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1

Noncanonical and canonical splice sites: a novel mutation at the rare noncanonical splice-donor cut site (IVS4+1A>G) of SEDL causes variable splicing isoforms in X-linked spondyloepiphyseal dysplasia tarda  

Microsoft Academic Search

X-linked spondyloepiphyseal dysplasia tarda can be caused by mutations in the SEDL gene. This study describes an interesting novel mutation (IVS4+1A>G) located exactly at the rare noncanonical AT–AC consensus splicing donor point of SEDL, which regained the canonical GT–AG consensus splicing junction in addition to several other rarer noncanonical splice patterns. The mutation activated several cryptic splice sites and generated

Feng Xiong; Jianjun Gao; Jun Li; Yun Liu; Guoyin Feng; Wenli Fang; Hongfen Chang; Jiang Xie; Haitao Zheng; Tingyu Li; Lin He; J Gao; T Li; L He

2009-01-01

2

Noncanonical and canonical splice sites: a novel mutation at the rare noncanonical splice-donor cut site (IVS4+1A>G) of SEDL causes variable splicing isoforms in X-linked spondyloepiphyseal dysplasia tarda.  

PubMed

X-linked spondyloepiphyseal dysplasia tarda can be caused by mutations in the SEDL gene. This study describes an interesting novel mutation (IVS4+1A>G) located exactly at the rare noncanonical AT-AC consensus splicing donor point of SEDL, which regained the canonical GT-AG consensus splicing junction in addition to several other rarer noncanonical splice patterns. The mutation activated several cryptic splice sites and generated the production of seven erroneous splicing isoforms, which we confirmed by sequencing of RT-PCR products and resequencing of cDNA clones. All the practical splice donors/acceptors were further assessed using FSPLICE 1.0 and SPL(M) Platforms to predict potential splice sites in genomic DNA. Subsequently, the expression levels of SEDL among the affected patients, carriers and controls were estimated using real-time quantitative PCR. Expression analyses showed that the expression levels of SEDL in both patients and carriers were decreased. Taken together, these results illustrated how disruption of the AT donor site in a rare AT-AC intron, leading to a canonical GT donor site, resulted in a multitude of aberrant transcripts, thus impairing exon definition. The unexpected splicing patterns resulting from the special mutation provide additional challenges and opportunities for understanding splicing mechanisms and specificity. PMID:19002213

Xiong, Feng; Gao, Jianjun; Li, Jun; Liu, Yun; Feng, Guoyin; Fang, Wenli; Chang, Hongfen; Xie, Jiang; Zheng, Haitao; Li, Tingyu; He, Lin

2009-04-01

3

Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties  

Microsoft Academic Search

There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it

Ya-li ZHANG; Ming-hui XU; Ya-wen ZENG; Chun-xin YAO; Shan-na CHEN

2007-01-01

4

Genome-wide activation of latent donor splice sites in stress and disease  

PubMed Central

Sequences that conform to the 5? splice site (5?SS) consensus are highly abundant in mammalian introns. Most of these sequences are preceded by at least one in-frame stop codon; thus, their use for splicing would result in pre-maturely terminated aberrant mRNAs. In normally grown cells, such intronic 5?SSs appear not to be selected for splicing. However, under heat shock conditions aberrant splicing involving such latent 5?SSs occurred in a number of specific gene transcripts. Using a splicing-sensitive microarray, we show here that stress-induced (e.g. heat shock) activation of latent splicing is widespread across the human transcriptome, thus highlighting the possibility that latent splicing may underlie certain diseases. Consistent with this notion, our analyses of data from the Gene Expression Omnibus (GEO) revealed widespread activation of latent splicing in cells grown under hypoxia and in certain cancers such as breast cancer and gliomas. These changes were found in thousands of transcripts representing a wide variety of functional groups; among them are genes involved in cell proliferation and differentiation. The GEO analysis also revealed a set of gene transcripts in oligodendroglioma, in which the level of activation of latent splicing increased with the severity of the disease. PMID:23002147

Nevo, Yuval; Kamhi, Eyal; Jacob-Hirsch, Jasmine; Amariglio, Ninette; Rechavi, Gideon; Sperling, Joseph; Sperling, Ruth

2012-01-01

5

A novel splice donor site at nt 1534 is required for long-term maintenance of HPV31 genomes  

SciTech Connect

Human papillomaviruses (HPV) are small double-stranded DNA viruses that replicate as low copy number nuclear plasmids during the persistent phase. HPV only possess nine open reading frames but extend their coding capabilities by alternative RNA splicing. We have identified in cell lines with replicating HPV31 genomes viral transcripts that connect the novel splice donor (SD) sites at nt 1426 and 1534 within the E1 replication gene to known splice acceptors at nt 3295 or 3332 within the E2/E4 region. These transcripts are polyadenylated and are present at low amounts in the non-productive and productive phase of the viral life cycle. Mutation of the novel splice sites in the context of HPV31 genomes revealed that the inactivation of SD1534 had only minor effects in short-term replication assays but displayed a low copy number phenotype in long-term cultures which might be due to the expression of alternative E1 circumflex E4 or yet unknown viral proteins. This suggests a regulatory role for minor splice sites within E1 for papillomavirus replication.

Poppelreuther, Sven; Iftner, Thomas [Sektion Experimentelle Virologie, Institut fuer Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universitaetsklinikum Tuebingen, Elfriede-Aulhorn-Str. 6, 72076 Tuebingen (Germany); Stubenrauch, Frank [Sektion Experimentelle Virologie, Institut fuer Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universitaetsklinikum Tuebingen, Elfriede-Aulhorn-Str. 6, 72076 Tuebingen (Germany)], E-mail: frank.stubenrauch@med.uni-tuebingen.de

2008-01-05

6

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

SciTech Connect

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

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

1994-09-01

7

Use of a cryptic splice donor site in the chloramphenicol acetyltransferase (CAT)-SV40 small-t antigen cassette generates alternative transcripts in transgenic rats.  

PubMed

The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9 kb), we identified an abundant 1.5 kb transcript which derives from an alternative splicing event that utilises a cryptic splice donor site located within the CAT gene. The native CAT open reading frame (ORF) is lost in the 1.5 kb transcript, and a western analysis has shown that protein deriving from an aberrant open reading frame is not expressed at detectable levels. PMID:10853270

Burke, Z D; Wells, T; Carter, D A; Baler, R

2000-02-01

8

Escape variants of the XPR1 gammaretrovirus receptor are rare due to reliance on a splice donor site and a short hypervariable loop.  

PubMed

Entry determinants in the XPR1 receptor for the xenotropic/polytropic mouse leukemia viruses (XP-MLVs) lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor and is evolutionarily conserved in vertebrate XPR1 genes; 2 of the 3 rare replacement mutations at this site destroy this receptor determinant. The 13 residue ECL4 is hypervariable, and replacement mutations carrying an intact ECL3 site alter but do not abolish receptor activity, including replacement of the entire loop with that of a jellyfish (Cnidaria) XPR1. Because ECL4 deletions are found in all X-MLV-infected Mus subspecies, we deleted each ECL4 residue to determine if deletion-associated restriction is residue-specific or is effected by loop size. All deletions influence receptor function, although different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations severely limits the likelihood of host escape mutations. PMID:25151060

Lu, Xiaoyu; Martin, Carrie; Bouchard, Christelle; Kozak, Christine A

2014-11-01

9

Our favourite alternative splice site.  

PubMed

Alternative splicing is a widespread mechanism in mammals that generates several mRNAs from one gene, thereby creating genetic diversity of the genome. Variant splice patterns are often specific to different stages of development or particular tissues, and alternative splicing defects are being more frequently detected in genetic diseases and cancers. The increasingly important role of alternative splicing in the function and the regulation of cellular process makes it critical to have an easy-to-use data repository for the biological and medical research communities. We have compared web resources that give access to information on alternatively spliced genes, and the FAST DB (Friendly Alternative Splicing and Transcripts DataBase) site came out as our favourite. PMID:16611169

Lerivray, Hubert; Méreau, Agnès; Osborne, H Beverley

2006-05-01

10

Complete androgen insensitivity caused by a splice donor site mutation in intron 2 of the human androgen receptor gene resulting in an exon 2-lacking transcript with premature stop-codon and reduced expression  

Microsoft Academic Search

Various mutations within the human androgen receptor gene have been documented to cause defective sexual differentiation in karyotypic male individuals. In this study, we report a previously undescribed point mutation at the donor splice-site of the second intron of the androgen receptor gene in a patient with a completely female phenotype. The sequence alteration was detected by single-strand-conformation-analysis-PCR and genomic

Olaf José-Carlos Hellwinkel; Kerstin Bull; Paul-Martin Holterhus; Nicole Homburg; Dagmar Struve; Olaf Hiort

1999-01-01

11

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype  

SciTech Connect

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.

Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X. [Cancer Research Institute, Barcelona (Spain); Doerk, T.; Will, K. [Medizinische Hochschule Hannover (Germany); Fonknechten, N. [Institut Cochin de Genetique Moleculaire, Paris (France)

1995-03-01

12

iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition  

PubMed Central

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called “iSS-PseDNC” was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called “pseudo dinucleotide composition” (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing. PMID:24967386

Feng, Peng-Mian; Chou, Kuo-Chen

2014-01-01

13

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

PubMed Central

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

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

2014-01-01

14

Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.  

PubMed Central

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs. Images PMID:8417357

Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W

1993-01-01

15

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.  

PubMed Central

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon. PMID:7623794

Humphrey, M B; Bryan, J; Cooper, T A; Berget, S M

1995-01-01

16

Synonymous polymorphisms at splicing regulatory sites are associated with CpGs in neurodegenerative disease-related genes.  

PubMed

Neuronal plasticity is associated with alternative splicing and epigenetic modulation. Recent evidence reveals the association of cytosine methylation with alternative splicing and splicing regulatory mechanisms. Single nucleotide polymorphisms (SNPs) are generally less frequent in conserved coding regions and probably in splice sites, compared to non-coding regions. CpG polymorphisms in coding regions and splice sites and their association with splicing regulatory elements have not been investigated till presently. We currently analyzed the CpG variability in 28 genes (361 constitutive and 105 alternative exons and the corresponding splice sites) associated with neurodegenerative diseases (ND). CpG polymorphisms in the splice sites of these genes are particularly frequent when compared to those at AG sequences. Moreover, in both constitutive and alternative exons, polymorphisms in CpGs are more frequent than in AG, GT sequences. On the contrary, in the polypyrimidine acceptor sequence C/T conservation is prominent indicating that in this locus the sequence of cytosines and thymines is preserved. Bioinformatic analysis of the splicing-associated regulatory elements in these exons and splice sites reveals that 18 out of a total of 39 SNPs which could strongly affect splicing (>1.5 score difference) contain CpG sequences. Cytosines are considerably more frequent and variable than expected at the position preceding the GT splice donors, while sites of epigenetic modification are absent from acceptors. The high CpG frequency in polymorphic splicing-associated sites implicates the involvement of epigenetic mechanisms in splicing selection decisions regulated by these sites, and indicates the complexity of genetic studies involving these, tentatively critical, polymorphisms in ND. PMID:20077034

Karambataki, Maria; Malousi, Andigoni; Maglaveras, Nicos; Kouidou, Sofia

2010-09-01

17

Nursing care of donor site wounds.  

PubMed

The list of ideal donor site characteristics includes many items related to nursing care such as the ability of the dressing to minimize pain, permit patient mobility, and simplify postoperative care. Biobrane must adhere to the donor site wound bed and be allowed to dry without fluid accumulation. Coarse-mesh gauze wraps applied over the Biobrane in the operating room help maintain contact between Biobrane and the wound bed, protect the donor site from traumatic dislodgment in the early postoperative period, and serve to wick wound drainage in the first 24 hours. Twenty-four hours after surgery the nurse removes the outer dressing. The Biobrane is usually adherent to the wound, and the site is left open to dry. The primary goal of nursing care is to maintain sufficient airflow around the site. Nursing care of Biobrane-covered donor sites is uncomplicated but requires adherence to certain procedures to promote optimal donor site healing. PMID:7673321

Hansbrough, W

1995-01-01

18

Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3' Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF\\/SF2 through Activation of the Phosphatidylinositol 3Kinase\\/Akt Pathway  

Microsoft Academic Search

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation- specific manner: the late leader 5 splice site alternatively splices to a proximal 3 splice site (at nucleotide 3225) to express L2 or to a distal 3 splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF\\/SF2 (alternative splicing factor\\/splicing

Xuefeng Liu; Akila Mayeda; Mingfang Tao; Zhi-Ming Zheng

2003-01-01

19

Splicing proofreading at 5' splice sites by ATPase Prp28p.  

PubMed

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS. PMID:23462954

Yang, Fei; Wang, Xiu-Ye; Zhang, Zhi-Min; Pu, Jia; Fan, Yu-Jie; Zhou, Jiahai; Query, Charles C; Xu, Yong-Zhen

2013-04-01

20

Splicing proofreading at 5? splice sites by ATPase Prp28p  

PubMed Central

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5? splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5?SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5?SS. Prp28p is thought to facilitate the disruption of 5?SS–U1 snRNA pairing to allow for 5?SS–U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5?SS proofreading by Prp28p is dependent on competition with the stability of the 5?SS:U6 duplex, but not the 5?SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5?SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5?SS. PMID:23462954

Yang, Fei; Wang, Xiu-Ye; Zhang, Zhi-Min; Pu, Jia; Fan, Yu-Jie; Zhou, Jiahai; Query, Charles C.; Xu, Yong-Zhen

2013-01-01

21

A statistical approach for 5¿ splice site prediction using short sequence motifs and without encoding sequence data.  

PubMed

BackgroundMost of the approaches for splice site prediction are based on machine learning techniques. Though, these approaches provide high prediction accuracy, the window lengths used are longer in size. Hence, these approaches may not be suitable to predict the novel splice variants using the short sequence reads generated from next generation sequencing technologies. Further, machine learning techniques require numerically encoded data and produce different accuracy with different encoding procedures. Therefore, splice site prediction with short sequence motifs and without encoding sequence data became a motivation for the present study.ResultsAn approach for finding association among nucleotide bases in the splice site motifs is developed and used further to determine the appropriate window size. Besides, an approach for prediction of donor splice sites using sum of absolute error criterion has also been proposed. The proposed approach has been compared with commonly used approaches i.e., Maximum Entropy Modeling (MEM), Maximal Dependency Decomposition (MDD), Weighted Matrix Method (WMM) and Markov Model of first order (MM1) and was found to perform equally with MEM and MDD and better than WMM and MM1 in terms of prediction accuracy.ConclusionsThe proposed prediction approach can be used in the prediction of donor splice sites with higher accuracy using short sequence motifs and hence can be used as a complementary method to the existing approaches. Based on the proposed methodology, a web server was also developed for easy prediction of donor splice sites by users and is available at http://cabgrid.res.in:8080/sspred. PMID:25420551

Meher, Prabina; Sahu, Tanmaya; Rao, Atmakuri; Wahi, Sant

2014-11-25

22

Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition  

PubMed Central

Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes. PMID:17881373

Královi?ová, Jana; Vo?echovský, Igor

2007-01-01

23

Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization.  

PubMed Central

A tripartite motif located in the centre of the 7.5 kb exon 26 of apolipoprotein B (apoB) mRNA directs editosome assembly and site-specific cytidine-to-uridine editing at nucleotide 6666. apoB mRNA editing is a post-transcriptional event, occurring primarily at the time exon 26 is spliced or at a time after splicing, but before nuclear export. We show, through reporter RNA constructs, that RNA splice sites suppress editing of precursor RNAs when placed proximal or distal to the editing site. Processed RNAs were edited more efficiently than precursor RNAs. Mutation of both the splice donor and acceptor sites was necessary for RNAs to be edited efficiently. The results suggested that commitment of pre-mRNA to the splicing and/or nuclear-export pathways may play a role in regulating editing-site utilization. The HIV-1 Rev-Rev response element ('RRE') interaction was utilized to uncouple the commitment of precursor RNAs to the spliceosome assembly pathway and associated nuclear-export pathway. Under these conditions, unspliced reporter RNAs were edited efficiently. We propose that pre-mRNA passage through the temporal or spatial restriction point where they become committed to spliceosome assembly contributes regulatory information for subsequent editosome activity. PMID:11672445

Sowden, M P; Smith, H C

2001-01-01

24

Computational discovery of human coding and non-coding transcripts with conserved splice sites  

E-print Network

Computational discovery of human coding and non-coding transcripts with conserved splice sites computationally. Results: We introduce an approach to predict spliced lncRNAs in vertebrate genomes combining comparative genomics and machine learning. It is based on detecting signatures of characteristic splice site

Will, Sebastian

25

A novel splice site mutation in the EDAR gene underlies autosomal recessive hypohidrotic ectodermal dysplasia in a Pakistani family.  

PubMed

Hypohidrotic ectodermal dysplasia is a rare congenital disorder that results in abnormalities in the structures of ectodermal origin: hair, teeth, and eccrine sweat glands. DNA sequence analysis of EDAR gene in a Pakistani family, demonstrating autosomal recessive form of hypohidrotic ectodermal dysplasia, identified a novel homozygous mutation affecting splice donor site of exon 5 [IVS5+1G > or = C] of the gene. PMID:20199431

Wasif, Naveed; Tariq, Muhammad; Ali, Ghazanfar; Hassan, Muhammad Jawad; Ahmad, Wasim

2010-01-01

26

Detection of Splice Sites Using Support Vector Machine  

NASA Astrophysics Data System (ADS)

Automatic identification and annotation of exon and intron region of gene, from DNA sequences has been an important research area in field of computational biology. Several approaches viz. Hidden Markov Model (HMM), Artificial Intelligence (AI) based machine learning and Digital Signal Processing (DSP) techniques have extensively and independently been used by various researchers to cater this challenging task. In this work, we propose a Support Vector Machine based kernel learning approach for detection of splice sites (the exon-intron boundary) in a gene. Electron-Ion Interaction Potential (EIIP) values of nucleotides have been used for mapping character sequences to corresponding numeric sequences. Radial Basis Function (RBF) SVM kernel is trained using EIIP numeric sequences. Furthermore this was tested on test gene dataset for detection of splice site by window (of 12 residues) shifting. Optimum values of window size, various important parameters of SVM kernel have been optimized for a better accuracy. Receiver Operating Characteristic (ROC) curves have been utilized for displaying the sensitivity rate of the classifier and results showed 94.82% accuracy for splice site detection on test dataset.

Varadwaj, Pritish; Purohit, Neetesh; Arora, Bhumika

27

Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency.  

PubMed Central

Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8178821

Arredondo-Vega, F. X.; Santisteban, I.; Kelly, S.; Schlossman, C. M.; Umetsu, D. T.; Hershfield, M. S.

1994-01-01

28

Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus  

PubMed Central

Background Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. Results By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. Conclusion We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus. PMID:17617899

Sørensen, Annette Balle; Lund, Anders H; Kunder, Sandra; Quintanilla-Martinez, Leticia; Schmidt, Jörg; Wang, Bruce; Wabl, Matthias; Pedersen, Finn Skou

2007-01-01

29

Negative pressure wound dressing of the radial forearm donor site  

Microsoft Academic Search

Donor site complications of the radial forearm are a significant cause of post-operative morbidity. 15 patients had radial forearm free tissue donor sites treated with split skin grafts and a negative pressure dressing. All grafts showed 100% take at 5 days. The advantages of this technique include rapid healing at an unfavourable graft recipient site, increased graft take and decreased

C. Avery; J. Pereira; A. Moody; M. Gargiulo; I. Whitworth

2000-01-01

30

Statistical analysis of DNA sequences in the neighborhood of splice sites  

Microsoft Academic Search

Prediction of gene sequences and their exon-intron structure in large eukaryotic genomic sequences is one of the central problems\\u000a of mathematical biology. Solving this problem involves, in particular, high-accuracy splice site recognition. Using statistical\\u000a analysis of a splice site-containing human gene fragment database, some characteristic features were described for nucleotide\\u000a sequences in the splicing site neighborhood, the frequencies of all

O. M. Korzinov; T. V. Astakhova; P. K. Vlasov; M. A. Roytberg

2008-01-01

31

Epidermolytic palmoplantar keratoderma caused by activation of a cryptic splice site in KRT9.  

PubMed

Epidermolytic palmoplantar keratoderma (EPPK) is caused by mutations in KRT9 and less often, KRT1. All known mutations in KRT9 have been found in regions of the gene encoding the conserved central ?-helix rod domain. In the present study, we investigated the molecular basis of EPPK in a patient of Ashkenazi Jewish origin. The patient was found to carry a novel missense mutation in KRT9, resulting in the substitution of a poorly conserved leucine for valine at position 11 of the amino acid sequence. Despite its unusual location, the mutation was shown to be pathogenic through activation of a cryptic donor splice site, resulting in the deletion of 162 amino acids. The present data indicate the need to screen keratin genes in their entirety, as mutations altering domains of lesser functional importance may exert their deleterious effect at the transcriptional level. PMID:23397986

Fuchs-Telem, D; Padalon-Brauch, G; Sarig, O; Sprecher, E

2013-03-01

32

Determinants of the inherent strength of human 5? splice sites  

PubMed Central

We previously showed that the authentic 5? splice site (5?ss) of the first exon in the human ?-globin gene is intrinsically stronger than a cryptic 5?ss located 16 nucleotides upstream. Here we examined by mutational analysis the contribution of individual 5?ss nucleotides to discrimination between these two 5?ss. Based on the in vitro splicing efficiencies of a panel of 26 wild-type and mutant substrates in two separate 5?ss competition assays, we established a hierarchy of 5?ss and grouped them into three functional subclasses: strong, intermediate, and weak. Competition between two 5?ss from different subclasses always resulted in selection of the 5?ss that belongs to the stronger subclass. Moreover, each subclass has different characteristic features. Strong and intermediate 5?ss can be distinguished by their predicted free energy of base-pairing to the U1 snRNA 5? terminus (?G). Whereas the extent of splicing via the strong 5?ss correlates well with the ?G, this is not the case for competition between intermediate 5?ss. Weak 5?ss were used only when the competing authentic 5?ss was inactivated by mutation. These results indicate that extensive complementarity to U1 snRNA exerts a dominant effect for 5?ss selection, but in the case of competing 5?ss with similarly modest complementarity to U1, the role of other 5?ss features is more prominent. This study reveals the importance of additional submotifs present in certain 5?ss sequences, whose characterization will be critical for understanding 5?ss selection in human genes. PMID:15840817

ROCA, XAVIER; SACHIDANANDAM, RAVI; KRAINER, ADRIAN R.

2005-01-01

33

U2AF1 mutations alter splice site recognition in hematological malignancies  

PubMed Central

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3? splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3? splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1’s zinc finger domains. PMID:25267526

Ilagan, Janine O.; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E.; Zebari, Ahmad S.

2015-01-01

34

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.  

