergo alternative splicing during development, generating differentially expressed variants with presumably unique ligand-binding and signalling properties. Here human cDNA clones isolated from a fetal heart lambda gt10 cDNA library encoded the complete sequence of the alpha 7 subunit and hybridised to a single major 4.4 kb alpha 7 subunit transcript abundantly expressed in human skeletal muscle, moderately expressed in heart, and weakly expressed in most other tissues. One clone out of four contained a novel 225-nucleotide in-frame deletion corresponding to 75 amino acids in the C-terminal region of the extracellular domain. The variant, whose expression appears to be tissue-specific, is created by alternative splicing at sites flanking an intron in the alpha 7 gene. A related mouse form was identified in P19 embryonal carcinoma cells. Deletion of the spliced region, which either contains or is in very close proximity to the major ADP-ribosylation site of the alpha 7 subunit, may serve to modulate the effects of ADP-ribosylation, or alternatively molecular associations, and receptor-ligand affinity. PMID:9473524

Leung, E; Lim, S P; Berg, R; Yang, Y; Ni, J; Wang, S X; Krissansen, G W

1998-02-01

358

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays  

Microsoft Academic Search

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for

Charles W. Sugnet; Karpagam Srinivasan; Tyson A. Clark; Georgeann O'Brien; Melissa S. Cline; Hui Wang; Alan Williams; David Kulp; John E. Blume; David Haussler; Manuel Ares

2006-01-01

359

Comprehensive analysis of alternative splicing in Digitalis purpurea by strand-specific RNA-Seq.  

PubMed

Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health. PMID:25167195

Wu, Bin; Suo, Fengmei; Lei, Wanjun; Gu, Lianfeng

2014-01-01

360

Comprehensive Analysis of Alternative Splicing in Digitalis purpurea by Strand-Specific RNA-Seq  

PubMed Central

Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health. PMID:25167195

Wu, Bin; Suo, Fengmei; Lei, Wanjun; Gu, Lianfeng

2014-01-01

361

The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa (black cottonwood) populations  

PubMed Central

Background Alternative splicing (AS) of genes is an efficient means of generating variation in protein structure and function. AS variation has been observed between tissues, cell types, and different treatments in non-woody plants such as Arabidopsis thaliana (Arabidopsis) and rice. However, little is known about AS patterns in wood-forming tissues and how much AS variation exists within plant populations. Results Here we used high-throughput RNA sequencing to analyze the Populus trichocarpa (P. trichocarpa) xylem transcriptome in 20 individuals from different populations across much of its range in western North America. Deep transcriptome sequencing and mapping of reads to the P. trichocarpa reference genome identified a suite of xylem-expressed genes common to all accessions. Our analysis suggests that at least 36% of the xylem-expressed genes in P. trichocarpa are alternatively spliced. Extensive AS was observed in cell-wall biosynthesis related genes such as glycosyl transferases and C2H2 transcription factors. 27902 AS events were documented and most of these events were not conserved across individuals. Differences in isoform-specific read densities indicated that 7% and 13% of AS events showed significant differences between individuals within geographically separated southern and northern populations, a level that is in general agreement with AS variation in human populations. Conclusions This genome-wide analysis of alternative splicing reveals high levels of AS in P. trichocarpa and extensive inter-individual AS variation. We provide the most comprehensive analysis of AS in P. trichocarpa to date, which will serve as a valuable resource for the plant community to study transcriptome complexity and AS regulation during wood formation. PMID:23718132

2013-01-01

362

The canine kallikrein-related peptidase 14: structural characterization, alternative splicing and differential expression in mammary cancer.  