PubMed

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events. PMID:7513051

Carr, B; Anderson, P

1994-05-01

35

Prediction of Splice Sites in Plant Pre-mRNA from Sequence Properties  

E-print Network

Prediction of Splice Sites in Plant Pre-mRNA from Sequence Properties Volker Brendel1 *, Ju of this contrast factor in recently developed statistical methods for splice site prediction from sequence inspection signi®cantly improved prediction accuracy. We applied the prediction tools to re-analyze exper

Brendel, Volker

36

Characterisation of three novel splice site mutations in introns 11, 18 and 30 of the NF-1 gene  

SciTech Connect

Identification and characterization of germline mutations within the NF-1 gene was carried out in 25 unrelated NF-1 patients, in whom we have detected three splice site mutations which cause exon skipping. Our detection strategy incorporated both RNA and DNA as templates for PCR, chemical mismatch cleavage and direct sequencing. The first mutation was detected in the splice donor sequence of intron 11 (1721+3A{r_arrow}G), which results in the skipping of exon 11 and causes a shift in the translational reading frame and the creation of a premature stop codon at position 560. This is predicted to result in the synthesis of a shorter protein product of 559 amino acids instead of 2818, with loss of the NF-1 GAP related domain. The patient is a familial case of NF-1 with neurological complications and no evidence of malignancy. She has an affected son who has inherited the same mutation and has skeletal complications. The second mutation was detected at the splice donor site in intron 18 (3113+1G{r_arrow}A) and caused the skipping of exon 18. This did not cause a shift in the reading frame but resulted in the exclusion of 41 amino acids from the predicted protein product and was seen in a familial case of NF-1 with neurological complications. The third mutation, at the splice donor site in intron 30 (5749+2T{r_arrow}G), caused the skipping of exon 30, shifting the translational reading frame and creating a premature stop codon at position 1851. The predicted protein product is reduced from the normal 2818 to 1850 amino acids. This patient is a sporadic case of NF-1, has neurological and skeletal complications and no evidence of malignancy. Thus in our analysis of 25 patients, the strategy of using RT-PCR to amplify the NF-1 cDNA greatly facilitated the detection of these errors of splicing, each of which is predicted to cause a major distruption of the protein product neurofibromin.

Purandare, S.M.; Lanyon, W.G.; Arngrimsson, R. [Univ. of Glasgow (United Kingdom)] [and others

1994-09-01

37

The strength of the HIV-1 3' splice sites affects Rev function  

PubMed Central

Background The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. Results In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression. Conclusion Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs. PMID:17144911

Kammler, Susanne; Otte, Marianne; Hauber, Ilona; Kjems, Jørgen; Hauber, Joachim; Schaal, Heiner

2006-01-01

38

Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity at human glycophorin (GYP) locus.  

PubMed

The glycophorin locus (GYP) on the long arm of chromosome 4 encodes antigens of the MNSs blood group system and displays considerable allelic variation among human populations. The genomic structure and organization of a variant glycophorin allele specifying a novel Miltenberger (Mi)-related phenotype, MiX, were examined. This variant probably arose from a gene conversion event involving a direct repeat of the acceptor splice site. Southern blot analysis indicated that MiX gene derived its 5' and 3' portions from glycophorin B or delta gene but its internal part from glycophorin A or alpha gene. Genomic sequences encompassing the rearranged regions of the MiX gene were amplified by single copy polymerase chain reaction. Direct DNA sequencing showed that during the formation of MiX gene, a short stretch of alpha exon III with a donor splice site has replaced a silent sequence in the delta gene containing a cryptic acceptor splice site. The upstream delta-alpha breakpoint is flanked by the direct repeats of the acceptor splice site, whereas the down-stream alpha-delta breakpoint is located in the adjacent intron. This segmental transfer produced a new composite exon whose expression not only transactivated a portion of silent sequence but also created intraexon and interexon hybrid junctions that characterize the antigenic specificities of MiX glycophorin. The identification of MiX as yet another delta-alpha-delta hybrid different from MiIII and MiVI in gene conversion sites suggests that shuffling of expressed and unexpressed sequences through particular genomic DNA motifs has been an important mechanism for shaping the antigenic diversity of MNSs blood group system during evolution. PMID:1737789

Huang, C H; Kikuchi, M; McCreary, J; Blumenfeld, O O

1992-02-15

39

Identifying potential kidney donors using social networking web sites.  

PubMed

Social networking sites like Facebook may be a powerful tool for increasing rates of live kidney donation. They allow for wide dissemination of information and discussion and could lessen anxiety associated with a face-to-face request for donation. However, sparse data exist on the use of social media for this purpose. We searched Facebook, the most popular social networking site, for publicly available English-language pages seeking kidney donors for a specific individual, abstracting information on the potential recipient, characteristics of the page itself, and whether potential donors were tested. In the 91 pages meeting inclusion criteria, the mean age of potential recipients was 37 (range: 2-69); 88% were US residents. Other posted information included the individual's photograph (76%), blood type (64%), cause of kidney disease (43%), and location (71%). Thirty-two percent of pages reported having potential donors tested, and 10% reported receiving a live-donor kidney transplant. Those reporting donor testing shared more potential recipient characteristics, provided more information about transplantation, and had higher page traffic. Facebook is already being used to identify potential kidney donors. Future studies should focus on how to safely, ethically, and effectively use social networking sites to inform potential donors and potentially expand live kidney donation. PMID:23600791

Chang, Alexander; Anderson, Emily E; Turner, Hang T; Shoham, David; Hou, Susan H; Grams, Morgan

2013-01-01

40

Alternative splicing of mouse IL-15 is due to the use of an internal splice site in exon 5.  

PubMed

IL-15 is a pleiotropic cytokine modulating growth and differentiation of several hematopoietic cell types. Recently, we have demonstrated that mouse microglial cells, the brain macrophages, express both IL-15 and IL-15/IL-2 receptors. Based on single-cell RT-PCR data, we describe here an alternatively spliced IL-15 mRNA variant found in a small subpopulation of mouse microglia (5%, 3 out of 60 cells expressing IL-15 transcripts). PCR cycle sequencing of this larger transcript revealed the mouse homologue of the alternatively spliced exon A as it is known from the human IL-15 gene. Analysis of the corresponding mouse IL-15 gene region shows that the larger IL-15 transcript contains an yet unidentified 5' sequence of exon 5 while the shorter transcript uses an internal splice acceptor site. The mouse exon 5A segment has a length of 136 nt (17 nt longer than the human exon A). It contains five in-frame stop codons at its 5' end and a new translation initiation site at its 3' end. This new start site is surrounded by a favourable Kozak consensus sequence suggesting a more efficient translation rate. Further translational control by stem-loop binding factors is inferred by a predicted RNA stem-loop structure around the start site. Insertion of exon 5A would lead to an IL-15 polypeptide with a shortened leader sequence of 26 amino acids, as compared to the 48 amino acid leader sequence encoded by the transcript lacking exon 5A. Thus, the final IL-15 protein of the two splice variants is identical; different leader sequences could, however, lead to differences in the intracellular sorting, processing and/or secretion of IL-15. PMID:9838089

Prinz, M; Hanisch, U K; Kettenmann, H; Kirchhoff, F

1998-12-10

41

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant.  

PubMed

Changes at the invariable donor splice site +1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6+1G>T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6+1G>T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6+1G>T mutation is compatible with the synthesis of functional FVII molecules (~0.01% of normal, 1pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55nM) measured in plasma from an IVS6+1G>T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition. PMID:22426302

Cavallari, Nicola; Balestra, Dario; Branchini, Alessio; Maestri, Iva; Chuamsunrit, Ampaiwan; Sasanakul, Werasak; Mariani, Guglielmo; Pagani, Franco; Bernardi, Francesco; Pinotti, Mirko

2012-07-01

42

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant  

PubMed Central

Changes at the invariable donor splice site + 1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6 + 1G > T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6 + 1G > T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30 bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6 + 1G > T mutation is compatible with the synthesis of functional FVII molecules (~ 0.01% of normal, 1 pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55 nM) measured in plasma from an IVS6 + 1G > T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition. PMID:22426302

Cavallari, Nicola; Balestra, Dario; Branchini, Alessio; Maestri, Iva; Chuamsunrit, Ampaiwan; Sasanakul, Werasak; Mariani, Guglielmo; Pagani, Franco; Bernardi, Francesco; Pinotti, Mirko

2012-01-01

43

Secondary structure of splice sites in adenovirus mRNA precursors.  

PubMed Central

In order to investigate the possible role of RNA secondary structure in determining the efficiency and specificity of mRNA splicing, the structures of sequences at three acceptor splice sites in adenovirus were studied. Transcripts spanning intron-exon junctions were synthesized using SP6 RNA polymerase and analyzed using single and double-strand specific nucleases. Distinctive patterns of nuclease cleavage were observed for each of the 3 sites examined. At both sites in the E2a region sequences adjacent to the splice sites were particularly susceptible to digestion with T1 and S1 nucleases. In contrast, a splice site for hexon mRNA was largely resistant to these nucleases. The results obtained suggest that the conformation of the RNA at some, but not all, acceptor sites may enhance the accessibility of these sites to factors involved in splicing nuclear RNA and confirm the presence of a large, previously predicted hairpin structure centered on the acceptor site at 67 map units. Images PMID:6095200

Munroe, S H

1984-01-01

44

Allele-specific recognition of the 3? splice site of INS intron 1  

PubMed Central

Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689), which rapidly expanded in modern humans, renders the 3? splice site of this intron more dependent on the auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF). This interaction required both zinc fingers of the 35-kD U2AF subunit (U2AF35) and was associated with repression of a competing 3? splice site in INS exon 2. Systematic mutagenesis of reporter constructs showed that intron 1 removal was facilitated by conserved guanosine-rich enhancers and identified additional splicing regulatory motifs in exon 2. Sequencing of intron 1 in primates revealed that relaxation of its 3? splice site in Hominidae coevolved with the introduction of a short upstream open reading frame, providing a more efficient coupled splicing and translation control. Depletion of SR proteins 9G8 and transformer-2 by RNA interference was associated with exon 2 skipping whereas depletion of SRp20 with increased representation of transcripts containing a cryptic 3? splice site in the last exon. Together, these findings reveal critical interactions underlying the allele-dependent INS expression and INS-mediated risk of T1D and suggest that the increased requirement for U2AF35 in higher primates may hinder thymic presentation of autoantigens encoded by transcripts with weak 3? splice sites. Electronic supplementary material The online version of this article (doi:10.1007/s00439-010-0860-1) contains supplementary material, which is available to authorized users. PMID:20628762

Kralovicova, Jana

2010-01-01

45

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5? splice site and branch site  

PubMed Central

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5? splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites. PMID:23569281

Crawford, Daniel J.; Hoskins, Aaron A.; Friedman, Larry J.; Gelles, Jeff; Moore, Melissa J.

2013-01-01

46

Pick one, but be quick: 5? splice sites and the problems of too many choices  

PubMed Central

Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5? Splice sites (5?ss) are the critical elements at the 5? end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5?ss in the human transcriptome. Most 5?ss are recognized by base-pairing with the 5? end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5?ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5?ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5?ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5?ss might lead to selection of a single 5?ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5?ss selection. PMID:23348838

Roca, Xavier; Krainer, Adrian R.; Eperon, Ian C.

2013-01-01

47

Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter  

PubMed Central

Background Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated. Results A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences. Conclusion The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors. PMID:17941994

Braun, Stephen E; Shi, Xuanling; Qiu, Gang; Wong, Fay Eng; Joshi, Pheroze J; Prasad, Vinayaka R; Johnson, R Paul

2007-01-01

48

The 5' splice site: phylogenetic evolution and variable geometry of association with U1RNA.  

PubMed Central

The 5' splice site sequences of 3294 introns from various organisms (1-672) were analyzed in order to determine the rules governing evolution of this sequence, which may shed light on the mechanism of cleavage at the exon-intron junction. The data indicate that, currently, in all organisms, a common sequence 1GUAAG6U and its derivatives are used as well as an additional sequence and its derivatives, which differ in metazoa (G/1GUgAG6U), lower eucaryotes (1GUAxG6U) and higher plants (AG/1GU3A). They all partly resemble the prototype sequence AG/1GUAAG6U whose 8 contigous nucleotides are complementary to the nucleotides 4-11 of U1RNA, which are perfectly conserved in the course of phylogenetic evolution. Detailed examination of the data shows that U1RNA can recognize different parts of 5' splice sites. As a rule, either prototype nucleotides at position -2 and -1 or at positions 4, 5 or 6 or at positions 3-4 are dispensable provided that the stability of the U1RNA-5' splice site hybrid is conserved. On the basis of frequency of sequences, the optimal size of the hybridizable region is 5-7 nucleotides. Thus, the cleavage at the exon-intron junction seems to imply, first, that the 5' splice site is recognized by U1RNA according to a "variable geometry" program; second, that the precise cleavage site is determined by the conserved sequence of U1RNA since it occurs exactly opposite to the junction between nucleotides C9 and C10 of U1RNA. The variable geometry of the U1RNA-5' splice site association provides flexibility to the system and allows diversification in the course of phylogenetic evolution. PMID:2704616

Jacob, M; Gallinaro, H

1989-01-01

49

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

50

Splicing mediates the activity of four putative cellular internal ribosome entry sites.  

PubMed

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity. PMID:18326627

Baranick, Brian T; Lemp, Nathan A; Nagashima, Jill; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R

2008-03-25

51

Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery  

SciTech Connect

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. The authors report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2[1/2] years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for a splice-donor-site mutation in IVS 1 (+1GT[yields]CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P<.0.02, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy. 57 refs., 4 figs., 2 tabs.

Hirschhorn, R.; Yang, D.R.; Israni, A.; Huie, M.L. (New York Univ. Medical Center, NY (United States)); Ownby, D.R. (Henry Ford Hospital, Detroit, MI (United States))

1994-07-01

52

Splice-acceptor site mutation in p53 gene of hu888 zebrafish line.  

PubMed

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response, which has been a subject of intense research for over 30 years. Recently, a zebrafish line which carries splice site mutation (G>T) in intron 8 of p53 gene (p53 (hu888) ), encoding the p53 paralogue, was developed (The Zebrafish Mutation Project). To uncover molecular effects of the mutation, we raised hu888 zebrafish line to adulthood and analyzed DNA, mRNA data, and protein levels of p53 to assess their potential contribution in molecular mechanisms of the mutant fish. To obtain zebrafish individuals homozygous for the point mutation, p53 (hu888) carriers were repeatedly incrossed but only heterozygous mutants (p53 (hu888/+) ) or p53-wild type hu888 zebrafish (p53 (+/+) ) were identified in their progeny. By evaluation of p53 expression changes in the liver of mutant and wild type hu888 zebrafish as well as of Tübingen reference strain, we demonstrated that two types of splicing occurred in each case: a classical one and the alternative splicing which involves the activation of cryptic splice-acceptor site in the exon 9 of zebrafish p53 pre-mRNA. The alternative splicing event results in a deletion 12 nucleotides in the mature mRNA, and produces a shortened variant of p53 protein. Interestingly, expression of p53 protein in liver of both heterozygous and wild type hu888 zebrafish was highly reduced compared to that in the reference strain. PMID:25183022

Piasecka, Alicja; Brzuzan, Pawe?; Wo?ny, Maciej; Ciesielski, S?awomir; Kaczmarczyk, Dariusz

2015-02-01

53

Splicing fidelity  

PubMed Central

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5? splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5? splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5? splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5? splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell. PMID:23770752

Koodathingal, Prakash; Staley, Jonathan P.

2013-01-01

54

A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signaling pathway and causes Lenz microphthalmia syndrome  

PubMed Central

Introduction Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. Methods and results Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T?A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. Conclusions We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway. PMID:24431331

Esmailpour, Taraneh; Riazifar, Hamidreza; Liu, Linan; Donkervoort, Sandra; Huang, Vincent H; Madaan, Shreshtha; Shoucri, Bassem M; Busch, Anke; Wu, Jie; Towbin, Alexander; Chadwick, Robert B; Sequeira, Adolfo; Vawter, Marquis P; Sun, Guoli; Johnston, Jennifer J; Biesecker, Leslie G; Kawaguchi, Riki; Sun, Hui; Kimonis, Virginia; Huang, Taosheng

2014-01-01

55

Haemostatic effects of adrenaline-lidocaine subcutaneous infiltration at donor sites.  

PubMed

This study sought methods in burn surgery to reduce postoperative pain and blood loss at donor sites. A prospective, randomised, controlled, blinded trial included 56 people undergoing burn surgery, divided into two groups. Both groups received subcutaneous infiltration at donor sites, with either 1:500,000 adrenaline solution containing added lidocaine or with 0.45% normal saline (controls). Outcome measurements included amount of intraoperative bleeding, need for electrocautery, days the hydrocolloid dressing remained on donor sites, percentage of re-epithelialised skin at donor sites 1 week after surgery and viability of skin grafts. Results indicated that subcutaneous adrenaline-lidocaine infiltration at donor sites reduced intraoperative bleeding, decreased postoperative pain, shortened the duration of surgery and general anaesthesia and accelerated re-epithelialisation at the donor site. The overall graft take in both groups was similar. PMID:18950945

Gacto, P; Miralles, F; Pereyra, J J; Perez, A; Martínez, E

2009-05-01

56

De novo SCN2A splice site mutation in a boy with Autism spectrum disorder  

PubMed Central

Background SCN2A is a gene that codes for the alpha subunit of voltage-gated, type II sodium channels, and is highly expressed in the brain. Sodium channel disruptions, such as mutations in SCN2A, may play an important role in psychiatric disorders. Recently, de novo SCN2A mutations in autism spectrum disorder (ASD) have been identified. The current study characterizes a de novo splice site mutation in SCN2A that alters mRNA and protein products. Case presentation We describe results from clinical and genetic characterizations of a seven-year-old boy with ASD. Psychiatric interview and gold standard autism diagnostic instruments (ADOS and ADI-R) were used to confirm ASD diagnosis, in addition to performing standardized cognitive and adaptive functioning assessments (Leiter-R and Vineland Adaptive Behavior Scale), and sensory reactivity assessments (Sensory Profile and Sensory Processing Scales). Genetic testing by whole exome sequencing revealed four de novo events, including a splice site mutation c.476?+?1G?>?A in SCN2A, a missense mutation (c.2263G?>?A) causing a p.V755I change in the TLE1 gene, and two synonymous mutations (c.2943A?>?G in the BUB1 gene, and c.1254 T?>?A in C10orf68 gene). The de novo SCN2A splice site mutation produced a stop codon 10 amino acids downstream, possibly resulting in a truncated protein and/or a nonsense-mediated mRNA decay. The participant met new DSM-5 criteria for ASD, presenting with social and communication impairment, repetitive behaviors, and sensory reactivity issues. The participant’s adaptive and cognitive skills fell in the low range of functioning. Conclusion This report indicates that a splice site mutation in SCN2A might be contributing to the risk of ASD. Describing the specific phenotype associated with SCN2A mutations might help to reduce heterogeneity seen in ASD. PMID:24650168

2014-01-01

57

A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment  

PubMed Central

The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

Gandía, Marta; del Castillo, Francisco J.; Rodríguez-Álvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderón, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

2013-01-01

58

Biased exon/intron distribution of cryptic and de novo 3? splice sites  

PubMed Central

We compiled sequences of previously published aberrant 3? splice sites (3?ss) that were generated by mutations in human disease genes. Cryptic 3?ss, defined here as those resulting from a mutation of the 3?YAG consensus, were more frequent in exons than in introns. They clustered in ?20 nt region adjacent to authentic 3?ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3?ss that were induced by mutations outside the 3?YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3?ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3?ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro–Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3?ss. Finally, AG-creating mutations in the PPT that produced aberrant 3?ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3?ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects. PMID:16141195

Královi?ová, Jana; Christensen, Mikkel B.; Vo?echovský, Igor

2005-01-01

59

Using multiple donor sites for enhanced flood estimation in ungauged catchments  

NASA Astrophysics Data System (ADS)

new generalized method is presented enabling the use of multiple donor sites when predicting an index flood variable in an ungauged catchment using a hydrological regression model. The method is developed from the premise of having an index flood prediction with minimum variance, which results in a set of optimal weights assigned to each donor site. In the model framework presented here, the weights are determined by the geographical distance between the centroids of the catchments draining to the subject site and the donor sites. The new method was applied to a case study in the United Kingdom using annual maximum series of peak flow from 602 catchments. Results show that the prediction error of the index flood is reduced by using donor sites until a minimum of six donors have been included, after which no or marginal improvements in prediction accuracy are observed. A comparison of these results is made with a variant of the method where donor sites are selected based on connectivity with the subject site through the river network. The results show that only a marginal improvement is obtained by explicitly considering the network structure over spatial proximity. The evaluation is carried out based on a new performance measure that accounts for the sampling variability of the index flood estimates at each site. Other results compare the benefits obtained by adding relevant catchment descriptors to a simple regression model with those obtained by transferring information from local donor sites.

Kjeldsen, T. R.; Jones, D. A.; Morris, D. G.

2014-08-01

60

Achieving Direct Closure of the Anterolateral Thigh Flap Donor Site—An Algorithmic Approach  

PubMed Central

Background: Minimizing donor-site morbidity after free flap harvest is of paramount importance. In this article, we share our experience with achieving primary closure of 58 anterolateral thigh (ALT) free flap donor sites using a simple algorithm in cases where primary closure would otherwise have not been possible. Methods: Between 2004 and 2010, 58 patients who underwent free ALT flap reconstruction were included in the study. The inclusion criteria were those who had flap width requirements that were wider than 16% of the thigh circumference and had achieved direct primary closure of the donor site by the use of our technique. Results: Primary closure of the donor sites was facilitated in all cases by the use of 3 distinct techniques. This included the use of the V-Y advancement technique in 13 patients, split skin paddle technique in 7 patients, and the tubed skin paddle design in 38 patients. No episodes of postoperative wound dehiscence at the donor site were encountered; however, 2 cases were complicated by superficial wound infections that settled with a course of antibiotics. Conclusions: Direct primary closure of the ALT donor site can be facilitated by the use of our simple algorithm. Certain strategies need to be adopted at the design stage; however, the techniques used are simple and reliable, produce superior cosmetic results at the donor site, save time, and spare the patient the morbidity associated with the harvest of a skin graft. PMID:25426349

Pachón Suárez, Jaime Eduardo; Sadigh, Parviz Lionel; Shih, Hsiang-Shun; Hsieh, Ching-Hua

2014-01-01

61

Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles.  

PubMed Central

The effect of heat shock on the packaging and splicing of nuclear CAD pre-mRNA, a transcript expressed constitutively from a non heat-inducible promoter, was studied in vivo in Syrian hamster cells. While mild heat shock did not affect significantly the packaging of CAD RNA in 200S InRNP particles, it caused perturbation to splicing. First, the heat shock inhibited splicing of CAD pre-mRNA. Second, it affected 5' splice site selection by activating cleavage at a cryptic 5' splice site; yet ligation of the cryptic exon to the downstream proximal exon was not observed. Base complementarities of the cryptic site with U1, U5, or U6 snRNAs are comparable, or even better, than those with the neighboring normal site. Hence, the exclusion of the cryptic site under normal growth conditions cannot be attributed to weaker base pairing with these snRNAs. On the other hand, these results imply the involvement of a heat labile factor in the selection of the 5' cleavage site. The exclusion of the cryptic site at 37 degrees C and the aborted splicing at this site after heat shock may also be explained by a proposed nuclear checking mechanism that detects in-frame stop codons upstream of the 5' splice site, and aborts splicing at such sites to prevent the production of a defective message. Images PMID:7915031

Miriami, E; Sperling, J; Sperling, R

1994-01-01

62

Splice-Site A Choice Targets Plasma-Membrane Ca2+-ATPase Isoform 2 to Hair Bundles  

PubMed Central

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport. PMID:16763025

Hill, Jennifer K.; Williams, Diane E.; LeMasurier, Meredith; Dumont, Rachel A.; Strehler, Emanuel E.; Gillespie, Peter G.

2007-01-01

63

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures  

SciTech Connect

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. 16 refs., 3 tabs.

Hunter, T.C.; Albertini, R.J.; O`Neill, J.P. [Univ. of Vermont Genetics Lab., Burlington, VT (United States)] [and others

1996-03-01

64

Chironomus tentans-Repressor Splicing Factor Represses SR Protein Function Locally on Pre-mRNA Exons and Is Displaced at Correct Splice Sites  

PubMed Central

Chironomus tentans-repressor splicing factor (Ct-RSF) represses the activation of splicing by SR proteins in vitro. Ct-RSF colocalizes with the Ser-Arg-rich (SR) protein hrp45 in interchromatin granule clusters and coimmunoprecipitates with hrp45 in nuclear extracts. Ct-RSF and hrp45 can also interact directly in vitro. Ct-RSF and hrp45 are recruited together to transcribing genes and associate with growing pre-mRNAs. Ct-RSF and hrp45 colocalize at a large number of gene loci. Injection of anti-Ct-RSF antibodies into nuclei of living cells blocks association of both Ct-RSF and hrp45 with the growing pre-mRNA, whereas binding of U2 small nuclear ribonucleoprotein particle (snRNP) to the pre-mRNA is unaffected. On the intron-rich Balbiani ring (BR) 3 pre-mRNA, hrp45 as well as U1 and U2 snRNPs bind extensively, whereas relatively little Ct-RSF is present. In contrast, the BR1 and BR2 pre-mRNAs, dominated by exon sequences, bind relatively much Ct-RSF compared with hrp45 and snRNPs. Our data suggest that Ct-RSF represses SR protein function at exons and that the assembly of spliceosomes at authentic splice sites displaces Ct-RSF locally. PMID:16236800

Björk, Petra; Wetterberg-Strandh, Ingela; Baurén, Göran; Wieslander, Lars

2006-01-01

65

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites  

SciTech Connect

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.

Bodaghi, Sohrab; Jia Rong [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Zheng Zhiming [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)], E-mail: zhengt@exchange.nih.gov

2009-03-30

66

Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.  

PubMed

Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis. PMID:23239986

Wappenschmidt, Barbara; Becker, Alexandra A; Hauke, Jan; Weber, Ute; Engert, Stefanie; Köhler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K

2012-01-01

67

Proceedings of International Joint Conference on Neural Networks, Montreal, Canada, July 31 -August 4, 2005 Characterizing Human Gene Splice Sites Using  

E-print Network

Natural Science Foundation ofChina (Nos.60472 111 and 60405002) and Chinese Human Liver Proteome Project - August 4, 2005 Characterizing Human Gene Splice Sites Using Evolved Regular Expressions+ Jing-Jing Li to characterize and predict human gene splice sites without any prior knowledge is described. In contrast

Hefei Institute of Intelligent Machines

68

Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity  

PubMed Central

Background: Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity. Methods: The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG). Results: After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the “fish-net” patterning caused by STSG. Conclusion: Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing. PMID:25289241

Wang, Ying; Farinelli, William A.; Jiménez-Lozano, Joel; Franco, Walfre; Sakamoto, Fernanda H.; Cheung, Evelyn J.; Purschke, Martin; Doukas, Apostolos G.; Anderson, R. Rox

2013-01-01

69

Haemostatic effects of adrenaline–lidocaine subcutaneous infiltration at donor sites  

Microsoft Academic Search

This study sought methods in burn surgery to reduce postoperative pain and blood loss at donor sites. A prospective, randomised, controlled, blinded trial included 56 people undergoing burn surgery, divided into two groups. Both groups received subcutaneous infiltration at donor sites, with either 1:500,000 adrenaline solution containing added lidocaine or with 0.45% normal saline (controls). Outcome measurements included amount of

P. Gacto; F. Miralles; J. J. Pereyra; A. Perez; E. Martínez

2009-01-01

70

Clinical and genetic studies in a family with a new splice-site mutation in the choroideremia gene  

PubMed Central

Purpose To describe the clinical and molecular findings of an Italian family with a new mutation in the choroideremia (CHM) gene. Methods We performed a comprehensive ophthalmologic examination, fundus photography, macular optical coherence tomography, perimetry, electroretinography, and fluorescein angiography in an Italian family. The clinical diagnosis was supported by western blot analysis of lymphoblastoid cell lines from patients with CHM and carriers, using a monoclonal antibody against the 415 C-terminal amino acids of Rab escort protein-1 (REP-1). Sequencing of the CHM gene was undertaken on genomic DNA from affected men and carriers; the RNA transcript was analyzed with reverse transcriptase-PCR. Results The affected men showed a variability in the rate of visual change and in the degree of clinical and functional ophthalmologic involvement, mainly age-related, while the women displayed aspecific areas of chorioretinal degeneration. Western blot did not show a detectable amount of normal REP-1 protein in affected men who were hemizygous for a novel mutation, c.819+2T>A at the donor splicing site of intron 6 of the CHM gene; the mutation was confirmed in heterozygosity in the carriers. Conclusions Western blot of the REP-1 protein confirmed the clinical diagnosis, and molecular analysis showed the new in-frame mutation, c.819+2T>A, leading to loss of function of the REP-1 protein. These results emphasize the value of a diagnostic approach that correlates genetic and ophthalmologic data for identifying carriers in families with CHM. An early diagnosis might be crucial for genetic counseling of this type of progressive and still untreatable disease. PMID:24672218

Piane, Maria; Cascone, Nikhil C.; Pasquale, Nadia; Ciarnella, Angela; Recupero, Santi M.; Chessa, Luciana

2014-01-01

71

A newly identified splice site mutation in ZMPSTE24 causes restrictive dermopathy in the Middle East.  

PubMed

Restrictive dermopathy (RD) is a severe neonatal inherited skin syndrome of which children die shortly after birth. Clinical features include intrauterine growth retardation, taut translucent and easily eroded skin, multiple joint ankylosis and distinct facial features. RD is usually caused by homozygous or compound heterozygous mutations in ZMPSTE24, predicted to cause loss of function of the encoded zinc metalloproteinase STE24. ZMPSTE24 is essential for the processing of the nuclear intermediate filament protein prelamin A. We report two distantly related children from the United Arab Emirates with RD. Remarkably, they lived up to 2 months, suggesting some residual function of the mutant protein. We sought to confirm the diagnosis by thorough microscopic analysis of patient skin, to identify the causative mutation and to study its functional consequences. A skin biopsy was obtained and processed for light and electron microscopy. Peripheral blood leucocytes were used for DNA and RNA isolation, and detection of prelamin A by immunofluorescence. Analysis of the skin confirmed the earlier reported densely packed collagen bundles and lack of elastin fibres. In both patients a homozygous splice site mutation c.627+1G>C in ZMPSTE24 was identified. Analysis of the ZMPSTE24 mRNA revealed an in-frame exon 5 skipping. Accumulation of prelamin A could be detected at the nuclear envelope of patient blood lymphocytes. We thus report the first splice site mutation in ZMPSTE24, which is likely to be a founder mutation in the United Arab Emirates. The accumulation of prelamin A at the nuclear periphery is consistent with defective ZMPSTE24 function. Interestingly, a regular blood sample can be used to investigate prelamin A accumulation. PMID:18671782

Sander, C S; Salman, N; van Geel, M; Broers, J L V; Al-Rahmani, A; Chedid, F; Hausser, I; Oji, V; Al Nuaimi, K; Berger, T G; Verstraeten, V L R M

2008-09-01

72

Exon sequences at the splice junctions affect splicing fidelity and alternative splicing  

PubMed Central

Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3? splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18?CR, and the strength of an exon sequence in Prp18?CR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3? splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3? splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected. PMID:19855008

Crotti, Luciana B.; Horowitz, David S.

2009-01-01

73

Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation.  

PubMed

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by an accelerated aging phenotype and an average life span of 13years. Patients typically exhibit extensive pathophysiological vascular alterations, eventually resulting in death from stroke or myocardial infarction. A silent point mutation at position 1824 (C1824T) of the LMNA gene, generating a truncated form of lamin A (progerin), has been shown to be the cause of most cases of HGPS. Interestingly, this mutation induces the use of an internal 5' cryptic splice site within exon 11 of the LMNA pre-mRNA, leading to the generation of progerin via aberrant alternative splicing. The serine-arginine rich splicing factor 1 (SRSF1 or ASF/SF2) has been shown to function as an oncoprotein and is upregulated in many cancers and other age-related disorders. Indeed, SRSF1 inhibition results in a splicing ratio in the LMNA pre-mRNA favoring lamin A production over that of progerin. It is our hypothesis that activation of AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, may lead to a reduction in SRSF1 and thus a decrease in the use of the LMNA 5' cryptic splice site in exon 11 through upregulation of p32, a splicing factor-associated protein and putative mitochondrial chaperone that has been shown to inhibit SRSF1 and enhance mitochondrial DNA (mtDNA) replication and oxidative phosphorylation. AMPK activation by currently available compounds such as metformin, resveratrol, and berberine may thus have wide-ranging implications for disorders associated with increased production and accumulation of progerin. PMID:25216752

Finley, Jahahreeh

2014-11-01

74

A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family.  

PubMed

With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI. PMID:25296581

Siddiqi, Saima; Ismail, Muhammad; Oostrik, Jaap; Munawar, Saba; Mansoor, Atika; Kremer, Hannie; Qamar, Raheel; Schraders, Margit

2014-12-01

75

Probabilistic simple splicing systems  

NASA Astrophysics Data System (ADS)

A splicing system, one of the early theoretical models for DNA computing was introduced by Head in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings of DNA molecules at the specific recognition sites and attaches the prefix of the first string to the suffix of the second string, and the prefix of the second string to the suffix of the first string, thus yielding the new strings. For a specific type of splicing systems, namely the simple splicing systems, the recognition sites are the same for both strings of DNA molecules. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions have been considered for splicing systems in order to increase their computational power. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic simple splicing systems are investigated. We prove that probabilistic simple splicing systems can also increase the computational power of the splicing languages generated.

Selvarajoo, Mathuri; Heng, Fong Wan; Sarmin, Nor Haniza; Turaev, Sherzod

2014-06-01

76

Donor site morbidity in oral mucosa graft urethroplasty: implications of tobacco consumption  

PubMed Central

Background The purpose of this prospective study was to evaluate the donor site morbidity in patients who have undergone oral mucosa graft urethroplasty for stricture of the urethra. The impact of smoking and oral consumption of tobacco and/or paan masala on the donor site was also assessed. This study is probably the first of its kind where the affect of smoking, paan masala and tobacco chewing on the donor site morbidity has been documented. Methods Forty-eight patients suffering from stricture of the urethra underwent oral mucosa graft urethroplasty between July 2005 and December 2007. The patients were divided into two groups (users or non-users) based on tobacco consumption and oral hygiene. The donor site was evaluated at frequent intervals for pain, swelling, numbness, bleeding, salivation and tightness of mouth. Results Donor site morbidity was more in users with poor oral hygiene. Pain scores were higher amongst the users and the morbidity persisted longer in the users compared to non-users with good oral hygiene. Conclusion Patients who consume tobacco and have poor oral hygiene should be warned regarding poorer outcomes after oral mucosa graft urethroplasty. PMID:19772567

Sinha, Rahul Janak; Singh, Vishwajeet; Sankhwar, SN; Dalela, Divakar

2009-01-01

77

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

PubMed Central

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

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

2013-01-01

78

Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.  

PubMed

We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate. Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA. Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate. The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step. Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step. The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur. PMID:8849776

Kim, C H; Abelson, J

1996-10-01

79

Comparative clinical study of Bactigras and Telfa AMD for skin graft donor-site dressing.  

PubMed

The Bactigras(®) paraffin tulle coated with chlorhexidine is normally used for the treatment of donor-site wounds in burn patients who received split-thickness skin grafts in several centers. It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD(®), a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras(®) or the Telfa AMD(®) wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (?90%) was significantly shorter in patients treated with the Telfa AMD(®) compared to the Bactigras(®) group (14.00 ± 3.05 vs. 9.25 ± 1.88 days for Bactigras(®) and Telfa AMD(®) groups, respectively, p < 0.001). The average pain score was also significantly lower in the Telfa AMD(®) group (1.57 ± 0.55 vs. 4.70 ± 1.16, p < 0.001). There was no difference in the cost of treatment between the groups (4.64 ± 1.97 vs. 5.72 ± 2.54 USD, p = 0.19). This study indicated that the Telfa AMD(®) was an effective dressing for the treatment of donor-site wounds. PMID:21954342

Muangman, Pornprom; Nitimonton, Sooksan; Aramwit, Pornanong

2011-01-01

80

Comparative Clinical Study of Bactigras and Telfa AMD for Skin Graft Donor-Site Dressing  

PubMed Central

The Bactigras® paraffin tulle coated with chlorhexidine is normally used for the treatment of donor-site wounds in burn patients who received split-thickness skin grafts in several centers. It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD®, a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras® or the Telfa AMD® wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (?90%) was significantly shorter in patients treated with the Telfa AMD® compared to the Bactigras® group (14.00 ± 3.05 vs. 9.25 ± 1.88 days for Bactigras® and Telfa AMD® groups, respectively, p < 0.001). The average pain score was also significantly lower in the Telfa AMD® group (1.57 ± 0.55 vs. 4.70 ± 1.16, p < 0.001). There was no difference in the cost of treatment between the groups (4.64 ± 1.97 vs. 5.72 ± 2.54 USD, p = 0.19). This study indicated that the Telfa AMD® was an effective dressing for the treatment of donor-site wounds. PMID:21954342

Muangman, Pornprom; Nitimonton, Sooksan; Aramwit, Pornanong

2011-01-01

81

The missing puzzle piece: splicing mutations  

PubMed Central

Proper gene splicing is highly dependent on the correct recognition of exons. Among the elements allowing this process are the “cis” (conserved sequences) and “trans” (snRNP, splicing factors) elements. Splicing mutations are related with a number of genetic disorders and usually induce exon skipping, form new exon/intron boundaries or activate new cryptic exons as a result of alterations at donor/acceptor sites. They constitute more than 9% of the currently published mutations, but this value is highly underestimated as many of the potential mutations are located in the “cis” elements and should be confirmed experimentally. The most commonly detected splicing mutations are located at donor (5’) and acceptor (3’) sites. Mutations at the branch point are rare (only over a dozen are known to date), and are mostly searched and detected when no alteration has been detected in the sequenced exons and UTRs. Polypyrimidine tract mutations are equally rare. High throughput technologies, as well as traditional Sanger sequencing, allow detection of many changes in intronic sequences and intron/exon boundaries. However, the assessment whether a mutation affects exon recognition and results in a genetic disorder has to be conducted using molecular biology methods: in vitro transcription of the sequence of interest cloned into a plasmid, with and without alterations, or mutation analysis via a hybrid minigene system. Even though microarrays and new generation sequencing methods pose difficulties in detecting novel branch point mutations, these tools seem appropriate to expand the mutation detection panel especially for diagnostic purposes. PMID:24294354

Lewandowska, Marzena A

2013-01-01

82

A novel splice site mutation in the EYA1 gene in a Korean family with branchio-oto (BO) syndrome.  

PubMed

Branchio-oto-renal (BOR) and branchio-oto (BO) syndromes are autosomal dominant hereditary disorders characterized by the presence of hearing loss and branchial fistulae and cysts, with (BOR syndrome) or without (BO syndrome) renal malformations of varying degrees of severity. Mutations in the human homologous of the Drosophila eyes absent (EYA1) gene are frequently the cause of BOR/BO syndrome. Here we describe a Korean family with BO syndrome; the proband had preauricular pit, cup-shaped auricles, branchial fistula, and hearing loss, without renal involvement. Molecular genetic study revealed a novel mutation occurring in the consensus acceptor splice site of intron 8 (c.868-2A > G) in the EYA1 gene. To the best of our knowledge, this is the first report of a splice site mutation in a family with BO syndrome without renal involvement, further extending the phenotypic-genotypic heterogeneity of BOR/BO syndrome. PMID:18763178

Kwon, Min-Jung; Boo, Sung Hyun; Kim, Hee-Jin; Cho, Yang-Sun; Chung, Won-Ho; Hong, Sung Hwa

2009-06-01

83

An Additional Option for Split-Thickness Skin Graft Donors: The Previous Free Flap Sites.  

PubMed

Free flap reconstruction is the best choice for soft-tissue defect. However, there are often accompanying problems such as partial flap loss, donor-site skin problems, and loss of previous skin grafts surrounding the flap site. This is especially true when dealing with multiple trauma, complex defects, and large skin flaps. Because of the simplicity of the procedure involved, split-thickness skin grafts are usually used for reconstructing skin and soft-tissue defects. These are also a good choice when there is a need for further procedures because of defects from several potential causes. Pain and the loss of healthy donor tissue are major concerns in such operations. Hence, we thought that the previous skin flap area might be a good alternative area for split-thickness skin grafts accompanying procedures subsequent to free flap reconstruction. Because this donor area is no longer sensitive, local anesthesia can be used during harvesting, and there is no loss of healthy donor tissue. Therefore, this procedure is an economical means of obtaining tissue for soft-tissue reconstruction. We describe 9 examples of flap reconstruction done in this way and suggest that this is a useful option for donor site. PMID:24691323

Kim, Sang Wha; Choi, Seung Hyup; Kim, Jeong Tae; Kim, Youn Hwan

2014-03-28

84

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.  

PubMed

BackgroundMutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.MethodsIn this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234¿+¿1G¿>¿A, c.633¿+¿1G¿>¿A and c.1542¿+¿4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A¿>¿G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234¿+¿1G¿>¿A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.ConclusionsWe have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications. PMID:25491247

Matos, Liliana; Canals, Isaac; Dridi, Larbi; Choi, Yoo; Prata, Maria; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Pshezhetsky, Alexey V; Grinberg, Daniel; Alves, Sandra; Vilageliu, Lluïsa

2014-12-10

85

Reduction of Donor Site Morbidity of Free Radial Forearm Flaps: What Level of Evidence Is Available?  

PubMed Central

Background: The radial forearm free flap (RFFF) is the most commonly used free flap in head and neck reconstructive surgery. However, despite excellent results with respect to the site of reconstruction, donor site morbidity cannot be neglected. This review summarizes the current state of knowledge and analyzes the level of evidence with regard to perioperative management of the reduction of RFFF donor site morbidity. Methods: The medical Internet source PubMed was screened for relevant articles. All relevant articles were tabulated according to the levels of scientific evidence, and the available methods for reduction of donor site morbidity are discussed. Results: Classification into levels of evidence reveals 3 publications (1.5%) with level I (randomized controlled trials), 29 (14.0%) with level II (experimental studies with no randomization, cohort studies, or outcome research), 3 (1.5%) with level III (systematic review of case-control studies or individual case-control studies), 121 (58.7%) with level IV (nonexperimental studies, such as cross-sectional trials, case series, case reports), and 15 (7.3%) with level V (narrative review or expert opinion without explicit critical appraisal). Thirty-five (17.0%) articles could not be classified, because they focused on a topic other than donor site morbidity of the RFFF. Conclusions: Although great interest has been expressed with regard to reducing the donor site morbidity of the workhorse flap in microvascular reconstruction procedures, most publications fail to provide the hard facts and solid evidence characteristic of high-quality research. PMID:22331991

Loeffelbein, Denys J.; Al-Benna, Sammy; Steinsträßer, Lars; Satanovskij, Robin M.; Rohleder, Nils H.; Mücke, Thomas; Wolff, Klaus-Dietrich; Kesting, Marco R.

2012-01-01

86

A Novel Splice-Site Mutation in ALS2 Establishes the Diagnosis of Juvenile Amyotrophic Lateral Sclerosis in a Family with Early Onset Anarthria and Generalized Dystonias  

PubMed Central

The diagnosis of childhood neurological disorders remains challenging given the overlapping clinical presentation across subgroups and heterogeneous presentation within subgroups. To determine the underlying genetic cause of a severe neurological disorder in a large consanguineous Pakistani family presenting with severe scoliosis, anarthria and progressive neuromuscular degeneration, we performed genome-wide homozygosity mapping accompanied by whole-exome sequencing in two affected first cousins and their unaffected parents to find the causative mutation. We identified a novel homozygous splice-site mutation (c.3512+1G>A) in the ALS2 gene (NM_020919.3) encoding alsin that segregated with the disease in this family. Homozygous loss-of-function mutations in ALS2 are known to cause juvenile-onset amyotrophic lateral sclerosis (ALS), one of the many neurological conditions having overlapping symptoms with many neurological phenotypes. RT-PCR validation revealed that the mutation resulted in exon-skipping as well as the use of an alternative donor splice, both of which are predicted to cause loss-of-function of the resulting proteins. By examining 216 known neurological disease genes in our exome sequencing data, we also identified 9 other rare nonsynonymous mutations in these genes, some of which lie in highly conserved regions. Sequencing of a single proband might have led to mis-identification of some of these as the causative variant. Our findings established a firm diagnosis of juvenile ALS in this family, thus demonstrating the use of whole exome sequencing combined with linkage analysis in families as a powerful tool for establishing a quick and precise genetic diagnosis of complex neurological phenotypes. PMID:25474699

Vu, Anthony; Azim, Saad; Silver, David L.; Mansoor, Atika; Tay, Stacey Kiat Hong; Abbasi, Sumiya; Hashmi, Asraf Hussain; Janjua, Jamal; Khalid, Sumbal; Tai, E. Shyong; Yeo, Gene W.; Khor, Chiea Chuen

2014-01-01

87

Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice.  

PubMed

Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5' and 3' alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate. PMID:20467437

Llères, David; Denegri, Marco; Biggiogera, Marco; Ajuh, Paul; Lamond, Angus I

2010-06-01

88

A simple model to explain evolutionary trends of eukaryotic gene architecture and expression: how competition between splicing and cleavage/polyadenylation factors may affect gene expression and splice-site recognition in eukaryotes.  

PubMed

Enormous phylogenetic variation exists in the number and sizes of introns in protein-coding genes. Although some consideration has been given to the underlying role of the population-genetic environment in defining such patterns, the influence of the intracellular environment remains virtually unexplored. Drawing from observations on interactions between co-transcriptional processes involved in splicing and mRNA 3'-end formation, a mechanistic model is proposed for splice-site recognition that challenges the commonly accepted intron- and exon-definition models. Under the suggested model, splicing factors that outcompete 3'-end processing factors for access to intronic binding sites concurrently favor the recruitment of 3'-end processing factors at the pre-mRNA tail. This hypothesis sheds new light on observations such as the intron-mediated enhancement of gene expression and the negative correlation between intron length and levels of gene expression. PMID:23568225

Catania, Francesco; Lynch, Michael

2013-06-01

89

Gracilis myocutaneous flap: evaluation of potential risk factors and long-term donor-site morbidity.  