PubMed

Human kallikrein-related peptidases (KLKs) represent a family of 15 serine proteases with diverse roles in many physiological and pathological processes, including carcinogenesis. In the dog, only two KLK genes are known; dKLK1 and canine arginine esterase. Recently, 12 other genes have been predicted using computational methods, but none of them has ever been experimentally validated in canine tissues. In this study we investigated the expression of Canis familiaris KLK14, (CANFA)KLK14, in normal and cancerous mammary tissues. First, it was demonstrated that the in-silico determined canine KLK14 mRNA (GenBank accession no: XM_541464) has been wrongfully predicted on its 5'-end (nucleotides 1-88). The (CANFA)KLK14 mRNA sequence presented here, has high homology to its human counterpart and exhibits all defining-KLK features. In addition to the classical form of the gene, five splice variants were also identified. The splicing events involved 5'-truncation or complete elimination of exon 4 and/or retention of intron I. All encoded protein products of the splice variants were predicted to be truncated and catalytically inactive. The classical form and variant 3 were almost ubiquitously expressed in both normal and neoplastic tissues. Variant 1 was predominantly detected in normal tissues. The classical form and variants 1 and 2 exhibited lower expression levels in tumor compared to normal tissues. Moreover, an Ile155Asn polymorphism was identified. This is the first report on the structural characterization, alternative splicing and tissue expression of canine KLK14 mRNA. These findings may form the basis for the establishment of comparative studies investigating KLK functions in health and disease using the dog as a model. PMID:19619623

Angelopoulou, Katerina; Prassas, Ioannis; Yousef, George M

2009-10-15

363

Targeting RNA Splicing for Disease Therapy  

PubMed Central

Splicing of pre-messenger RNA into mature messenger RNA is an essential step for expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics. PMID:23512601

Havens, Mallory A.; Duelli, Dominik M.

2013-01-01

364

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing  

PubMed Central

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18?496 isoforms, out of 19?008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons. PMID:23792425

Sun, Wei; You, Xintian; Gogol-Döring, Andreas; He, Haihuai; Kise, Yoshiaki; Sohn, Madlen; Chen, Tao; Klebes, Ansgar; Schmucker, Dietmar; Chen, Wei

2013-01-01

365

Rhythmic U2af26 alternative splicing controls PERIOD1 stability and the circadian clock in mice.  

PubMed

The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. Here we describe a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator TIMELESS. This new U2AF26 variant destabilizes PERIOD1 protein, and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions. PMID:24837677

Preußner, Marco; Wilhelmi, Ilka; Schultz, Astrid-Solveig; Finkernagel, Florian; Michel, Monika; Möröy, Tarik; Heyd, Florian

2014-05-22

366

Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides  

PubMed Central

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, that were isolated from Rhizopus oryzae as the first cellulase genes from a member of the subdivision Zygomycota. In this study, two cDNAs homologous to the rce1 gene, designated the mce1 and mce2 cDNAs, were cloned from Mucor circinelloides, a member of the subdivision Zygomycota. The mce1 cDNA encoded an endoglucanase (family 45 glycoside hydrolase) having one carbohydrate-binding module (CBM), designated MCE1, and the mce2 cDNA encoded the same endoglucanase having two tandem repeated CBMs, designated MCE2. The two cDNAs contained the same sequences but with a 147-bp insertion. The corresponding genomic mce gene consisted of four exons. The mce1 cDNA was created from exons 1, 3, and 4, and the mce2 cDNA was created from exons 1, 2, 3, and 4. These results indicate that the mce1 and mce2 cDNAs were created from one genomic mce gene by alternative splicing. MCE1 and MCE2, purified to apparent homogeneity from the culture supernatant of M. circinelloides, had molecular masses of 43 and 47 kDa, respectively. The carboxymethyl cellulase specific activity of MCE2 was almost the same as that of MCE1, whereas the Avicelase specific activity of MCE2 was two times higher than that of MCE1. Furthermore, MCE2, whose two tandem CBMs might be more effective for degradation of crystalline cellulose than one CBM, was secreted only at an early culture stage when crystalline cellulose was abundant. PMID:15838031

Baba, Yuko; Shimonaka, Atsushi; Koga, Jinichiro; Kubota, Hidetoshi; Kono, Toshiaki

2005-01-01

367