PubMed

This study reviewed our experience with the gracilis myocutaneous (GMC) flap, potential risk factors for flap necrosis, and long-term morbidity at the donor-site. From 1993 to 2002, 29 GMC flaps were harvested from 27 patients (pedicled n = 21 and free n = 8). The overall incidence of flap necrosis was 13.79% (partial (n = 2) and total (n = 2) necrosis). Flap necrosis was correlated with body mass index >25 (P = 0.022), with smoking (P = 0.04 9) and with radiation therapy at the recipient site (P = 0.020). The long-term morbidity at the donor-site was low, except for scar appearance (17.24%), thigh contour deformity (58.62%), and hypoesthesia (17.24%). Significant age and gender differences were seen for ranking of scar ugliness, with females (P = 0.0061) and younger patients (age ?55) (P = 0.046) assigned higher values. Significant age differences were seen for ranking of thigh contour deformity, with younger patients assigned higher values (P = 0.0012). In conclusion, patient overweight, smoking, and previous radiation therapy at the recipient site may be the "potential risk factors" for flap necrosis. The long-term morbidity at the donor-site was low, which was in agreement with previous reported studies. A larger series would be the subject of a future study. PMID:21898880

Papadopoulos, Othon; Konofaos, Petros; Georgiou, Panos; Chrisostomidis, Chrisostomos; Tsantoulas, Zacharias; Karypidis, Dimitrios; Kostakis, Alkiviadis

2011-09-01

90

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

91

The properties of the “ideal” donor site dressing: results of a worldwide online survey  

PubMed Central

Summary Split skin grafting is a widely used technique for reconstructing skin defects. Although a vast number of different coverage options for donor sites have become available in daily clinical practice, no optimum dressing material has been found to date. For this reason, we conducted a globally-distributed online survey to poll for the properties of such an “ideal” donor site dressing, possibly leading to an improved clinically-driven direction of future wound dressing developments. A total of 69 respondents from 34 countries took part in the questionnaire, resulting in a response rate of 13.8% (69/500) over a 1-month period. The majority of respondents rated the characteristics of an “ideal” donor site dressing to be either “essential” or “desirable” as follows: lack of adhesion to the wound bed (“essential”: 31/69, 44.9%; “desirable”: 30/69, 43.5%); pain-free dressing changes (“essential”: 38/69, 55.1%; “desirable”: 30/69, 43.5%); absorbency (“essential”: 27/69, 39.1%; “desirable”: 33/69, 47.8%); ease of removal (“essential”: 37/69, 53.6%; “desirable”: 27/69, 39.13%). With regard to the desired frequency of dressing changes, respondents preferred “no dressing change until the donor site has healed” (51/69, 73.9%) in the majority of cases, followed by “twice weekly” (10/69, 14.5%), “alternate days” (5/69, 7.2%) and “daily” (3/69, 4.3%). With regard to the design of the dressing material, the majority of participants preferred a one-piece (composite) dressing product (44/69, 63.8%). The majority of respondents also denied the current availability of an “ideal” donor site dressing (49/69, 71%). The strength of this study was the remarkable geographic distribution of responses; all parts of the world were included and participated. We believe that this globally conducted online survey has polled for the properties of the “ideal” donor site dressing and possibly will lead to an improved clinically-driven direction of future wound dressing development. PMID:24563639

Lars, P. Kamolz L.P.; Giretzlehner, M.; Trop, M.; Parvizi, D.; Spendel, S.; Schintler, M.; Justich, I.; Wiedner, M.; Laback, C.; Lumenta, D.B..

2013-01-01

92

Comparison of scalp and abdomen as split-thickness skin graft donor sites for aural stenosis repair.  

PubMed

To evaluate and compare the scalp and the abdomen as split-thickness skin graft donor sites for aural stenosis repair. A total of 34 patients with aural stenosis were included in the study. All the patients underwent meatoplasty using split-thickness skin grafts. Among them, the skin graft donor site was the scalp in 11 patients and the abdomen in the other 23 patients. The surgical team followed the patients in the outpatient department for at least 6 months after surgery. Evaluations concerned healing of the donor site, hair regeneration of the donor site, survival of split-thickness skin grafts, reoccurrence of aural stenosis and hair growth in the ear canal. The incidences of reoccurrence of aural stenosis in the two groups were compared. Subjective scar evaluation of the donor sites was performed using the Patient Scar Assessment Scale (PASA). The scale items were pain, itching, color, stiffness, thickness and irregularity. All the scalp and abdominal donor sites healed well with no sign of infection. Hair regrowth and reepithelialization was observed at all the scalp donor sites. Pink discoloration was observed at the scalp donor sites in six patients 2-3 months after surgery and disappeared 6-9 months after surgery. Scars were observed at the scalp donor sites in two patients 6 months after surgery. No alopecia was observed at the scalp donor sites. The scars and pink discoloration were hidden in the hair. Scars and/or discoloration were observed at all the abdominal donor sites 12 months after surgery. All the scalp and abdominal skin grafts survived with no sign of infection. Hair growth was observed in the ear canals in two patients in the scalp group. The incidences of reoccurrence of aural stenosis were 0 % (0/23) in the abdominal group and 9.1 % (1/11) in the scalp group, respectively (Chi square test, p > 0.05). The PASA values about color, stiffness, thickness and irregularity were higher in the abdominal group than in the scalp group (Mann-Whitney U test, p < 0.001). The PASA values about pain and itching were the same (Mann-Whitney U test, p > 0.05). The scalp meets most requirements of an ideal donor site of skin grafts for aural stenosis. The advantages of scalp as a donor site include easy accessibility in the operative field, simple postoperative care, low risk of infection, rapid wound healing, minimal interference with rehabilitation, and minimal scar formation. PMID:24057102

Du, Qiang; Zhang, Tianyu

2014-08-01

93

Successful Introduction of Laparoendoscopic Single-Site Donor Nephrectomy After Experience with Laparoscopic Single-Site Plus-One Trocar Donor Nephrectomy.  

PubMed

Abstract Purpose: To assess the feasibility, safety, and efficacy of the laparoendoscopic single-site (LESS) donor nephrectomy (LESSDN) procedure after experience with the LESS-plus-one-trocar donor nephrectomy (LEPODN) procedure. Patients and Methods: From 2009 to 2014, 126 left laparoscopic donor nephrectomies (LDNs) were performed, including 59 Standard (Std)-LDN, 30 LEPODN, and 37 LESSDN. In the LEPODN procedure, a 5-mm trocar was added as a right-hand working trocar to the LESSDN procedure. A GelPOINT(®) platform was applied on a pararectal single incision in both LEPODN and LESSDN procedures. After performing the LEPODN procedure several times, each surgeon performed the LESSDN procedure. Results: Std-LDN, LEPODN, and LESSDN procedures were performed by 10, 10, and 7 surgeons, respectively. The mean operative time, estimated blood loss, warm ischemia time, time to ambulation, and length of postoperative hospital stay were the shortest for the LESSDN procedure (P<0.012, P=0.007, P<0.001, P=0.027, and P=0.001, respectively). No significant difference in the complication rate, delayed graft function rate, and mean 7-day post-transplant serum creatinine levels was observed among the three procedures. Individual results of the operative time and estimated blood loss for the LESSDN procedure were not significantly inferior to those of Std-LDN and LEPODN procedures for each surgeon. Conclusions: The LESSDN procedure can be introduced safely and effectively without compromising the operative time, complication rate, and graft function after experience with the LEPODN procedure among multiple surgeons. The LEPODN procedure may be an effective bridge from standard multiport LDN to LESSDN. PMID:25286117

Inoue, Takamitsu; Tsuchiya, Norihiko; Narita, Shintaro; Tsuruta, Hiroshi; Akihama, Susumu; Saito, Mitsuru; Satoh, Shigeru; Habuchi, Tomonori

2014-11-14

94

An abnormal mRNA produced by a novel PMP22 splice site mutation associated with HNPP  

PubMed Central

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating neuropathy. Point mutations in the PMP22 gene are a rare cause of HNPP. A novel PMP22 splice site mutation (c.179+1 G?C) is reported in an HNPP family. By reverse transcriptase?polymerase chain reaction experiments, this mutation was shown to cause the synthesis of an abnormal mRNA in which a premature stop codon probably produces a truncated non?functional protein. PMID:16199442

Bellone, E; Balestra, P; Ribizzi, G; Schenone, A; Zocchi, G; Maria, E Di; Ajmar, F; Mandich, P

2006-01-01

95

Solid phase electron donors control denitrification in groundwater at agricultural sites  

NASA Astrophysics Data System (ADS)

Increased concentrations of nitrate in groundwater caused by agricultural use of chemical and organic fertilizers are a concern because of possible risks to environmental and human health. At many sites, these problems are mitigated by natural attenuation of nitrate as a result of microbially mediated denitrification of nitrate to nitrogen gas. Recent studies have clarified the factors affecting the rates and extents of denitrification in groundwater in agricultural areas. Intensive studies were conducted by the US Geological Survey to study agricultural chemicals in California, Nebraska, Washington, and Maryland using laboratory analyses, field measurements, and flow and transport modeling for monitoring well transects (0.5 to 2.5 km in length) and vertical profiles (0 to 50 m in depth). Groundwater analyses included major ion chemistry, dissolved gases, nitrogen and oxygen stable isotopes, and atmospheric age-tracers. Sediments were analyzed for concentrations of potential electron donors for denitrification, including reduced iron and sulfur, and organic carbon. Geochemical data and mass balance calculations indicated that solid-phase electron donors were an important factor controlling denitrification at these sites. To examine the generality of this result, a mathematical model of vertical flux of water, oxygen, and nitrate was developed and applied at these study sites along with 2 new study sites in Iowa and Mississippi and 8 additional sites from previous studies in Nebraska, Texas, Minnesota, Wisconsin, North Carolina, Maryland (2 sites), and New York. Model results confirmed the importance of solid phase electron donors. The normalized reaction rates on an electron flux basis tended to increase with depth from the shallow oxygen reduction zone to the underlying nitrate reduction zone. The pattern of higher rates at depth is consistent with a reaction rate controlled by solid phase donors that are depleted under oxidizing conditions near the surface and in greater supply at depth. The eventual depth and rate of migration of nitrate and oxygen in aquifers will depend on the concentrations and reactivities of solid electron donor phases currently in the reduced zones.

Green, C. T.; Liao, L.; Bekins, B. A.; Bohlke, J. K.

2011-12-01

96

Iatrogenic implantation of giant cell tumor at bone graft donor site and clinical recommendations to prevent "a rare avoidable complication".  

PubMed

The treatment of giant cell tumor of bone is directed toward local control without sacrificing joint function. This is achieved by intralesional curettage. When autograft is used for the reconstruction of the curetted cavity, there is always a theoretical risk of contamination of graft donor site. We report a case of iatrogenic implantation of giant cell tumor at the bone graft donor site after intralesional curettage and bone grafting of giant cell tumor of distal femur. Patient was treated with repeat intralesional curettage and excision of implantation lesion at bone graft donor site. We recommend precautionary measures to prevent this avoidable complication. PMID:23412188

Gulia, Ashish; Puri, Ajay; Salunke, Abhijeet; Desai, Subhash; Jambhekar, N A

2013-08-01

97

Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5? Splice Site Choice  

PubMed Central

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5? and 3? splice site (5?ss and 3?ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5?ss (u5?ss and d5?ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5?ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5?ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5?ss and d5?ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5?ss is intrinsically weaker than d5?ss, with a similar tendency observed for other genes with Ptbp1-induced u5?ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5?ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism. PMID:25375251

Hamid, Fursham M.; Makeyev, Eugene V.

2014-01-01

98

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

99

Deletions in cox2 mRNA Result in Loss of Splicing and RNA Editing and Gain of Novel RNA Editing Sites  

PubMed Central

As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites. PMID:24324745

Kumar, Abhishek; Kempken, Frank

2013-01-01

100

Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.  

PubMed

U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites. PMID:25555158

Kondo, Yasushi; Oubridge, Chris; van Roon, Anne-Marie M; Nagai, Kiyoshi

2015-01-01

101

Efficacy of Quilting Sutures and Fibrin Sealant Together for Prevention of Seroma in Extended Latissimus Dorsi Flap Donor Sites  

PubMed Central

Background The extended latissimus dorsi flap is important for breast reconstruction. Unfortunately, donor site seroma is the most common complication of extended latissimus dorsi flap for breast reconstruction. Although using fibrin sealant in the donor site reduces the rate of seroma formation, donor site seroma remains a troublesome complication. The purpose of this study was to analyze the effectiveness of the combination of quilting sutures and fibrin sealant in the latissimus dorsi donor site for the prevention of seroma. Methods Forty-six patients who underwent breast reconstruction with extended latissimus flap were enrolled in the study. The patients received either fibrin sealant (group 1, n=25) or a combination of fibrin sealant and quilting sutures (group 2, n=21) in the extended latissimus dorsi donor site. Outcome measures were obtained from the incidence, volume of postoperative seroma, total drainage amount, indwelling period of drainage, and duration of hospital stay. Results The incidence of seroma was 76% in group 1 and 42.9% in group 2 (P=0.022). We also found significant reductions in seroma volume (P=0.043), total drainage amount (P=0.002), indwelling period of drainage (P=0.01), and frequency of aspiration (P=0.043). The quilting sutures did not affect the rate of drainage, tube reinsertion, or hospital stay. Conclusions The use of quilting sutures combined with fibrin sealant on the latissimus dorsi flap donor site is helpful for reducing the overall seroma volume, frequency of aspiration, and total drainage amount. PMID:23094247

Shin, In Soo; Lee, Dong Won

2012-01-01

102

Computer-based planning of optimal donor sites for autologous osseous grafts  

NASA Astrophysics Data System (ADS)

Bone graft surgery is often necessary for reconstruction of craniofacial defects after trauma, tumor, infection or congenital malformation. In this operative technique the removed or missing bone segment is filled with a bone graft. The mainstay of the craniofacial reconstruction rests with the replacement of the defected bone by autogeneous bone grafts. To achieve sufficient incorporation of the autograft into the host bone, precise planning and simulation of the surgical intervention is required. The major problem is to determine as accurately as possible the donor site where the graft should be dissected from and to define the shape of the desired transplant. A computer-aided method for semi-automatic selection of optimal donor sites for autografts in craniofacial reconstructive surgery has been developed. The non-automatic step of graft design and constraint setting is followed by a fully automatic procedure to find the best fitting position. In extension to preceding work, a new optimization approach based on the Levenberg-Marquardt method has been implemented and embedded into our computer-based surgical planning system. This new technique enables, once the pre-processing step has been performed, selection of the optimal donor site in time less than one minute. The method has been applied during surgery planning step in more than 20 cases. The postoperative observations have shown that functional results, such as speech and chewing ability as well as restoration of bony continuity were clearly better compared to conventionally planned operations. Moreover, in most cases the duration of the surgical interventions has been distinctly reduced.

Krol, Zdzislaw; Chlebiej, Michal; Zerfass, Peter; Zeilhofer, Hans-Florian U.; Sader, Robert; Mikolajczak, Pawel; Keeve, Erwin

2002-05-01

103

The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA  

PubMed Central

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize. PMID:23739895

Regulski, Michael; Lu, Zhenyuan; Kendall, Jude; Donoghue, Mark T.A.; Reinders, Jon; Llaca, Victor; Deschamps, Stephane; Smith, Andrew; Levy, Dan; McCombie, W. Richard; Tingey, Scott; Rafalski, Antoni; Hicks, James; Ware, Doreen; Martienssen, Robert A.

2013-01-01

104

The evolution of mRNA splicing in mammals  

E-print Network

In this thesis, I describe investigations into the evolution of splicing in mammals. I first investigate a small class of alternative splicing events, tandem splice sites, and show how they are used to introduce and remove ...

Merkin, Jason Jay

2014-01-01

105

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients  

Microsoft Academic Search

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are

R. G. Geissler; J. Muth; A. Maurer; U. Mentzel; M. Mag; J. W. Engels; D. Hoelzer; A. Ganser

1995-01-01

106

Fat-plug myringoplasty of ear lobule vs abdominal donor sites.  

PubMed

The purpose of this study is to compare the success rates of fat-graft myringoplasties harvesting adipose grafts from different donor sites (ear lobule vs abdomen). The clinical records of 61 patients (24 males and 37 females) who underwent fat-plug myringoplasty (FPM) were reviewed retrospectively. Fat from ear lobule (FEL) and abdominal fat were used as graft materials. The impact of age, gender, systemic diseases, topography of the perforation, utilization of fat graft materials of different origin on the tympanic membrane closure rate and the effect of FPM on hearing gain was analyzed. Our tympanic membrane (TM) closure rate was 82 %. No statistical significant difference was observed regarding age, gender, comorbidities (septal deviation, hypertension and diabetes mellitus) or habits (smoking). Posterior TM perforations had significantly lower healing rate. The change in TM closure rate considering different adipose tissue donor sites was not statistically significant. The hearing gain of the patients was mostly below 20 dB. Fat-plug myringoplasty (FPM) is a safe, cost-effective and easy operation for selected patients. Abdominal fat graft is as effective as ear lobe fat graft on tympanic membrane healing, has cosmetic advantages and should be taken into consideration when planning fat as the graft source. PMID:24469028

Acar, Mustafa; Yaz?c?, Demet; San, Turhan; Muluk, Nuray Bayar; Cingi, Cemal

2014-01-28

107

Gene expression profiling of negative-pressure-treated skin graft donor site wounds.  

PubMed

Negative-pressure wound therapy (NPWT) is widely used to improve skin wound healing. Although NPWT has been studied as a treatment for wound closure and healing, the molecular mechanisms explaining its therapeutic effects remain unclear. To investigate the effect of NPWT on gene expression, and to discover the genes most dominantly responding to this treatment during skin wound healing, we applied negative pressure on split-thickness skin graft donor sites from the first postoperative day (POD) to the seventh POD. Biopsies were collected from 4 NPWT-treated and 2 control patients. Two biopsy samples were taken from each patient: one from intact skin before graft harvesting, and one on the seventh POD from the donor site wound. Genome-wide microarrays were performed on all samples. Gene expression changes on the seventh POD were compared between NPWT and control patients, and were analyzed for statistical significance. In addition, we analyzed wound exudates for volume, and for concentrations of leukocytes, erythrocytes, and haemoglobin. NPWT induced major changes in gene expression during healing. These changes ranged from 10-fold induction to 27-fold suppression. The genes most induced were associated with cell proliferation and inflammation, and the most down-regulated genes were linked to epidermal differentiation. Our results provide the first insight into the molecular mechanisms behind NPWT, and suggest that NPWT enhances specific inflammatory gene expression at the acute phase associated with epithelial migration and wound healing. However, its continued use may inhibit epithelial differentiation. PMID:23141686

Nuutila, Kristo; Siltanen, Antti; Peura, Matti; Harjula, Ari; Nieminen, Tapio; Vuola, Jyrki; Kankuri, Esko; Aarnio, Pertti

2013-06-01

108

Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase.  

PubMed

The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs. PMID:9020165

Liu, Y; George, C X; Patterson, J B; Samuel, C E

1997-02-14

109

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.  

PubMed Central

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing. PMID:8608445

Reyes, J L; Kois, P; Konforti, B B; Konarska, M M

1996-01-01

110

Results of an internet survey on the treatment of partial thickness burns, full thickness burns, and donor sites.  

PubMed

The objective of this study was to analyze which materials and methods are used for the management of partial and full thickness burns, as well as donor sites. An Internet survey was used to poll directors of burn centers around the world on their preferences for local treatment of different types of burns and donor sites. Results were tabulated and expressed as a percentage of the total number of answers for a given indication. Although many new wound care materials have been launched in the last decade, few of these actually are used widely. The most commonly used materials for partial thickness burns and donor sites are still silver sulphadiazine 1% cream, other antimicrobial ointments and creams and impregnated gauze type dressings. Of the newly available treatment modalities, only two silver dressings were chosen frequently as a primary option for the management of partial thickness burns and donor sites. For full thickness burns, the primary choice is excision and grafting. The diversity of dressings and techniques indicated as preferred in this survey, including many that are known to have side effects, indicates that there is no consensus on topical treatment of partial thickness burns and donor sites. Many respondents prefer "tried and true" materials over newer dressings, particularly if the latter have not been tested in a clinical trial. PMID:17925651

Hermans, Michel H E

2007-01-01

111

Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 2'-OH in catalysis.  

PubMed

The ai5gamma group II intron from yeast excises itself from precursor transcripts in the absence of proteins. When a shortened form of the intron containing all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing. As this tripartite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting. We use this framework to characterize the metal ion specificity switch observed previously upon sulfur substitution of the 3'-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis. The results suggest that both the 3'-oxygen leaving group and the neighboring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond. Metal specificity switch experiments combined with quantitative analysis show that the Mn(2+) that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raising the possibility that a single metal ion mediates interactions with the 2'- and 3'-oxygen atoms at the 3' splice site. PMID:11041859

Gordon, P M; Sontheimer, E J; Piccirilli, J A

2000-10-24

112

Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.  

PubMed

The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore(®) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function. PMID:25462814

Bury, Daniel; Dahmane, Ismahene; Derouaux, Adeline; Dumbre, Shrinivas; Herdewijn, Piet; Matagne, André; Breukink, Eefjan; Mueller-Seitz, Erika; Petz, Michael; Terrak, Mohammed

2015-01-15

113

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.  

PubMed Central

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4. PMID:11333022

Kammler, S; Leurs, C; Freund, M; Krummheuer, J; Seidel, K; Tange, T O; Lund, M K; Kjems, J; Scheid, A; Schaal, H

2001-01-01

114

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing – a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites  

PubMed Central

Background Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC?+?K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. Results Healing time was reduced from 13.9?±?0.5 days (mean?±?SEM) in the control group to 7.2?±?0.2 days in the PC group (P?

2013-01-01

115

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5? splice site  

PubMed Central

Recognition of the 5? splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem–loop of domain 1 and a complementary sequence at the 3? end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5? intron, we probed the solution structure of the ID3 stem–loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5? and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5? splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1–IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5? and O.i. introns and may help to fine-tune elements of recognition in group II introns. PMID:24243113

Popovi?, Milena; Greenbaum, Nancy L.

2014-01-01

116

Integrated Study of Ice-Rafted Debris, Temperaturess, and Stable Isotopes on a Spliced Record (piston cores and ODP Site 177-1090) From the South Atlantic  

Microsoft Academic Search

We have conducted an integrated study of ice-rafted debris (IRD) and stable isotopes on a spliced record (TN057-6-PC4\\/ODP Site 177-1090, about 43° S, 9° E) raised on the Agulhas Ridge, in the South Atlantic. The site is just north of the northern boundary of the present-day Polar-Front Zone (PFZ), and is in a very sensitive location to record both ice-rafting

D. A. Warnke; L. Teitler; S. Becquey; R. Gersonde; K. Venz; D. A. Hodell

2003-01-01

117

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.  

PubMed

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics. PMID:24574459

Adamia, Sophia; Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Tenen, Daniel G; Stone, Richard M; Griffin, James D

2014-05-01

118

Alternative splicing and retinal degeneration  

PubMed Central

Alternative splicing is highly regulated in tissue-specific and development-specific patterns, and it has been estimated that 15% of disease-causing point mutations affect pre-mRNA splicing. In this review, we consider the cis-acting splice site and trans-acting splicing factor mutations that affect pre-mRNA splicing and contribute to retinal degeneration. Numerous splice site mutations have been identified in retinitis pigmentosa and various cone-rod dystrophies. For example, mutations in alternatively spliced retina-specific exons of the widely expressed RPGR and COL2A1 genes lead primarily to X-linked retinitis pigmentosa and ocular variants of Stickler Syndrome, respectively. Furthermore, mutations in general pre-mRNA splicing factors, such as PRPF31, PRPF8, and PRPF3, predominantly cause autosomal dominant retinitis pigmentosa. These findings suggest an important role for pre-mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule based therapies, has the potential to lead to the development of new treatments for retinal degenerative diseases. PMID:23647439

Liu, Melissa M.; Zack, Donald J.

2014-01-01

119

A randomized, prospective, parallel group study of laparoscopic versus laparoendoscopic single site donor nephrectomy for kidney donation.  

PubMed

Few prospective, randomized studies have assessed the benefits of laparoendoscopic single site donor nephrectomy (LESS-DN) over laparoscopic donor nephrectomy (LDN). Our center initiated such a trial in January 2011, following subjects randomized to LESS-DN versus LDN from surgery through 5 years postdonation. Subjects complete recovery/satisfaction questionnaires at 2, 6 and 12 months postdonation; transplant recipient outcomes are also recorded. One hundred subjects (49 LESS-DN, 51 LDN) underwent surgery; donor demographics were similar between groups, and included a predominance of female, living-unrelated donors, mean age of 47 years who underwent left donor nephrectomy. Operative parameters (overall time, time to extraction, warm ischemia time, blood loss) were similar between groups. Conversion to hand-assist laparoscopy was required in 3 LESS-DN (6.1%) versus 2 LDN (3.9%; p?=?0.67). Questionnaires revealed that 97.2% of LESS-DN versus 79.5% of LDN (p?=?0.03) were 100% recovered by 2 months after donation. No significant difference was seen in satisfaction scores between the groups. Recipient outcomes were similar between groups. Our randomized trial comparing LESS donor nephrectomy to LDN confirms that LESS-DN offers a safe alternative to conventional LDN in terms of intra- and post-operative complications. LDN and LESS-DN offer similar recovery and satisfaction after donation. PMID:24934732

Aull, M J; Afaneh, C; Charlton, M; Serur, D; Douglas, M; Christos, P J; Kapur, S; Del Pizzo, J J

2014-07-01

120

Evolution of splicing regulatory networks in Drosophila  

PubMed Central

The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5? splice sites, and alternative 3? splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3? splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ?80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ?50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation. PMID:24515119

McManus, C. Joel; Coolon, Joseph D.; Eipper-Mains, Jodi; Wittkopp, Patricia J.; Graveley, Brenton R.

2014-01-01

121

Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5' splice site within Col2a1 exon 2.  

PubMed

This study describes a new mechanism controlling the production of alternatively spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5' splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5' splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the ?3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue. PMID:24735995

Hering, Thomas M; Wirthlin, Louisa; Ravindran, Soumya; McAlinden, Audrey

2014-06-01

122

MutPred Splice: machine learning-based prediction of exonic variants that disrupt splicing  

PubMed Central

We have developed a novel machine-learning approach, MutPred Splice, for the identification of coding region substitutions that disrupt pre-mRNA splicing. Applying MutPred Splice to human disease-causing exonic mutations suggests that 16% of mutations causing inherited disease and 10 to 14% of somatic mutations in cancer may disrupt pre-mRNA splicing. For inherited disease, the main mechanism responsible for the splicing defect is splice site loss, whereas for cancer the predominant mechanism of splicing disruption is predicted to be exon skipping via loss of exonic splicing enhancers or gain of exonic splicing silencer elements. MutPred Splice is available at http://mutdb.org/mutpredsplice. PMID:24451234

2014-01-01

123

Two Siblings with Homozygous Pathogenic Splice-Site Variant in Mitochondrial Asparaginyl-tRNA Synthetase (NARS2).  

PubMed

A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl-tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood, whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In-gel activity staining after blue native-polyacrylamide gel electrophoresis confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole-exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3' splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV-transformed lymphoblasts, a specific decrease in the amount of charged mt-tRNA(Asn) was demonstrated as compared with controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease-associated aaRS2. PMID:25385316

Vanlander, Arnaud V; Menten, Björn; Smet, Joél; De Meirleir, Linda; Sante, Tom; De Paepe, Boel; Seneca, Sara; Pearce, Sarah F; Powell, Christopher A; Vergult, Sarah; Michotte, Alex; De Latter, Elien; Vantomme, Lies; Minczuk, Michal; Van Coster, Rudy

2015-02-01

124

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Al= of a 3' Splice Site In Vivo  

E-print Network

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential ...

Ma, Long

125

Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron  

PubMed Central

Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved ‘peripheral’ domains that buttress the core through predicted interhelical contacts, while smaller introns use loop–helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA–RNA and RNA–magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core. PMID:15107495

Lupták, Andrej; Doudna, Jennifer A.

2004-01-01

126

SLaP mapper: A webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes  

PubMed Central

The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ?39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website. PMID:25111964

Fiebig, Michael; Gluenz, Eva; Carrington, Mark; Kelly, Steven

2014-01-01

127

SLaP mapper: a webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes.  

PubMed

The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ?39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website. PMID:25111964

Fiebig, Michael; Gluenz, Eva; Carrington, Mark; Kelly, Steven

2014-09-01

128

Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation  

PubMed Central

Background Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups. Case presentation In this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475?+?1G?>?C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa. Conclusion This report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling. PMID:23506628

2013-01-01

129

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging  

PubMed Central

Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments. PMID:24935247

2014-01-01

130

Expanded alternative splice isoform profiling of the mouse Cav3.1/?1G T-type calcium channel  

PubMed Central

Background Alternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models. Results Transcript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/?1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns. Conclusion The determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/?1G channels does not explain the enhancement of T-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of T-type channels through this mechanism may influence other developmental neurological phenotypes. PMID:19480703

Ernst, Wayne L; Noebels, Jeffrey L

2009-01-01

131

Histone Deacetylase Activity Modulates Alternative Splicing  

PubMed Central

There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ?700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications. PMID:21311748

Hnilicová, Jarmila; Hozeifi, Samira; Dušková, Eva; Icha, Jaroslav; Tománková, Tereza; Stan?k, David

2011-01-01

132

Histone deacetylase activity modulates alternative splicing.  

PubMed

There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ?700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications. PMID:21311748

Hnilicová, Jarmila; Hozeifi, Samira; Dušková, Eva; Icha, Jaroslav; Tománková, Tereza; Stan?k, David

2011-01-01

133

Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.  

PubMed

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. PMID:24361966

Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

2014-03-01

134

A randomised controlled pilot study comparing Mepitel(®) and SurfaSoft(®) on paediatric donor sites treated with Recell(®).  

PubMed

This randomized controlled pilot study examined the effects of a silicone net dressing (Mepitel(®)) and a monofilament polyamide woven dressing (SurfaSoft(®)) on the rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal on paediatric donor sites treated with epithelial cell suspension (ReCell(®)). Fifteen children (1-15 years) admitted for acute or reconstructive burns procedures in a tertiary referral hospital in Australia were randomly assigned to the experimental group, Mepitel(®) (n=8) and to the control group, SurfaSoft(®) (n=7). All donor sites were treated with ReCell(®) and covered with the assigned dressing. Measurements of rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal were recorded every two days until the wound was healed. Results showed that there was no difference in the rate of epidermal maturation between the two groups. Less pain and force to remove the dressing was shown in the Mepitel(®) group when compared to SurfaSoft(®). The rate of epithelialisation was found to be an unreliable measure. Although additional research is required to support the results of this study, these results suggest that Mepitel's(®) pliable, self-adhesive and atraumatic properties may improve healing of ReCell(®) treated donor sites with less pain at dressing changes. This pilot study provides a strong base for further research in this area. PMID:21982622

Campanella, S D; Rapley, P; Ramelet, A-S

2011-12-01

135

Objective and subjective evaluation of donor-site morbidity after nipple sharing for nipple areola reconstruction.  

PubMed

Nipple reconstruction is of importance in achieving the best possible aesthetic outcome after breast reconstruction. Nipple sharing is a common technique; this study focused on the potential morbidity at the donor nipple. Between 2008 and 2012, 26 patients underwent nipple sharing at our institution. The donor nipple was examined before and after the procedure (mean follow-up of 21 months). Sensitivity, projection, diameter, and patient satisfaction were evaluated. The sensitivity in the donor nipple decreased, albeit insignificantly, from 1.2 g/mm(2) (0.8-1.6) to 1.8 g/mm(2) (0.8-4.8) (p = 0.054, n = 26). The projection due to graft removal decreased from 8.0 mm (6.8-10.0) to 4.5 mm (4.0-5.0) (p = 0.001). Of the patients, 88% were "very satisfied" or "somewhat satisfied" with the sensitivity and 89% with the symmetry between the donor and reconstructed nipple. At least 60% of the patients were "very satisfied" with all aesthetic outcome parameters (projection, appearance, naturalness, color, and shape). All patients would agree to undergo this procedure again, if necessary. Nipple sharing was associated with minimal morbidity at the donor nipple. The postoperative projection was adequate. Regardless of whether simultaneous mastopexy was performed, the loss of sensitivity was minimal and presumably imperceptible to the patient. By using no sutures after graft removal and letting the donor nipple heal spontaneously, scarring was minimized and the natural appearance and good sensitivity of the donor nipple were preserved. PMID:25465146

Haslik, W; Nedomansky, J; Hacker, S; Nickl, S; Schroegendorfer, K F

2015-02-01

136

Genotype-phenotype associations in neurofibromatosis type 1 (NF1): an increased risk of tumor complications in patients with NF1 splice-site mutations?  

PubMed Central

Neurofibromatosis type 1 (NF1) is a complex neurocutaneous disorder with an increased susceptibility to develop both benign and malignant tumors but with a wide spectrum of inter and intrafamilial clinical variability. The establishment of genotype-phenotype associations in NF1 is potentially useful for targeted therapeutic intervention but has generally been unsuccessful, apart from small subsets of molecularly defined patients. The objective of this study was to evaluate the clinical phenotype associated with the specific types of NF1 mutation in a retrospectively recorded clinical dataset comprising 149 NF1 mutation-known individuals from unrelated families. Each patient was assessed for ten NF1-related clinical features, including the number of café-au-lait spots, cutaneous and subcutaneous neurofibromas and the presence/absence of intertriginous skin freckling, Lisch nodules, plexiform and spinal neurofibromas, optic gliomas, other neoplasms (in particular CNS gliomas, malignant peripheral nerve sheath tumors (MPNSTs), juvenile myelomonocytic leukemia, rhabdomyosarcoma, phaechromocytoma, gastrointestinal stromal tumors, juvenile xanthogranuloma, and lipoma) and evidence of learning difficulties. Gender and age at examination were also recorded. Patients were subcategorized according to their associated NF1 germ line mutations: frame shift deletions (52), splice-site mutations (23), nonsense mutations (36), missense mutations (32) and other types of mutation (6). A significant association was apparent between possession of a splice-site mutation and the presence of brain gliomas and MPNSTs (p?=?0.006). If confirmed, these findings are likely to be clinically important since up to a third of NF1 patients harbor splice-site mutations. A significant influence of gender was also observed on the number of subcutaneous neurofibromas (females, p?=?0.009) and preschool learning difficulties (females, p?=?0.022). PMID:23244495

2012-01-01

137

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects  

PubMed Central

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5?ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5?ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5?ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition. PMID:22362925

Fernandez Alanis, Eugenio; Pinotti, Mirko; Dal Mas, Andrea; Balestra, Dario; Cavallari, Nicola; Rogalska, Malgorzata E.; Bernardi, Francesco; Pagani, Franco

2012-01-01

138

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects.  

PubMed

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5'ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5'ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5'ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition. PMID:22362925

Fernandez Alanis, Eugenio; Pinotti, Mirko; Dal Mas, Andrea; Balestra, Dario; Cavallari, Nicola; Rogalska, Malgorzata E; Bernardi, Francesco; Pagani, Franco

2012-06-01

139

A sequence compilation and comparison of exons that are alternatively spliced in neurons.  

PubMed Central

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation. PMID:8202349

Stamm, S; Zhang, M Q; Marr, T G; Helfman, D M

1994-01-01

140

DNA donors for sequencing at Celera, Craig VenterSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Craig Venter DNAi Location:Genome>the project>players>private project The private project's DNA donors Craig Venter, the leader of the private genome effort at Celera Genomics, talks about the sources of the DNA used in their sequence.

2008-10-06

141

Expression studies of a novel splice site mutation in the LIPH gene identified in a Japanese patient with autosomal recessive woolly hair.  

PubMed

Autosomal recessive woolly hair (ARWH) is characterized by short and tightly curled scalp hair without any obvious complications. The disease is known to be caused by either lipase H (LIPH) or LPAR6 genes. Proteins encoded by these two genes are closely related to each other in a lipid-signaling pathway that is believed to play crucial roles in hair follicle development and hair growth. In the Japanese population, most affected individuals with ARWH have been shown to carry two prevalent founder mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.742C>A (p.His248Asn), while other LIPH mutations have been occasionally identified. In this study, we analyzed a Japanese patient with ARWH, and identified compound heterozygous mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.982+5G>T. The latter one was a novel splice site mutation in intron 7. Expression studies using blood-derived RNA from the patient detected the LIPH transcript from the c.736T>A mutant allele, but not from the c.982+5G>T mutant allele. Furthermore, in vitro transcription assay in cultured cells showed that the mutation c.982+5G>T caused an aberrant splicing event, leading to a frame-shift and a premature termination codon (p.Met328Serfs*41). To the best of our knowledge, this is the second splice site mutation in the LIPH gene, and our findings further expand the spectrum of the LIPH mutations underlying ARWH. PMID:25271093

Hayashi, Ryota; Inui, Shigeki; Farooq, Muhammad; Ito, Masaaki; Shimomura, Yutaka

2014-10-01

142

Multiple U2AF65 binding sites within SF3b155: Thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors  

PubMed Central

Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA Auxiliary Factor (U2AF65) with the Splicing Factor 1 (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF homology motif (UHM) of U2AF65. SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF65-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF65-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF65 interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF65 complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF65 binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF65 interacting domains is stabilized by 3.3 kcal mol?1 relative to the complex of the SF3b155/U2AF65 interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF65 binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)nXRW(DE) consensus sequence for predicting U2AF65-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding. PMID:16376933

Thickman, Karen R.; Swenson, Matthew; Kabogo, Joseph M.; Gryczynski, Zygmunt; Kielkopf, Clara L.

2007-01-01

143

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome.  

PubMed

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40% of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1. PMID:24590738

Kim, Hui Ram; Song, Mee Hyun; Kim, Min-A; Kim, Ye-Ri; Lee, Kyu-Yup; Sonn, Jong Kyung; Lee, Jaetae; Choi, Jae Young; Kim, Un-Kyung

2014-07-01

144

Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17  

PubMed Central

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splice-donor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10. PMID:10393977

Varani, Luca; Hasegawa, Masato; Spillantini, Maria Grazia; Smith, Michael J.; Murrell, Jill R.; Ghetti, Bernardino; Klug, Aaron; Goedert, Michel; Varani, Gabriele

1999-01-01

145

Donor-site morbidity after pedicled TRAM breast reconstruction: a comparison of two different types of mesh.  

PubMed

Many different approaches have been used to minimize the risk of bulge or hernia formation when using autologous abdominal tissue for breast reconstruction. Studies have shown that further reinforcement of the abdominal wall using a mesh may decrease the complication rate.The current study included 40 consecutive patients having unilateral breast reconstruction with the pedicled transverse rectus abdominus musculocutaneous flap. The defect in the abdominal fascia was closed primarily and further reinforced using a Prolene mesh (Ethicon), n = 20, or using a self-fixating Parietex ProGrip mesh (Covidien), n = 20. The patients were examined at an outpatient consultation, with a minimum follow-up of 1 year and questioned about donor-site symptoms using a standardized questionnaire.Of the 20 patients in the Prolene group, 2 (10%) developed abdominal wall bulging, and of the 20 patients in the ProGrip group, 11 (55%) developed abdominal wall bulging (P = 0.006). In both the Prolene and the ProGrip group, most patients reported having continued donor-site symptoms at the time of the follow-up (70% and 80%, respectively); 15% and 30%, respectively, reported having symptoms that influenced their daily or physical activities (not a significant difference). All but 1 patient in our study reported being very happy with the reconstruction and would have done it again, had they known what they did at the time of the follow-up.We conclude that the self-gripping properties of the Parietex ProGrip mesh are not sufficient in withstanding the abdominal wall tension at the donor site after transverse rectus abdominus musculocutaneous-flap harvest and do not recommend using the Parietex ProGrip mesh without fixating sutures for this procedure. PMID:23392261

Sværdborg, Mille; Damsgaard, Tine Engberg

2013-11-01

146

Management of pediatric skin-graft donor sites: a randomized controlled trial of three wound care products.  

PubMed

Skin grafts are used to treat many types of skin defects in children, including burns, traumatic wounds, and revision of scars. The objective of this prospective randomized controlled trial was to compare the effectiveness of three dressing types for pediatric donor sites: foam, hydrofiber, and calcium alginate. Children attending a pediatric Burns & Plastics Service from October 2010 to March 2013, who required a split-skin graft, were recruited to the trial. Patients were randomly assigned to the two experimental groups, foam or hydrofiber, and to the control group, calcium alginate. Data were gathered on the management of exudate, assessment of pain, time to healing, and infection. Fifty-seven children aged 1 to 16 years (mean = 4.9 years) were recruited to the trial. Fifty-six patients had evaluable data and one participant from the control group was lost to follow-up. Most children required skin grafting for a burn injury (78%). The median size of the donor site was 63.50 cm (8-600 cm). There was a statistically significant difference in time to healing across the three dressing groups (x [2, n = 56] = 6.59, P = .037). The calcium alginate group recorded a lower median value of days to healing (median = 7.5 days) compared to the other two groups, which recorded median values of 8 days (hydrofiber) and 9.5 days (foam). The greatest leakage of exudate, regardless of dressing type, occurred on day 2 after grafting. No statistically significant difference was found in leakage of exudate, pain scores, or infection rates across the three groups. Calcium alginate emerged as the optimum dressing for pediatric donor site healing in this trial. PMID:25185932

Brenner, Maria; Hilliard, Carol; Peel, Glynis; Crispino, Gloria; Geraghty, Ruth; O?Callaghan, Gill

2015-01-01

147

Skin Thickness of the Anterior, Anteromedial, and Anterolateral Thigh: A Cadaveric Study for Split-Skin Graft Donor Sites  

PubMed Central

Background The depth of graft harvest and the residual dermis available for reepithelization primarily influence the healing of split-skin graft donor sites. When the thigh region is chosen, the authors hypothesize based on thickness measurements that the anterolateral region is the optimal donor site. Methods Full-thickness skin specimens were sampled from the anteromedial, anterior, and anterolateral regions of human cadavers. Skin specimens were cut perpendicularly with a custom-made precision apparatus to avoid the overestimation of thickness measurements. The combined epidermal and dermal thicknesses (overall skin thickness) were measured using a digital calliper. The specimens were histologically stained to visualize their basement membrane, and microscopy images were captured. Since the epidermal thickness varies across the specimen, a stereological method was used to eliminate observer bias. Results Epidermal thickness represented 2.5% to 9.9% of the overall skin thickness. There was a significant difference in epidermal thickness from one region to another (P<0.05). The anterolateral thigh region had the most consistent and highest mean epidermal thickness (60±3.2 µm). We observed that overall skin thickness increased laterally from the anteromedial region to the anterior and anterolateral regions of the thigh. The overall skin thickness measured 1,032±435 µm in the anteromedial region compared to 1,220±257 µm in the anterolateral region. Conclusions Based on skin thickness measurements, the anterolateral thigh had the thickest epidermal and dermal layers. We suggest that the anterolateral thigh region is the optimal donor site for split-skin graft harvests from the thigh. PMID:25396179

Ward, John; Quondamatteo, Fabio; Dockery, Peter; Kelly, John L

2014-01-01

148

Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?  

PubMed

To date the Simian Virus 40 (SV40) is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event. PMID:25210599

Poddar, Sushmita; Eul, Joachim; Patzel, Volker

2014-06-01

149

Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?  

PubMed Central

To date the Simian Virus 40 (SV40) is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event. PMID:25210599

Poddar, Sushmita; Eul, Joachim; Patzel, Volker

2014-01-01

150

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

PubMed Central

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

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

1993-01-01

151

Brain magnetic resonance imaging findings and auditory brainstem response in a child with spastic paraplegia 2 due to a PLP1 splice site mutation.  

PubMed

A boy with spastic paraplegia type 2 (SPG2) due to a novel splice site mutation of PLP1 presented with progressive spasticity of lower limbs, which was first observed during late infancy, when he gained the ability to walk with support. His speech was slow and he had dysarthria. The patient showed mildly delayed intellectual development. Subtotal dysmyelination in the central nervous system was revealed, which was especially prominent in structures known to be myelinated during earlier period, whereas structures that are myelinated later were better myelinated. These findings on the brain magnetic resonance imaging were unusual for subjects with PLP1 mutations. Peaks I and II of the auditory brainstem response (ABR) were normally provoked, but peaks III-V were not clearly demarcated, similarly to the findings in Pelizaeus-Merzbacher disease. These findings of brain MRI and ABR may be characteristic for a subtype of SPG2 patients. PMID:24685771

Kubota, Kazuo; Saito, Yoshiaki; Ohba, Chihiro; Saitsu, Hirotomo; Fukuyama, Tetsuhiro; Ishiyama, Akihiko; Saito, Takashi; Komaki, Hirofumi; Nakagawa, Eiji; Sugai, Kenji; Sasaki, Masayuki; Matsumoto, Naomichi

2015-01-01

152

An RNA map predicting Nova-dependent splicing regulation  

Microsoft Academic Search

Nova proteins are a neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova's effect to inhibit or

Jernej Ule; Giovanni Stefani; Aldo Mele; Matteo Ruggiu; Xuning Wang; Bahar Taneri; Terry Gaasterland; Benjamin J. Blencowe; Robert B. Darnell

2006-01-01

153

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.  

PubMed

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions. PMID:7885825

Teigelkamp, S; Whittaker, E; Beggs, J D

1995-02-11

154

Recurrent mis-splicing of fibrillin exon 32 in two patients with neonatal Marfan syndrome.  

PubMed

The Marfan syndrome (MFS) is an autosomal dominant heritable disorder of connective tissue. Variable and pleiotropic clinical features are observed in the skeletal, ocular, and cardiovascular systems. The most severe end of the phenotypic spectrum of this disorder comprises a group of patients usually diagnosed at birth, who have a life expectancy of little more than a year. To distinguish this group of patients from those with classical MFS, we refer to them as neonatal Marfan syndrome (nMFS). These infants usually die of congestive heart failure rather than aortic aneurysmal disease, the most frequent cause of morbidity and mortality in classical MFS. Defects in fibrillin, an elastin-associated microfibrillar glycoprotein, are now known to cause both the classical and neonatal forms of MFS. Here we report the recurrent mis-splicing of fibrillin (FBN1) exon 32, a precursor EGF-like calcium binding domain, in two unrelated infants with nMFS. The mis-splicing, in one patient, was due to an A-->T transversion at the -2 position of the consensus acceptor splice site; while that in the second patient was caused by a G-->A transition at the +1 position of the donor splice site. Characterization of FBN1 mutations in individuals at the most severe end of the Marfan syndrome spectrum should provide greater understanding of the multiple domains and regions of fibrillin. PMID:7633409

Wang, M; Price, C; Han, J; Cisler, J; Imaizumi, K; Van Thienen, M N; DePaepe, A; Godfrey, M

1995-04-01

155

Cloning and Characterization of Buffalo NANOG Gene: Alternative Transcription Start Sites, Splicing, and Polyadenylation in Embryonic Stem Cell-Like Cells  

PubMed Central

NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5?-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5?-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3?-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3?-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5?-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at ?30 and ?139?bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter. PMID:22011250

Singh, Natwar; Sharma, Ruchi; George, Aman; Singla, Suresh K.; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S.

2012-01-01

156

Reduction of pain via platelet-rich plasma in split-thickness skin graft donor sites: a series of matched pairs  

PubMed Central

In the past decade, autologous platelet-rich plasma (PRP) therapy has seen increasingly widespread integration into medical specialties. PRP application is known to accelerate wound epithelialization rates, and may also reduce postoperative wound site pain. Recently, we observed an increase in patient satisfaction following PRP gel (Angel, Cytomedix, Rockville, MD) application to split-thickness skin graft (STSG) donor sites. We assessed all patients known to our university-based hospital service who underwent multiple STSGs up to the year 2014, with at least one treated with topical PRP. Based on these criteria, five patients aged 48.4±17.6 (80% male) were identified who could serve as their own control, with mean time of 4.4±5.1 years between operations. In both therapies, initial dressing changes occurred on postoperative day (POD) 7, with donor site pain measured by Likert visual pain scale. Paired t-tests compared the size and thickness of harvested skin graft and patient pain level, and STSG thickness and surface area were comparable between control and PRP interventions (p>0.05 for all). Donor site pain was reduced from an average of 7.2 (±2.6) to 3 (±3.7), an average reduction in pain of 4.2 (standard error 1.1, p=0.0098) following PRP use. Based on these results, the authors suggest PRP as a beneficial adjunct for reducing donor site pain following STSG harvest. PMID:25623477

Miller, John D.; Rankin, Timothy M.; Hua, Natalie T.; Ontiveros, Tina; Giovinco, Nicholas A.; Mills, Joseph L.; Armstrong, David G.

2015-01-01

157

Reduction of pain via platelet-rich plasma in split-thickness skin graft donor sites: a series of matched pairs.  

PubMed

In the past decade, autologous platelet-rich plasma (PRP) therapy has seen increasingly widespread integration into medical specialties. PRP application is known to accelerate wound epithelialization rates, and may also reduce postoperative wound site pain. Recently, we observed an increase in patient satisfaction following PRP gel (Angel, Cytomedix, Rockville, MD) application to split-thickness skin graft (STSG) donor sites. We assessed all patients known to our university-based hospital service who underwent multiple STSGs up to the year 2014, with at least one treated with topical PRP. Based on these criteria, five patients aged 48.4±17.6 (80% male) were identified who could serve as their own control, with mean time of 4.4±5.1 years between operations. In both therapies, initial dressing changes occurred on postoperative day (POD) 7, with donor site pain measured by Likert visual pain scale. Paired t-tests compared the size and thickness of harvested skin graft and patient pain level, and STSG thickness and surface area were comparable between control and PRP interventions (p>0.05 for all). Donor site pain was reduced from an average of 7.2 (±2.6) to 3 (±3.7), an average reduction in pain of 4.2 (standard error 1.1, p=0.0098) following PRP use. Based on these results, the authors suggest PRP as a beneficial adjunct for reducing donor site pain following STSG harvest. PMID:25623477

Miller, John D; Rankin, Timothy M; Hua, Natalie T; Ontiveros, Tina; Giovinco, Nicholas A; Mills, Joseph L; Armstrong, David G

2015-01-01

158

Next generation sequencing detection and characterization of a heterozygous novel splice junction mutation in the 2B domain of KRT1 in a family with diffuse palmoplantar keratoderma.  

PubMed

Diffuse palmoplantar keratoderma (DPPK) is an autosomal dominant genodermatosis characterized by restricted, uniform hyperkeratosis on the palm and sole epidermis. DPPK is normally associated with dominant-negative mutations in the keratin-encoding gene, KRT1. We report a heterozygous novel point mutation in the exon 6 splice donor site of KRT1 (c.1254G>C) by next generation sequencing, resulting in the formation of two alternative transcripts, that segregates with DPPK in a four generation Chinese family. This results in both the complete loss of exon 6 as well as the simultaneous utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent deletion of 42 amino acids and the insertion of 18 amino acids into the protein's 2B domain. This is the first report of a novel splice donor site mutation with aberrant splicing and the formation of two alternative transcripts causing DPPK. This study also demonstrates the value of next-generation sequencing in the identification of novel disease causing mutations. This article is protected by copyright. All rights reserved. PMID:25429721

Banerjee, Santasree; Ren, Yunqing; Wei, Tianying; Zhou, Zhongwei; Yu, Ping; Guan, Fenghui; Wei, Xiaonming; Ye, Sheng; Yan, Shaofeng; Zheng, Min; Raff, Michael; Qi, Ming

2014-11-28

159

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.  

PubMed

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. PMID:25470420

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2014-12-01

160

Conservation and sex-specific splicing of the transformer gene in the calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata.  

PubMed

Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs. PMID:23409170

Li, Fang; Vensko, Steven P; Belikoff, Esther J; Scott, Maxwell J

2013-01-01

161

Conservation and Sex-Specific Splicing of the transformer Gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata  

PubMed Central

Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3? end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a “male-only” strain for genetic control programs. PMID:23409170

Li, Fang; Vensko, Steven P.; Belikoff, Esther J.; Scott, Maxwell J.

2013-01-01

162

Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions.  

PubMed

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication. PMID:25014653

Schnerwitzki, Danny; Perner, Birgit; Hoppe, Beate; Pietsch, Stefan; Mehringer, Rebecca; Hänel, Frank; Englert, Christoph

2014-09-01

163

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements  

PubMed Central

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based ‘HEXplorer score’ as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide. PMID:25147205

Erkelenz, Steffen; Theiss, Stephan; Otte, Marianne; Widera, Marek; Peter, Jan Otto; Schaal, Heiner

2014-01-01

164

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

PubMed

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

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

2013-06-01

165

A Mouse Splice-Site Mutant and Individuals with Atypical Chromosome 22q11.2 Deletions Demonstrate the Crucial Role for Crkl in Craniofacial and Pharyngeal Development  

PubMed Central

The 22q11.2 deletion syndrome (22q11DS) is thought to be a contiguous gene syndrome caused by haploinsufficiency for a variable number of genes with overlapping function during the development of the craniofacial, pharyngeal and cardiac structures. The complexity of genetic and developmental anomalies resulting in 22q11DS has made attributing causation to specific genes difficult. The CRKL gene resides within the common 3-Mb region, most frequently affected in 22q11DS, and has been shown to play an essential role in the development of tissues affected in 22q11DS. Here, we report the characterisation of a mouse strain we named ‘snoopy’, harbouring a novel Crkl splice-site mutation that results in a loss of Crkl expression. The snoopy strain exhibits a variable phenotype that includes micrognathia, pharyngeal occlusion, aglossia and holoprosencephaly, and altered retinoic acid and endothelin signalling. Together, these features are reminiscent of malformations occurring in auriculocondylar syndrome and agnathia-otocephaly complex, 2 conditions not previously associated with the CRKL function. Comparison of the features of a cohort of patients harbouring small 22q11.2 deletions centred over the CRKL gene, but sparing TBX1, highlights the role of CRKL in contributing to the craniofacial features of 22q11DS. These analyses demonstrate the central role of Crkl in regulating signalling events in the developing oropharyngeal complex and its potential to contribute to dysmorphology.

Miller, Kerry A.; Tan, Tiong Y.; Welfare, Megan F.; White, Susan M.; Stark, Zornitza; Savarirayan, Ravi; Burgess, Trent; Heggie, Andrew A.; Caruana, Georgina; Bertram, John F.; Bateman, John F.; Farlie, Peter G.

2014-01-01

166

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3' Splice Site In Vivo  

E-print Network

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential ...

Ma, Long

167

Specificity in splicing.  

PubMed

Considerable information about the process of pre-mRNA splicing has accumulated, but the mechanism by which highly accurate splicing is achieved is unresolved. Fifteen years ago we proposed that accuracy in splicing might depend on small RNA molecules (splicer RNAs) which hybridise across adjacent exon termini, or intron termini. Gene expression, including alternative splicing, could be controlled by the transcription of specific splicer RNA genes. We re-assess our model here, in the light of subsequent developments. PMID:7980482

Holliday, R; Murray, V

1994-10-01

168

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties  

Microsoft Academic Search

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US

H. Frances J. Bligh; Patrick D. Larkin; Paul S. Roach; Christopher A. Jones; Hongyong Fu; William D. Park

1998-01-01

169

The 5'-terminal sequence of U1 RNA complementary to the consensus 5' splice site of hnRNA is single-stranded in intact U1 snRNP particles.  

PubMed Central

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing. Images PMID:6203096

Rinke, J; Appel, B; Blöcker, H; Frank, R; Lührmann, R

1984-01-01

170

Functional association between promoter structure and transcript alternative splicing.  

PubMed

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription. PMID:9326631

Cramer, P; Pesce, C G; Baralle, F E; Kornblihtt, A R

1997-10-14

171

Genetic analysis of autosomal recessive osteopetrosis in Chuvashiya: the unique splice site mutation in TCIRG1 gene spread by the founder effect  

PubMed Central

The rare malignant disorder autosomal recessive osteopetrosis (OPTB) is one of the most prevalent autosomal recessive diseases in the Chuvash Republic of Russia. The purpose of this study was to determine the underlying molecular cause of osteopetrosis in Chuvashiya and to reveal the factors causing the unusual high frequency of the disease in this region. Having assumed a founder effect, we performed linkage disequilibrium (LD) mapping of the OPTB locus at the TCIRG1 region and found a unique splice site mutation c.807+5G>A in all Chuvashian OPTB patients studied. We then analyzed the mutational change in mRNA and detected an intron insertion within the mutant transcript, resulting in a frameshift and premature stop-codon formation (p.Leu271AspfsX231). A decreased expression of the mutant transcript was also detected, which may have been the result of nonsense-mediated decay. Real-time qPCR and MLPA® melting curve analysis-based systems were designed and used for c.807+5G>A mutation screening. In addition to analyzing the gene frequency in Chuvashiya, we also estimated three other populations in the Volga-Ural region (Mari, Udmurt and Bashkir). We found a 1.68% prevalence in Chuvashiya (calculated disease frequency, 1/3500 newborns) and a 0.84% in the Mari population (1/14?000 newborns). The haplotype analysis revealed that all OPTB cases in Chuvashians and Marians originated from a single mutational event and the age of the mutation in Chuvashians was estimated to be approximately 890 years. PMID:19172990

Bliznetz, Elena A; Tverskaya, Svetlana M; Zinchenko, Rena A; Abrukova, Anna V; Savaskina, Ekaterina N; Nikulin, Maxim V; Kirillov, Alexander G; Ginter, Evgeny K; Polyakov, Alexander V

2009-01-01

172

Site-directed mutagenesis in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: Donor D is a tryosine residue in the D2 protein  

SciTech Connect

The chemical nature of electron donor(s) in photosystem II in photosynthetic membranes was analyzed by site-directed mutagenesis of the gene encoding the protein D2 of the photosystem II reaction center. Mutation of the Try-160 residue of the D2 protein into phenylalanine results in the disappearance of the electron paramagnetic resonance signal II{sub s} originating from D{sup +}, the oxidized form of the slow photosystem II electron donor D. Signal II{sub s} is still present if a neighboring residue in D2, Met-159, is mutated into arginine. Both mutants have normal rereduction kinetics of the oxidized primary electron donor, P680{sup +}, in octyl glucoside-extracted thylakoids, indicating that D is not directly involved in P680{sup +} reduction. However, overall photosystem II activity appears to be impaired in the Try-160-Phe mutant: photosystem II-dependent growth of this mutant is slowed down by a factor of 3-4, whereas photoheterotrophic growth rates in wild type and mutant are essentially identical. Binding studies of diuron, a photosystem II herbicide, show that there is no appreciable decrease in the number of photosystem II centers in the Tyr-160-Phe mutant. The decrease in photosystem II activity in this mutant may be interpreted to indicate a role of D in photoactivation, rather than one as an important redox intermediate in the photosynthetic electron-transport chain.

Vermaas, W.F.J.; Rutherford, A.W.; Hansson, O. (Arizona State Univ., Tempe (USA))

1988-11-01

173

Electron donor concentrations in sediments and sediment properties at the agricultural chemicals team research site near New Providence, Iowa, 2006-07  

USGS Publications Warehouse

The concentrations of electron donors in aquifer sediments are important to the understanding of the fate and transport of redox-sensitive constituents in groundwater, such as nitrate. For a study by the U.S. Geological Survey National Water-Quality Assessment Program, 50 sediment samples were collected from below the water table from 11 boreholes at the U.S. Geological Survey Agricultural Chemicals Team research site near New Providence, Iowa, during 2006-07. All samples were analyzed for gravel, sand (coarse, medium, and fine), silt, clay, Munsell soil color, inorganic carbon content, and for the following electron donors: organic carbon, ferrous iron, and inorganic sulfide. A subset of 14 sediment samples also was analyzed for organic sulfur, but all of these samples had concentrations less than the method detection limit; therefore, the presence of this potential electron donor was not considered further. X-ray diffraction analyses provided important semi-quantitative information of well-crystallized dominant minerals within the sediments that might be contributing electron donors.

Maharjan, Bijesh; Korom, Scott F.; Smith, Erik A.

2013-01-01

174

RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.  

PubMed

To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine. PMID:25525159

Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

2015-01-01

175

BRCA1 Exon 11, a CERES (Composite Regulatory Element of Splicing) Element Involved in Splice Regulation  

PubMed Central

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

Tammaro, Claudia; Raponi, Michela; Wilson, David I.; Baralle, Diana

2014-01-01

176

BRCA1 EXON 11, a CERES (composite regulatory element of splicing) element involved in splice regulation.  

PubMed

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

Tammaro, Claudia; Raponi, Michela; Wilson, David I; Baralle, Diana

2014-01-01

177

A novel mutation in the intron 1 splice donor site of the cholesterol ester transfer protein ( CETP) gene as a cause of hyperalphalipoproteinemia  

Microsoft Academic Search

The exchange of cholesterol ester (CE) between lipoproteins occurs through the action of cholesterol ester transfer protein (CETP). The human CETP gene is composed of 16 exons encompassing 25 kbp on chromosome 16q13. The objective of this study was to determine whether a mutation in the CETP gene accounted for severe hyperalphalipoproteinemia in an 80-year-old subject. As a secondary objective,

Tjin-Shing Jap; Yi-Chi Wu; Yi-Chu Tso; Chih-Yang Chiu

2002-01-01

178

Alternative splicing switches potassium channel sensitivity to protein phosphorylation.  

PubMed

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation. PMID:11244090

Tian, L; Duncan, R R; Hammond, M S; Coghill, L S; Wen, H; Rusinova, R; Clark, A G; Levitan, I B; Shipston, M J

2001-03-16

179

Pre-mRNA splicing: a complex picture in higher definition.  

PubMed

Intron excision from pre-mRNAs of higher eukaryotes requires a transition from splice-site recognition across short exons to organization of the spliceosome across long introns. Recently, insight into this transition has been provided and, in addition, it has been shown that an alternative splicing factor, the polypyrimidine-tract-binding protein, can exert its control on splice-site choice by blocking this key step in the assembly of the splicing machinery. PMID:18472266

Schellenberg, Matthew J; Ritchie, Dustin B; MacMillan, Andrew M

2008-06-01

180

A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs  

Microsoft Academic Search

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic

Kyoungha Han; Gene Yeo; Ping An; Christopher B. Burge; Paula J. Grabowski

2005-01-01

181

Laparoendoscopic single-site (LESS) vs laparoscopic living-donor nephrectomy: a systematic review and meta-analysis.  

PubMed

The aim of this study was to provide a systematic review and meta-analysis of reports comparing laparoendoscopic single-site (LESS) living-donor nephrectomy (LDN) vs standard laparoscopic LDN (LLDN). A systematic review of the literature was performed in September 2013 using PubMed, Scopus, Ovid and The Cochrane library databases. Article selection proceeded according to the search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-analyses criteria. Weighted mean differences (WMDs) were used to measure continuous variables and odds ratios (ORs) to measure categorical ones. Nine publications meeting eligibility criteria were identified, including 461 LESS LDN and 1006 LLDN cases. There were more left-side cases in the LESS LDN group (96.5% vs 88.6%, P < 0.001). Meta-analysis of extractable data showed that LLDN had a shorter operative time (WMD 15.06?min, 95% confidence interval [CI] 4.9-25.1; P = 0.003), without a significant difference in warm ischaemia time (WMD 0.41?min, 95% CI -0.02 to 0.84; P = 0.06). Estimated blood loss was lower for LESS LDN (WMD -22.09?mL, 95% CI -29.5 to -14.6; P < 0.001); however, this difference was not clinically significant. There was a greater likelihood of conversion for LESS LDN (OR 13.21, 95% CI 4.65-37.53; P < 0.001). Hospital stay was similar (WMD -0.11 days, 95% CI -0.33 to 0.12; P = 0.35), as well as the visual analogue pain score at discharge (WMD -0.31, 95% CI -0.96 to 0.35; P = 0.36), but the analgesic requirement was lower for LESS LDN (WMD -2.58?mg, 95% CI -5.01 to -0.15; P = 0.04). Moreover, there was no difference in the postoperative complication rate (OR 1.00, 95% CI 0.65-1.54; P = 0.99). Renal function of the recipient, as based on creatinine levels at 1 month, showed similar outcomes between groups (WMD 0.10?mg/dL, -0.09 to 0.29; P = 0.29). In conclusion, LESS LDN represents an emerging option for living kidney donation. This procedure offers comparable surgical and early functional outcomes to the conventional LLDN, with a lower analgesic requirement. However, it is more technically challenging than LLDN, as shown by a greater likelihood of conversion. The role of LESS LDN remains to be defined. PMID:24588876

Autorino, Riccardo; Brandao, Luis Felipe; Sankari, Bashir; Zargar, Homayoun; Laydner, Humberto; Akça, Oktay; De Sio, Marco; Mirone, Vincenzo; Chueh, Shih-Chieh J; Kaouk, Jihad H

2015-02-01

182

Translational control of intron splicing in eukaryotes.  

PubMed

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy. PMID:18202663

Jaillon, Olivier; Bouhouche, Khaled; Gout, Jean-François; Aury, Jean-Marc; Noel, Benjamin; Saudemont, Baptiste; Nowacki, Mariusz; Serrano, Vincent; Porcel, Betina M; Ségurens, Béatrice; Le Mouël, Anne; Lepère, Gersende; Schächter, Vincent; Bétermier, Mireille; Cohen, Jean; Wincker, Patrick; Sperling, Linda; Duret, Laurent; Meyer, Eric

2008-01-17

183

Changing Transcriptional Initiation Sites and Alternative 5'- and 3'-Splice Site Selection of the First Intron Deploys the Arabidopsis Protein Isoaspartyl Methyltransferase2 Variants to Different Subcellular Compartments  

Technology Transfer Automated Retrieval System (TEKTRAN)

Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT), genes encoding an enzyme (EC 2.1.1.77) capable of converting uncoded, L-isoaspartyl residues, arising spontaneously at L-asparaginyl and L-aspartyl sites in proteins, to L-aspartate. PIMT2 produces at lea...

184

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences  

Microsoft Academic Search

A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that

Michael Krawczak; Jochen Reiss; David N. Cooper

1992-01-01

185

Evaluation of the effect of c.2946+1G>T mutation on splicing in the SCN1A gene.  

PubMed

Mutations in the SCN1A gene have commonly been associated with a wide range of mild to severe epileptic syndromes. They generate a wide spectrum of phenotypes ranging from the relatively mild generalized epilepsy with febrile seizures plus (GEFS+) to other severe epileptic encephalopathies, including myoclonic epilepsy in infancy (SMEI), cryptogenic focal epilepsy (CFE), cryptogenic generalized epilepsy (CGE) and a distinctive subgroup termed as severe infantile multifocal epilepsy (SIMFE). The present study was undertaken to investigate the potential effects of a transition in the first nucleotide at the donor splice site of intron 15 of the SCN1A gene leading to CGES. Functional analyses using site-directed mutagenesis by PCR and subsequent ex-vivo splicing assays, revealed that the c.2946+1G>T mutation lead to a total skipping of exon 15. The exclusion of this exon did not alter the reading frame but induced the deletion of the amino acids (853 Leu -971 Val) which are a major part in the fourth, fifth and sixth transmembrane segments of the SCN1A protein. The theoretical implications of the splice site mutations predicted with the bioinformatic tool human splice finder were investigated and compared with the results obtained by the cellular assay. PMID:25590135

Ben Mahmoud, Afif; Ben Mansour, Riadh; Driss, Fatma; Baklouti-Gargouri, Siwar; Siala, Olfa; Mkaouar-Rebai, Emna; Fakhfakh, Faiza

2015-02-01

186

Control of protein splicing by intein fragment reassembly.  

PubMed Central

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein. PMID:9463370

Southworth, M W; Adam, E; Panne, D; Byer, R; Kautz, R; Perler, F B

1998-01-01

187

Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes  

PubMed Central

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

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

2006-01-01

188

Splicing: HACking into the unfolded-protein response.  

PubMed

Unfolded proteins in the endoplasmic reticulum of Saccharomyces cerevisiae trigger a specialized RNA splicing event that allows the subsequent translation of the Hac1p transcription factor. This splicing can be reconstituted with Ire1p, a transmembrane kinase that has a site-specific RNase activity, and tRNA ligase. PMID:9501974

Shamu, C E

1998-02-12

189

LETTER TO THE EDITOR The effect of nonsense codons on splicing  

E-print Network

LETTER TO THE EDITOR The effect of nonsense codons on splicing: A genomic analysis XIANG ZHANG that the recognition of in-frame nonsense codons is used generally for exon identification during pre-mRNA splicing. However, nonsense codon frequencies in pseudo exons and in regions flanking 5 splice sites are no greater

Tong, Liang

190

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

PubMed Central

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3? splice sites (ss) of exon 2?/2. Here, we demonstrate that exon 1 and 2?/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3? ss and exon 1B to the proximal 3? ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2?/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2?/2 junction CAGAGAA, a sequence that overlaps the distal U2AF35-binding 3? ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3? ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3? ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network. PMID:19729518

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

2009-01-01

191

The Arabic allele: a single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia.  

PubMed

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3%±6.5 and 17.9%±8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14-18. We called this mutation "the Arabic allele". We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay. PMID:22129472

Shawar, Said M; Al-Drees, Mohammad A; Ramadan, Ahmad R; Ali, Najat H; Alfadhli, Suad M

2012-02-01

192

Mechanisms of the androgen receptor splicing in prostate cancer cells.  

PubMed

Prostate tumors develop resistance to androgen deprivation therapy (ADT) by multiple mechanisms, one of which is to express constitutively active androgen receptor (AR) splice variants lacking the ligand-binding domain. AR splice variant 7 (AR-V7, also termed AR3) is the most abundantly expressed variant that drives prostate tumor progression under ADT conditions. However, the molecular mechanism by which AR-V7 is generated remains unclear. In this manuscript, we demonstrated that RNA splicing of AR-V7 in response to ADT was closely associated with AR gene transcription initiation and elongation rates. Enhanced AR gene transcription by ADT provides a prerequisite condition that further increases the interactions between AR pre-mRNA and splicing factors. Under ADT conditions, recruitment of several RNA splicing factors to the 3' splicing site for AR-V7 was increased. We identified two RNA splicing enhancers and their binding proteins (U2AF65 and ASF/SF2) that had critical roles in splicing AR pre-mRNA into AR-V7. These data indicate that ADT-induced AR gene transcription rate and splicing factor recruitment to AR pre-mRNA contribute to the enhanced AR-V7 levels in prostate cancer cells. PMID:23851510

Liu, L L; Xie, N; Sun, S; Plymate, S; Mostaghel, E; Dong, X

2014-06-12

193

The clinical features of Ehlers-Danlos syndrome type VIIB resulting from a base substitution at the splice acceptor site of intron 5 of the COL1A2 gene.  

PubMed Central

The features of a 32 year old woman with Ehlers-Danlos syndrome type VIIB and affected members of her family, resulting from a mutation in one COL1A2 allele, were studied. Her dermal type I collagen contained alpha 2(I) chains and mutant pN-alpha 2(I) chains in which the amino-terminal propeptide remained attached to the alpha 2(I) chain. She was heterozygous for an AG-->AC mutation at the splice acceptor site of intron 5 of the COL1A2 gene. The mutation activated a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6 of the COL1A2 gene. In contrast to previous reports only five, rather than all 18, amino acids encoded by exon 6 were deleted in the proband. The deleted peptide removed the amino-proteinase cleavage site, but not the nearby lysine cross linking site in the amino-telopeptide of the alpha 2(I) chain. She was born with bilateral hip dislocations, knee subluxations, and generalised joint hypermobility. Bilateral inguinal herniae and an umbilical hernia were present at birth. Facial features included a depressed nasal bridge with prominent paranasal folds. The skin was soft, moderately hyperelastic, and sagged over the face. Skin fragility and easy bruising were apparent from childhood. Skin wounds healed slowly and with broad, paper thin scars. Throughout her life, she had multiple fractures of the small bones of her hands and feet following moderate trauma. Electron microscopy of the proband's dermis as well as deep fascia and hip joint capsule from her affected brother showed that collagen fibrils in transverse section were nearly circular but with irregular margins. Light microscopy of bone from her affected brother and son showed normal Haversian systems and lamellar bone. All of these tissues contained approximately equal amounts of the normal and mutant alpha2(I) chains. The findings of this study confirm that loss of the amino-proteinase cleavage site of the pro alpha2(I) collagen chains, owing to anomalous splicing of exon 6 sequences in the conversion of pre-mRNA to mRNA, produces the clinical features of Ehlers-Danlos syndrome type VIIB. The history of frequent fractures found in this family is atypical and indicates an overlap with osteogenesis imperfecta. Images PMID:8071956

Carr, A J; Chiodo, A A; Hilton, J M; Chow, C W; Hockey, A; Cole, W G

1994-01-01

194

RNA helicases in splicing  

PubMed Central

In eukaryotic cells, introns are spliced from pre-mRNAs by the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. There is evidence that the various helicases are functionally and physically connected with each other and with many other factors in the spliceosome. Understanding the dynamics of those interactions is essential to comprehend the mechanism and regulation of normal as well as of pathological splicing. This review focuses on recent advances in the characterization of the splicing helicases and their interactions, and highlights the deep integration of splicing helicases in global mRNP biogenesis pathways. PMID:23229095

Cordin, Olivier; Beggs, Jean D.

2013-01-01

195

A case of donor-site lymphoedema after lymph node-superficial circumflex iliac artery perforator flap transfer.  

PubMed

Vascularised lymph node transfer is a promising technique to treat limb lymphoedema, especially when caused by lymph node dissection. The most common approach is the transfer of superficial inguinal lymph nodes using groin flaps or superficial circumflex iliac artery perforator flaps. Lower-limb lymphatic sequelae are unexpected as these lymph nodes should drain lymph from the lower abdominal wall. Recently, Vignes et al. described two cases out of 26 cases of chronic lymphoedema after superficial inguinal lymph node harvest. From a series of 42 vascularised lymph node transfers performed at our centre, only one patient developed swelling in the donor thigh. The features of this patient who underwent a lymph node-containing superficial circumflex iliac artery perforator flap are reported herein. We recommend maximal accuracy in selecting the appropriate lymph nodes for transfer and provide some tips from our experience. PMID:23827445

Pons, Gemma; Masia, Jaume; Loschi, Pietro; Nardulli, Maria Luisa; Duch, Joan

2014-01-01

196

A Splice Mutation and mRNA Decay of EXT2 Provoke Hereditary Multiple Exostoses  

PubMed Central

Background Hereditary multiple exostoses (HME) is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2, which are responsible for over 70% of HME cases. However, the pathological significance of the majority of these mutations is often unclear. Methods In a Chinese family with HME, EXT1 and EXT2 genes were screened by direct sequencing. The consequence of a detected mutant was predicted by in silico analysis and confirmed by mRNA analysis. The EXT1 and EXT2 mRNA and protein levels and the HS patterns in the HME patients were compared with those in healthy controls. Results A heterozygous transition (c.743+1G>A) in the EXT2 gene, which co-segregated with the HME phenotype in this family, was identified. The G residue at position +1 in intron 4 of EXT2 was predicted to be a 5? donor splice site. The mRNA analysis revealed an alternative transcript with a cryptic splice site 5 bp downstream of the wild-type site, which harbored a premature stop codon. However, the predicted truncated protein was not detected by western blot analysis. Decay of the mutant mRNA was shown by clone sequencing and quantification analysis. The corresponding downregulation of the EXT2 mRNA will contribute to the abnormal EXT1/EXT2 ratio and HS pattern that were detected in the patients with HME. Conclusion The heterozygous mutation c.743+1G>A in the EXT2 gene causes HME as a result of abnormal splicing, mRNA decay, and the resulting haploinsufficiency of EXT2. PMID:24728384

Tian, Chen; Yan, Rengna; Wen, Shuzhen; Li, Xueling; Li, Tianfeng; Cai, Zhenming; Li, Xinxiu; Du, Hong; Chen, Huimei

2014-01-01

197

A UGT2B10 Splicing Polymorphism Common in African Populations May Greatly Increase Drug Exposure.  

PubMed

RO5263397 [(S)-4-(3-fluoro-2-methyl-phenyl)-4,5-dihydro-oxazol-2-ylamine], a new compound that showed promising results in animal models of schizophrenia, is mainly metabolized in humans by N-glucuronidation. Enzyme studies, using the (then) available commercial uridine 5'-diphosphate-glucuronosyltransferases (UGTs), suggested that UGT1A4 is responsible for its conjugation. In the first clinical trial, in which RO5263397 was administered orally to healthy human volunteers, a 136-fold above-average systemic exposure to the parent compound was found in one of the participants. Further administration in this trial identified two more such poor metabolizers, all three of African origin. Additional in vitro studies with recombinant UGTs showed that the contribution of UGT2B10 to RO5263397 glucuronidation is much higher than UGT1A4 at clinically relevant concentrations. DNA sequencing in all of these poor metabolizers identified a previously uncharacterized splice site mutation that prevents assembly of full-length UGT2B10 mRNA and thus functional UGT2B10 protein expression. Further DNA database analyses revealed the UGT2B10 splice site mutation to be highly frequent in individuals of African origin (45%), moderately frequent in Asians (8%) and almost unrepresented in Caucasians (<1%). A prospective study using hepatocytes from 20 individual African donors demonstrated a >100-fold lower intrinsic clearance of RO5263397 in cells homozygous for the splice site variant allele. Our results highlight the need to include UGT2B10 when screening the human UGTs for the enzymes involved in the glucuronidation of a new compound, particularly when there is a possibility of N-glucuronidation. Moreover, this study demonstrates the importance of considering different ethnicities during drug development. PMID:25503386

Fowler, Stephen; Kletzl, Heidemarie; Finel, Moshe; Manevski, Nenad; Schmid, Paul; Tuerck, Dietrich; Norcross, Roger D; Hoener, Marius C; Spleiss, Olivia; Iglesias, Victor A

2015-02-01

198

Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells  

PubMed Central

Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP. PMID:21326217

Glaus, Esther; Schmid, Fabian; Da Costa, Romain; Berger, Wolfgang; Neidhardt, John

2011-01-01

199

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.  

PubMed

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5' donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy. PMID:24013503

Bezzi, Marco; Teo, Shun Xie; Muller, Julius; Mok, Wei Chuen; Sahu, Sanjeeb Kumar; Vardy, Leah A; Bonday, Zahid Q; Guccione, Ernesto

2013-09-01

200

Some relations between two stages DNA splicing languages  

NASA Astrophysics Data System (ADS)

A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

2014-06-01

201

A prospective, randomised study of a novel transforming methacrylate dressing compared with a silver-containing sodium carboxymethylcellulose dressing on partial-thickness skin graft donor sites in burn patients.  

PubMed

This prospective, randomised study compares a new transforming methacrylate dressing (TMD) with a silver-containing carboxymethylcellulose dressing (CMC-Ag) after application to split-thickness skin graft (STSG) donor sites. This was an unblinded, non-inferiority, between-patient, comparison study that involved patients admitted to a single-centre burn unit who required two skin graft donor sites. Each patient's donor sites were covered immediately after surgery: one donor site with TMD and the other with CMC-Ag. The donor sites were evaluated until healing or until 24?days post-application, whichever came first. Study endpoints were time to healing, daily pain scores, number of dressing changes, patient comfort and physicians' and patients' willingness to use the dressings in the future. Nineteen patients had both the dressings applied. No statistically significant difference was noted in time to healing between the two dressings (14·2?days using TMD compared with 13·2?days using CMC-Ag). When pain scores were compared, TMD resulted in statistically significantly less pain at three different time periods (2-5?days, 6-10?days and 11-15?days; P?donor site management. PMID:23919667

Assadian, Ojan; Arnoldo, Brett; Purdue, Gary; Burris, Agnes; Skrinjar, Edda; Duschek, Nikolaus; Leaper, David J

2013-08-01

202

Copper-Sulfur Complexes Supported by N-Donor Ligands: Towards Models of the CuZ Site in Nitrous Oxide Reductase  

PubMed Central

The distinctive structure of the [(his)7Cu4(?-S)]n+ cluster in the “CuZ” active site of nitrous oxide reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper-sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper-sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper-sulfur bonding interactions in the complexes. PMID:19262681

York, John T.; Bar-Nahum, Itsik; Tolman, William B.

2008-01-01

203

Oxidation of substrates tethered to N-donor ligands for modeling non-heme diiron enzyme active sites  

E-print Network

Chapter 1. Modeling Carboxylate-Rich Diiron Sites of Dioxygen-Dependent Non-Heme Enzymes Carboxylate-bridged diiron centers are employed in a variety of biological systems to activate dioxygen for substrate oxidation, and ...

Carson, Emily Carrig, 1978-

2005-01-01

204

Modeling the active sites of non-heme diiron metalloproteins with sterically hindered carboxylates and syn N-Donor ligands  

E-print Network

Chapter 1. Different Synthetic Approaches to Modeling the Active Sites of Carboxylate-Bridged Non-Heme Diiron Enzymes Carboxylate-bridged non-heme diiron enzymes activate dioxygen to perform a variety of biological functions. ...

Friedle, Simone, 1976-

2009-01-01

205

Genome-wide discovery of human splicing branchpoints.  

PubMed

During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations. PMID:25561518

Mercer, Tim R; Clark, Michael B; Andersen, Stacey B; Brunck, Marion E; Haerty, Wilfried; Crawford, Joanna; Taft, Ryan J; Nielsen, Lars K; Dinger, Marcel E; Mattick, John S

2015-02-01

206

Biogeochemical Modeling of In Situ U(VI) Reduction and Immobilization with Emulsified Vegetable Oil as the Electron Donor at a Field Site in Oak Ridge, Tennessee  

NASA Astrophysics Data System (ADS)

A comprehensive biogeochemical model was developed to quantitatively describe the coupled hydrologic, geochemical and microbiological processes that occurred following injection of emulsified vegetable oil (EVO) as the electron donor to immobilize U(VI) at the Oak Ridge Integrated Field Research Challenge site (ORIFRC) in Tennessee. The model couples the degradation of EVO, production and oxidation of long-chain fatty acids (LCFA), glycerol, hydrogen and acetate, reduction of nitrate, manganese, ferrous iron, sulfate and uranium, and methanoganesis with growth of multiple microbial groups. The model describes the evolution of geochemistry and microbial populations not only in the aqueous phase as typically observed, but also in the mineral phase and therefore enables us to evaluate the applicability of rates from the literature for field scale assessment, estimate the retention and degradation rates of EVO and LCFA, and assess the influence of the coupled processes on fate and transport of U(VI). Our results suggested that syntrophic bacteria or metal reducers might catalyze LCFA oxidation in the downstream locations when sulfate was consumed, and competition between methanogens and others for electron donors and slow growth of methanogen might contribute to the sustained reducing condition. Among the large amount of hydrologic, geochemical and microbiological parameter values, the initial biomass, and the interactions (e.g., inhibition) of the microbial functional groups, and the rate and extent of Mn and Fe oxide reduction appear as the major sources of uncertainty. Our model provides a platform to conduct numerical experiments to study these interactions, and could be useful for further iterative experimental and modeling investigations into the bioreductive immobiliztion of radionuclide and metal contaminants in the subsurface.

Tang, G.; Parker, J.; Wu, W.; Schadt, C. W.; Watson, D. B.; Brooks, S. C.; Orifrc Team

2011-12-01

207

A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.  

PubMed

X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development. PMID:25443657

Gray, P E A; Logan, G J; Alexander, I E; Poulton, S; Roscioli, T; Ziegler, J

2015-02-01

208

Splice assembly tool and method of splicing  

DOEpatents

A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

Silva, Frank A. (Basking Ridge, NJ)

1980-01-01

209

SPA: A Probabilistic Algorithm for Spliced Alignment  

PubMed Central

Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5? and 3? ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice site that we also find in the mouse dataset. The SPA software package is available at http://www.biozentrum.unibas.ch/personal/nimwegen/cgi-bin/spa.cgi. PMID:16683023

van Nimwegen, Erik; Paul, Nicodeme; Sheridan, Robert; Zavolan, Mihaela

2006-01-01

210

Donor Tag Game  

MedlinePLUS

... Games > Donor Tag Game Printable Version Donor Tag Game This feature requires version 6 or later of ... Blood Donor Community Donor Stories Recipient Stories SleevesUp Games Facebook Fanbox Avatars and Badges Banners eCards Enter ...

211

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

212

Aberrant and alternative splicing in skeletal system disease.  

PubMed

The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. PMID:23800666

Fan, Xin; Tang, Liling

2013-10-01

213

Premature transcript termination, trans-splicing and DNA repair: a vicious path to cancer  

PubMed Central

So far, about 800 different chromosomal translocations have been characterized in hemato-malignant and solid tumors. Chromosomal translocations mostly result in the expression of chimeric fusion proteins associated with enhanced proliferation and/or malignant transformation. Here, we demonstrate that genes frequently involved in such genetic rearrangements exhibit a unique feature: premature transcriptional termination. These early-terminated RNA molecules have an abundance of 10-20% when compared to their cognate full-length transcripts. They exhibit an unsaturated splice donor site that gives rise to trans-splicing events, leading to RNAs displaying exon repetitions or chimeric fusion RNAs. These arbitrary fusion RNAs mimic the presence of a chromosomal translocation in genetically unaffected cells. Based on our and published data, we propose the hypothesis that these artificial “chimeric fusion transcripts” may influence DNA repair processes, resulting in the generation of de novo chromosomal translocations. This idea provides a rational explanation why different individuals suffer from nearly identical genetic rearrangements. PMID:22432062

Kowarz, Eric; Merkens, Jennifer; Karas, Michael; Dingermann, Theo; Marschalek, Rolf

2011-01-01

214

Techniques in laparoscopic donor nephrectomy.  

PubMed

What's known on the subject? and What does the study add? Innovations in laparoscopic surgery have provided transplant surgeons with a range of techniques as well as a vast array of minimally invasive instruments. Whilst randomized control trials have compared open and laparoscopic donor nephrectomy, there is a paucity of high quality data comparing different laparoscopic approaches. This article summarizes the main techniques of laparoscopic donor nephrectomy currently in use and reviews the evidence available for each. In addition, controversial aspects of donor nephrectomy are examined, including the technological advances applicable to this operation. Increasing numbers of living donor kidney transplants are being performed worldwide, and the majority of donor operations are now laparoscopic. Transperitoneal 'pure' and hand-assisted laparoscopic donor nephrectomy are the two most commonly performed procedures, although retroperitoneal approaches are advocated by some centres. Controversy persists with respect to the technical aspects of donor nephrectomy, including both the approach and the method of ligation of the hilar vessels. More recently, robot-assisted, laparo-endoscopic single site surgery (LESS) and natural orifice transluminal endoscopic surgery (NOTES) -assisted donor nephrectomy have also been performed, further increasing the number of options available, but creating uncertainty as to the ideal approach. PMID:22489654

Banga, Neal; Nicol, David

2012-11-01

215

The Rare TXNRD1_v3 (“v3”) Splice Variant of Human Thioredoxin Reductase 1 Protein Is Targeted to Membrane Rafts by N-Acylation and Induces Filopodia Independently of Its Redox Active Site Integrity*  

PubMed Central

The human selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene, is a key player in redox regulation. Alternative splicing generates several TrxR1 variants, one of which is v3 that carries an atypical N-terminal glutaredoxin domain. When overexpressed, v3 associates with membranes and triggers formation of filopodia. Here we found that membrane targeting of v3 is mediated by myristoylation and palmitoylation of its N-terminal MGC motif, through which v3 specifically targets membrane rafts. This was suggested by its localization in cholera toxin subunit B-stained membrane areas and also shown using lipid fractionation experiments. Utilizing site-directed mutant variants, we also found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting. These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment. PMID:23413027

Cebula, Marcus; Moolla, Naazneen; Capovilla, Alexio; Arnér, Elias S. J.

2013-01-01

216

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

217

Solution structure of the HIV-1 exon splicing silencer 3.  

PubMed

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.8±1.1 nM) complex with ESS3 via site-specific interactions with the loop. PMID:22154809

Levengood, Jeffrey D; Rollins, Carrie; Mishler, Clay H J; Johnson, Charles A; Miner, Grace; Rajan, Prashant; Znosko, Brent M; Tolbert, Blanton S

2012-01-27

218

Building robust transcriptomes with master splicing factors.  

PubMed

Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or "master splicing factors," respond to environmental cues to establish and maintain tissue transcriptomes during development. PMID:25417102

Jangi, Mohini; Sharp, Phillip A

2014-10-23

219

Genomic mRNA profiling reveals compensatory mechanisms for the requirement of the essential splicing factor U2AF.  

PubMed

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3' splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF(59) (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF(59) inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF(59)-insensitive introns have stronger 5' splice sites and higher A/U content. Most importantly, features that contribute to U2AF(59) insensitivity of an intron unexpectedly reside in its 5'-most 30 nucleotides. These include the 5' splice site, a guanosine at position 7, and the 5' splice site-to-branch point sequence context. A differential requirement (similar to U2AF(59)) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly. PMID:21149581

Sridharan, Vinod; Heimiller, Joseph; Singh, Ravinder

2011-02-01

220

Novel splice variants of the bovine PCK1 gene.  

PubMed

Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated. PMID:24089092

Zhang, Z B; Zhang, W; Li, R L; Li, J B; Zhong, J F; Zhao, Z S; Huang, J M

2013-01-01

221

Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder.  

PubMed

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV. PMID:25098561

Ahmed, Iltaf; Mittal, Kirti; Sheikh, Taimoor I; Vasli, Nasim; Rafiq, Muhammad Arshad; Mikhailov, Anna; Ohadi, Mehrnaz; Mahmood, Huda; Rouleau, Guy A; Bhatti, Attya; Ayub, Muhammad; Srour, Myriam; John, Peter; Vincent, John B

2014-11-01

222

Intron RNA editing is essential for splicing in plant mitochondria  

PubMed Central

Most plant mitochondria messenger RNAs (mRNAs) undergo editing through C-to-U conversions located mainly in exon sequences. However, some RNA editing events are found in non-coding regions at critical positions in the predicted secondary and tertiary structures of introns, suggesting that RNA editing could be important for splicing. Here, we studied the relationships between editing and splicing of the mRNA encoding the ribosomal protein S10 (rps10), which has a group II intron and five editing sites. Two of them, C2 and C3, predicted to stabilize the folded structure of the intron necessary for splicing, were studied by using rps10 mutants introduced into isolated potato mitochondria by electroporation. While mutations of C2 involved in EBS2/IBS2 interactions did not affect splicing, probably by the presence of an alternative EBS2? region in domain I of the intron, the edition of site C3 turned out to be critical for rps10 mRNA splicing; only the edited (U) form of the transcript was processed. Interestingly, RNA editing was strongly reduced in transcripts from two different intronless genes, rps10 from potato and cox2 from wheat, suggesting that efficient RNA processing may require a close interaction of factors engaged in different maturation processes. This is the first report linking editing and splicing in conditions close to the in vivo situation. PMID:20615898

Castandet, Benoît; Choury, David; Bégu, Dominique; Jordana, Xavier; Araya, Alejandro

2010-01-01

223

Intron RNA editing is essential for splicing in plant mitochondria.  

PubMed

Most plant mitochondria messenger RNAs (mRNAs) undergo editing through C-to-U conversions located mainly in exon sequences. However, some RNA editing events are found in non-coding regions at critical positions in the predicted secondary and tertiary structures of introns, suggesting that RNA editing could be important for splicing. Here, we studied the relationships between editing and splicing of the mRNA encoding the ribosomal protein S10 (rps10), which has a group II intron and five editing sites. Two of them, C2 and C3, predicted to stabilize the folded structure of the intron necessary for splicing, were studied by using rps10 mutants introduced into isolated potato mitochondria by electroporation. While mutations of C2 involved in EBS2/IBS2 interactions did not affect splicing, probably by the presence of an alternative EBS2' region in domain I of the intron, the edition of site C3 turned out to be critical for rps10 mRNA splicing; only the edited (U) form of the transcript was processed. Interestingly, RNA editing was strongly reduced in transcripts from two different intronless genes, rps10 from potato and cox2 from wheat, suggesting that efficient RNA processing may require a close interaction of factors engaged in different maturation processes. This is the first report linking editing and splicing in conditions close to the in vivo situation. PMID:20615898

Castandet, Benoît; Choury, David; Bégu, Dominique; Jordana, Xavier; Araya, Alejandro

2010-11-01

224

Committee of Donor Agencies for Small Enterprise Development  

NSDL National Science Digital Library

Established in 1979, the Committee of Donor Agencies promotes the development of small enterprise in developing countries. This site offers numerous working and research papers about the Committee of Donor Agencies and its members. Showcased on the site are the Committee's Donor Business Development Services Case Studies. The Case studies are browseable by several categories including Region, Country, Theme, and Member Agency. Also provided are the Donor Committee Guidelines and links to member agencies's sites.

225

Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.  

PubMed

Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation. PMID:25482510

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

2015-01-01

226

Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism  

USGS Publications Warehouse

The gene encoding IgH ? has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated ?-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory ? transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory ? transcript resulted in two ?-H chains, which incorporated C?1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory ? mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.

2012-01-01

227

Simultaneous detection of Hb constant spring (?142, TAA>CAA, ?2) and the ?2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran.  

PubMed

Hb Constant Spring (Hb CS, codon 142, TAA>CAA, ?2) (HBA2:c.427T>C) and ?2 IVS-I donor site (GAGGTGAGG>GAGG?- - - - -) (HBA2:c.95+2_95+6delTGAGG) are nondeletional ?-thalassemia (?-thal) mutations found all over the world. Identification of ?-thal genotypes in at-risk couples for severe anemia or in highly heterogeneous populations requires rapid, accurate and cost-effective genotyping methods. In this study, a pair of primers were used to specifically amplify an 883 bp fragment from the ?2-globin gene in order to simultaneously identify these two mutations by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We determined the genotypic frequencies of Hb CS and the ?2 IVS-I donor site mutations after amplification and enzymatic digestion with Tru9I in 238 northern Iranian samples referred for ?-thal testing. Hb CS and the ?2 IVS-I donor site mutations accounted for 21 (8.8%) and 29 (12.2%) of the nondeletional cases. This genotyping assay has proven to be a rapid, reliable and useful diagnostic tool for simultaneous detection of these two anomalies for genetic counseling or further prenatal diagnosis. PMID:22356652

Akhavan-Niaki, Haleh; Banihashemi, Ali; Mostafazadeh, Amrollah; Kholghi Oskooei, Vahid; Azizi, Mandana; Youssefi Kamangar, Reza; Elmi, Maryam Mitra

2012-01-01

228

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

PubMed Central

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

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

2014-01-01

229

Tissue-Specific Genetic Control of Splicing: Implications for the Study of Complex Traits  

PubMed Central

Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes. PMID:19222302

Cronin, Kenneth D; Maia, Jessica M; Shianna, Kevin V; Gabriel, Willow N; Welsh-Bohmer, Kathleen A; Hulette, Christine M; Denny, Thomas N; Goldstein, David B

2008-01-01

230

MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation.  

PubMed

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways, mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts, from several donors, into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2, CLSTN1, ATP2A1, PALM, ITGA6, KIF13A, FMNL3, PPIP5K1, MARK2 and FNIP1), implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks. PMID:24048253

Venables, Julian P; Lapasset, Laure; Gadea, Gilles; Fort, Philippe; Klinck, Roscoe; Irimia, Manuel; Vignal, Emmanuel; Thibault, Philippe; Prinos, Panagiotis; Chabot, Benoit; Abou Elela, Sherif; Roux, Pierre; Lemaitre, Jean-Marc; Tazi, Jamal

2013-01-01

231

In vitro splicing of simian virus 40 early pre mRNA.  

PubMed Central

The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery. Images PMID:3005968

Noble, J C; Prives, C; Manley, J L

1986-01-01

232

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

PubMed Central

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

2014-01-01

233

Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome  

PubMed Central

The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3?-sulfur substitution at the 5? splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3?-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3?-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems. PMID:10398685

Sontheimer, Erik J.; Gordon, Peter M.; Piccirilli, Joseph A.

1999-01-01

234

The Role of Polypyrimidine Tract-Binding Proteins and Other hnRNP Proteins in Plant Splicing Regulation  

PubMed Central

Alternative precursor mRNA splicing is a widespread phenomenon in multicellular eukaryotes and represents a major means for functional expansion of the transcriptome. While several recent studies have revealed an important link between splicing regulation and fundamental biological processes in plants, many important aspects, such as the underlying splicing regulatory mechanisms, are so far not well understood. Splicing decisions are in general based on a splicing code that is determined by the dynamic interplay of splicing–controlling factors and cis-regulatory elements. Several members of the group of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins are well known regulators of splicing in animals and the comparatively few reports on some of their plant homologs revealed similar functions. This also applies to polypyrimidine tract-binding proteins, a thoroughly investigated class of hnRNP proteins with splicing regulatory functions in both animals and plants. Further examples from plants are auto- and cross-regulatory splicing circuits of glycine-rich RNA binding proteins and splicing enhancement by oligouridylate binding proteins. Besides their role in defining splice site choice, hnRNP proteins are also involved in multiple other steps of nucleic acid metabolism, highlighting the functional versatility of this group of proteins in higher eukaryotes. PMID:22639666

Wachter, Andreas; Rühl, Christina; Stauffer, Eva

2012-01-01

235

Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy.  

PubMed

Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making. PMID:25652415

Martorell, L; Corrales, I; Ramirez, L; Parra, R; Raya, A; Barquinero, J; Vidal, F

2015-03-01

236

Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens1[W][OPEN  

PubMed Central

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

2014-01-01

237

A novel splicing mutation in the SEDL gene causes spondyloepiphyseal dysplasia tarda in a large Chinese pedigree.  

PubMed

The X-linked form of spondyloepiphyseal dysplasia tarda (SEDT, OMIM# 313400) is a rare osteochondrodysplasia caused by mutations in the SEDL (TRAPPC2, OMIM# 300202) gene. It is clinically characterized by disproportionate short stature, barrel-shaped chests and early development of degenerative joint disease. We report here a novel mutation in the intron 3 splice-donor site (c. 93+5G>C) segregated in an X-link pattern in a large Chinese family with SEDT. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mutation causes an aberrant splicing of exon 3, resulting in the elimination of 31 codons in the exon and a considerable loss function of the SEDL protein. This mutation was not detected in the 100 healthy controls. This novel mutation adds to the spectrum of previously-identified disease-causing mutations. Pre-symptomatic molecular diagnosis and prenatal diagnosis of the pregnant carriers could be helpful to families with SEDT. PMID:23876379

Wang, Hui; Wu, Weiqing; Xu, Zhiyong; Xie, Jiansheng

2013-10-21

238

Promoter-proximal polyadenylation sites reduce transcription activity  

PubMed Central

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ?500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

2012-01-01

239

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.

240

Laparoscopic donor nephrectomy.  

PubMed

Living donor nephrectomy has been developed and promoted as a method to address the shortfall in kidneys available for transplantation. The classical method to procure a kidney from a living donor is the open donor nephrectomy performed through a flank lumbotomy incision. However, this classical method has negative short- and long-term side effects for the donor. These disincentives are a drawback for possible donors to donate a kidney. Therefore, transplant surgeons were stimulated to develop new and less invasive techniques. In this review several new open and laparoscopic techniques are described. Compared with open donor nephrectomy, laparoscopic donor nephrectomy has shown superior results in terms of postoperative pain, cosmetics, convalescence, and return to normal daily activities. No significant differences exist between the two approaches in terms of complication rates, cost-effectiveness and graft function. Nowadays, laparoscopic donor nephrectomy has become the preferred method for procuring kidney grafts of living donors in many centres. PMID:20508268

Minnee, R C; Idu, M M

2010-05-01

241

Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.  

PubMed

Replacement of mRNA 5' UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3' of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare. PMID:20142326

Derelle, Romain; Momose, Tsuyoshi; Manuel, Michael; Da Silva, Corinne; Wincker, Patrick; Houliston, Evelyn

2010-04-01

242

Serum albumin binding sites properties in donors and in schizophrenia patients: the study of fluorescence decay of the probe K-35 using S-60 synchrotron pulse excitation  

NASA Astrophysics Data System (ADS)

The properties of serum albumin obtained from donors and from paranoid schizophrenia patients were studied with the fluorescent probe K-35 (N-carboxyphenylimide of dimethylaminonaphthalic acid) and time-resolved fluorescence spectroscopy on the SR beam station of the S-60 synchrotron of the Lebedev Physical Institute. The mean fluorescence quantum yield of K-35 in patients serum was decreased significantly by 25-60% comparing with donors. The analysis of pre-exponential factors of fluorescence decay using "amplitude standard" method has shown that in patient sera the fraction of K-35 molecules bound with albumin and inaccessible to fluorescence quenchers ("bright" K-35 molecules with ?1=8.0±0.4 ns) is 1.2-3 times less than in the donor sera. The fraction of K-35 molecules with partly quenched fluorescence ( ?2=1.44±0.22 ns) was significantly increased in schizophrenia patients. The results obtained suggest that the properties of binding region in serum albumin molecules of acute paranoid schizophrenia patients change significantly.

Gryzunov, Yu. A.; Syrejshchikova, T. I.; Komarova, M. N.; Misionzhnik, E. Yu; Uzbekov, M. G.; Molodetskich, A. V.; Dobretsov, G. E.; Yakimenko, M. N.

2000-06-01

243

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

244

Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica.  

PubMed

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

2012-06-01

245

Structure of the branched intermediate in protein splicing  

PubMed Central

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site. PMID:24778214

Liu, Zhihua; Frutos, Silvia; Bick, Matthew J.; Vila-Perelló, Miquel; Debelouchina, Galia T.; Darst, Seth A.; Muir, Tom W.

2014-01-01

246

Structure of the branched intermediate in protein splicing.  

PubMed

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site. PMID:24778214

Liu, Zhihua; Frutos, Silvia; Bick, Matthew J; Vila-Perelló, Miquel; Debelouchina, Galia T; Darst, Seth A; Muir, Tom W

2014-06-10

247

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

PubMed Central

Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle. PMID:9872965

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

1999-01-01

248

Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II.  

PubMed

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II. PMID:9047318

Coleman, W J; Mattioli, T A; Youvan, D C; Rutherford, A W

1997-02-25

249

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

250

Functional Characterization of NIPBL Physiological Splice Variants and Eight Splicing Mutations in Patients with Cornelia de Lange Syndrome  

PubMed Central

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ?E10, ?E12, ?E33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame. PMID:24918291

Teresa-Rodrigo, María E.; Eckhold, Juliane; Puisac, Beatriz; Dalski, Andreas; Gil-Rodríguez, María C.; Braunholz, Diana; Baquero, Carolina; Hernández-Marcos, María; de Karam, Juan C.; Ciero, Milagros; Santos-Simarro, Fernando; Lapunzina, Pablo; Wierzba, Jolanta; Casale, César H.; Ramos, Feliciano J.; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J.; Pié, Juan

2014-01-01

251

An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing  

PubMed Central

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

2010-01-01

252

Contacting My Donor Family  

MedlinePLUS

... Donor Family Newsroom Minorities Contacting My Donor Family Writing anything can be a challenge. Staring at a ... can take to get started. The process of writing your letter may take some time, but at ...

253

Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome.  

PubMed

Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. PMID:25586593

Madan, Vikas; Kanojia, Deepika; Li, Jia; Okamoto, Ryoko; Sato-Otsubo, Aiko; Kohlmann, Alexander; Sanada, Masashi; Grossmann, Vera; Sundaresan, Janani; Shiraishi, Yuichi; Miyano, Satoru; Thol, Felicitas; Ganser, Arnold; Yang, Henry; Haferlach, Torsten; Ogawa, Seishi; Koeffler, H Phillip

2015-01-01

254

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

255

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

256

Self-splicing of a group IIC intron: 5? exon recognition and alternative 5? splicing events implicate the stem–loop motif of a transcriptional terminator  

PubMed Central

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5? junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem–loop and IBS1 motifs, suggesting that the 5? exon is recognized by both elements. These results provide the first example of a group II intron that uses 5? splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5? exon definition, and that the terminator stem–loop substitutes in part for the missing IBS2–EBS2 (intron and exon binding sites 2) interaction. PMID:17130159

Toor, Navtej; Robart, Aaron R.; Christianson, Joshua; Zimmerly, Steven

2006-01-01

257

Alternative splicing in regulation of cholesterol homeostasis  

PubMed Central

Purpose of review With the advent of whole-transcriptome sequencing, or RNA-seq, we now know that alternative splicing is a generalized phenomenon, with nearly all multi-exonic genes subject to alternative splicing. In this review we highlight recent studies examining alternative splicing as a modulator of cellular cholesterol homeostasis, and as an underlying mechanism of dyslipidemia. Recent findings A number of key genes involved in cholesterol metabolism are known to undergo functionally relevant alternative splicing. Recently, we have identified coordinated changes in alternative splicing in multiple genes in response to alteration in cellular sterol content. We and others have implicated several splicing factors as regulators of lipid metabolism. Furthermore, a number of cis-acting human gene variants that modulate alternative splicing have been implicated in a variety of human metabolic diseases. Summary Alternative splicing is of importance in various types of genetically influenced dyslipidemias, and in the regulation of cellular cholesterol metabolism. PMID:23314925

Medina, Marisa W.; Krauss, Ronald M.

2013-01-01

258

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

259

GIFT AGREEMENT BETWEEN (NAME OF DONOR(S))  

E-print Network

1 GIFT AGREEMENT BETWEEN (NAME OF DONOR(S)) ("the Donor(s)") AND MCMASTER UNIVERSITY ("the University") I. THE GIFT (NAME OF DONOR(S)) have generously made a Gift of (X) to McMaster University. This generous Gift will be provided in the form (METHOD OF PAYMENT) according to the following schedule: II

Haykin, Simon

260

GIFT AGREEMENT BETWEEN NAME OF DONOR(S)  

E-print Network

1 GIFT AGREEMENT BETWEEN NAME OF DONOR(S) ("the Donor(s)") AND McMASTER UNIVERSITY ("the University") I. THE GIFT (NAME OF DONOR(S)) has generously made a Gift of (GIFT/PLEDGE $) to McMaster University (hereinafter the "Gift"). This Gift/Pledge will be provided in the form of (METHOD OF PAYMENT) according

Haykin, Simon

261

Detect Digital Image Splicing with Visual Cues  

E-print Network

Detect Digital Image Splicing with Visual Cues Zhenhua Qu1 , Guoping Qiu2 , and Jiwu Huang1 1. Image splicing detection has been considered as one of the most challenging problems in passive image authentication. In this paper, we propose an automatic detection framework to identify a spliced image

Aickelin, Uwe

262

Networking in an alternative splicing world.  

PubMed

Using Caenorhabditis elegans as a model system, Norris et al. (2014) define complex combinatorial regulation of alternative splicing at single-neuron resolution and illustrate functional coherence among components of a splicing regulatory network controlled by a neuronal splicing factor. PMID:24950376

Carstens, Russ P

2014-06-19

263

Influenza Viruses and mRNA Splicing: Doing More with Less  

PubMed Central

ABSTRACT During their nuclear replication stage, influenza viruses hijack the host splicing machinery to process some of their RNA segments, the M and NS segments. In this review, we provide an overview of the current knowledge gathered on this interplay between influenza viruses and the cellular spliceosome, with a particular focus on influenza A viruses (IAV). These viruses have developed accurate regulation mechanisms to reassign the host spliceosome to alter host cellular expression and enable an optimal expression of specific spliced viral products throughout infection. Moreover, IAV segments undergoing splicing display high levels of similarity with human consensus splice sites and their viral transcripts show noteworthy secondary structures. Sequence alignments and consensus analyses, along with recently published studies, suggest both conservation and evolution of viral splice site sequences and structure for improved adaptation to the host. Altogether, these results emphasize the ability of IAV to be well adapted to the host’s splicing machinery, and further investigations may contribute to a better understanding of splicing regulation with regard to viral replication, host range, and pathogenesis. PMID:24825008

Dubois, Julia

2014-01-01

264

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

PubMed Central

NAGNAG alternative splicing plays an essential role in biological processes and represents a highly adaptable system for posttranslational regulation of gene function. NAGNAG alternative splicing impacts a myriad of biological processes. Previous studies of NAGNAG largely focused on messenger RNA. To the best of our knowledge, this is the first study testing the hypothesis that NAGNAG alternative splicing is also operative in large intergenic noncoding RNA (lincRNA). The RNA-seq data sets from recent deep sequencing studies were queried to test our hypothesis. NAGNAG alternative splicing of human lincRNA was identified while querying two independent RNA-seq data sets. Within these datasets, 31 NAGNAG alternative splicing sites were identified in lincRNA. Notably, most exons of lincRNA containing NAGNAG acceptors were longer than those from protein-coding genes. Furthermore, presence of CAG coding appeared to participate in the splice site selection. Finally, expression of the isoforms of NAGNAG lincRNA exhibited tissue specificity. Together, this study improves our understanding of the NAGNAG alternative splicing in lincRNA. PMID:24995327

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

2014-01-01

265

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly  

PubMed Central

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7+ (spslu7+) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3? splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3? splice site distance, intron length, and the impact of its A/U content at the 5? end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly. PMID:23754748

Banerjee, Shataparna; Khandelia, Piyush; Melangath, Geetha; Bashir, Samirul; Nagampalli, Vijaykrishna

2013-01-01

266

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

267

Some Characterizations in Splicing Systems  

NASA Astrophysics Data System (ADS)

The splitting and recombinant of deoxyribonucleic acid or DNA by specified enzymes using concepts in Formal Language Theory was first mathematically modeled by Head in 1987. This splicing system, S can be presented as a set of initial string I over an alphabet A that acts upon 5' or 3' overhangs of restriction enzymes and can be simply viewed as S = (A, I, B, C). In this paper, a great interest in presenting some relations on certain types of splicing system namely null-context, uniform, simple, semi-simple, semi-null and Sk based on differentiating their rules are given as proposition, corollaries and counterexamples.

Sarmin, Nor Haniza; Yusof, Yuhani; Wan Heng, Fong

2010-11-01

268

Operational characteristics of trailing cable splices  

SciTech Connect

The US Bureau of Mines investigated the operational characteristics of spliced portable power cables. This research had a dual purpose: (1) to determine the thermal and mechanical performance of repaired trailing cables and compare them with undamaged cables, and (2) to gauge the impact of long-term localized heating on the insulating and jacketing materials contained in cable splice kits accepted or approved by the Mine Safety and Health Administration. The ranges of splice joint resistance and tensile breaking strength were determined from laboratory measurements. The choice of crimping tools affected the strength of the splice under tension. Thermal profiles of energized spliced cables were constructed, which showed that spliced cables were constructed, which showed that spliced conductor joints operated 5 to 20 C hotter than the intact cable at rated currents. Accelerated life tests of thermally-aged samples of splice kit insulation and jacket materials confirmed a deficiency in the thermal rating of the insulating tape. The recommendations in this paper may be utilized to revise splice kit design, splice kit acceptance criteria, and trailing cable loading guidelines. Characterizing the thermal operating limits of spliced trailing cables may help to minimize associated risks from explosions, fires, personnel burns, and shock.

Yenchek, M.R.; Schuster, K.C.; Hudson, A.J. [Bureau of Mines, Pittsburgh, PA (United States). Pittsburgh Research Center

1995-12-31

269

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

PubMed Central

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

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

2012-01-01

270

Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons  

PubMed Central

Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3? and 5? splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

2012-01-01

271

An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing  

Microsoft Academic Search

BACKGROUND: Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each

Keven R Johnson; Jessie Nicodemus-Johnson; Robert S Danziger

2010-01-01

272

Crowd Around: Expanding Your Donor Pool with Crowdfunding  

ERIC Educational Resources Information Center

At most institutions, annual fund-giving is down. Crowdfunding sites allow people with a great idea or worthy cause to bypass traditional funding methods and take their case directly to web-savvy investors and donors. This article describes how higher education institutions are expanding their donor pool through such crowdfunding sites as USEED,…

Jarrell, Andrea

2013-01-01

273

Digging up Classroom Dollars on DonorsChoose  

ERIC Educational Resources Information Center

Back in 2000, Charles Best was teaching at Wings Academy, an alternative high school in the Bronx, when he got the idea for a Web site where teachers could solicit donations for class projects. With help from his students, DonorsChoose.org soon was born. Last year, the site won Amazon.com's Nonprofit Innovation Award. So far, DonorsChoose has…

Curriculum Review, 2006

2006-01-01

274

Diverse splicing pathways of the membrane IgHM pre-mRNA in a Chondrostean, the Siberian sturgeon.  

PubMed

Teleosts and tetrapods have evolved different splice patterns to generate their membrane-bound IgM. In the tetrapod lineage, the first transmembrane exon is spliced to an internal cryptic site located close to the end of the fourth constant exon. Because teleosts lack this site they use the regular 3'-splice site of the CH3 exon instead. We characterized the mum splicing patterns in a Chondrostean, the Siberian sturgeon. We observed a surprising diversity of splice patterns, the TM1 exon being spliced to a cryptic site at the end of CH4, to a cryptic site in CH3 or to the 3'-end of CH1. These different pathways lead to mIGHM transcripts encoding four, two or one complete C-domain(s), respectively. The short variant CH1-TM1 was found only in VH2 positive transcripts, while the two other variants were observed for IgHM transcripts expressing all VH families. These results shed light on the evolution of IgM splicing pathways. PMID:19027787

Lundqvist, Mats; Strömberg, Siv; Bouchenot, Catherine; Pilström, Lars; Boudinot, Pierre

2009-04-01

275

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

PubMed Central

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation. PMID:19893621

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

2009-01-01

276

GIFT AGREEMENT BETWEEN (NAME OF DONOR(S))  

E-print Network

1 GIFT AGREEMENT BETWEEN (NAME OF DONOR(S)) ("the Donor(s)") AND MCMASTER UNIVERSITY ("the University") I. THE GIFT (DONORS NAME(S) have generously made a gift of (GIFT/PLEDGE AMOUNT) to McMaster University (hereinafter the "Gift"). This Gift/Pledge will be provided in the form of (METHOD OF PAYMENT

Haykin, Simon

277

Automatic detection of exonic splicing enhancers (ESEs) using SVMs  

PubMed Central

Background Exonic splicing enhancers (ESEs) activate nearby splice sites and promote the inclusion (vs. exclusion) of exons in which they reside, while being a binding site for SR proteins. To study the impact of ESEs on alternative splicing it would be useful to have a possibility to detect them in exons. Identifying SR protein-binding sites in human DNA sequences by machine learning techniques is a formidable task, since the exon sequences are also constrained by their functional role in coding for proteins. Results The choice of training examples needed for machine learning approaches is difficult since there are only few exact locations of human ESEs described in the literature which could be considered as positive examples. Additionally, it is unclear which sequences are suitable as negative examples. Therefore, we developed a motif-oriented data-extraction method that extracts exon sequences around experimentally or theoretically determined ESE patterns. Positive examples are restricted by heuristics based on known properties of ESEs, e.g. location in the vicinity of a splice site, whereas negative examples are taken in the same way from the middle of long exons. We show that a suitably chosen SVM using optimized sequence kernels (e.g., combined oligo kernel) can extract meaningful properties from these training examples. Once the classifier is trained, every potential ESE sequence can be passed to the SVM for verification. Using SVMs with the combined oligo kernel yields a high accuracy of about 90 percent and well interpretable parameters. Conclusion The motif-oriented data-extraction method seems to produce consistent training and test data leading to good classification rates and thus allows verification of potential ESE motifs. The best results were obtained using an SVM with the combined oligo kernel, while oligo kernels with oligomers of a certain length could be used to extract relevant features. PMID:18783607

Mersch, Britta; Gepperth, Alexander; Suhai, Sándor; Hotz-Wagenblatt, Agnes

2008-01-01

278

Rich Donors, Poor Countries  

ERIC Educational Resources Information Center

The shifting ideological winds of foreign aid donors have driven their policy towards governments in poor countries. Donors supported state-led development policies in poor countries from the 1940s to the 1970s; market and private-sector driven reforms during the 1980s and 1990s; and returned their attention to the state with an emphasis on…

Thomas, M. A.

2012-01-01

279

Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide.  

PubMed

Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3' splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts. PMID:21390052

Ijspeert, H; Lankester, A C; van den Berg, J M; Wiegant, W; van Zelm, M C; Weemaes, C M R; Warris, A; Pan-Hammarström, Q; Pastink, A; van Tol, M J D; van Dongen, J J M; van Gent, D C; van der Burg, M

2011-09-01

280

A general definition and nomenclature for alternative splicing events.  

PubMed

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

Sammeth, Michael; Foissac, Sylvain; Guigó, Roderic

2008-01-01

281

Alternative Splicing in Plant Immunity  

PubMed Central

Alternative splicing (AS) occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R) genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel. PMID:24918296

Yang, Shengming; Tang, Fang; Zhu, Hongyan

2014-01-01

282

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.  

PubMed

Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth. PMID:22172722

Amin, Elianna M; Oltean, Sebastian; Hua, Jing; Gammons, Melissa V R; Hamdollah-Zadeh, Maryam; Welsh, Gavin I; Cheung, Man-Kim; Ni, Lan; Kase, Satoru; Rennel, Emma S; Symonds, Kirsty E; Nowak, Dawid G; Royer-Pokora, Brigitte; Saleem, Moin A; Hagiwara, Masatoshi; Schumacher, Valérie A; Harper, Steven J; Hinton, David R; Bates, David O; Ladomery, Michael R

2011-12-13

283

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing  

PubMed Central

Summary Angiogenesis is regulated by the balance of pro-angiogenic VEGF165 and anti-angiogenic VEGF165b splice isoforms. Mutations in WT1, the Wilms’ tumour suppressor gene, suppress VEGF165b and cause abnormal gonadogenesis, renal failure and Wilms’ tumours. In WT1 mutant cells, reduced VEGF165b was due to lack of WT1 mediated transcriptional repression of the splicing factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding-site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF, and rendered WT1 mutant cells pro-angiogenic. Altered VEGF splicing was reversed by wildtype WT1, knockdown of SRSF1 or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumour growth. PMID:22172722

Amin, Elianna M; Oltean, Sebastian; Hua, Jing; Gammons, Melissa VR; Hamdollah-Zadeh, Maryam; Welsh, Gavin I; Cheung, Man-Kim; Ni, Lan; Kase, Satoru; Rennel, Emma S; Symonds, Kirsty E; Nowak, Dawid G; Pokora-Royer, Brigitte; Saleem, Moin A; Hagiwara, Masatoshi; Schumacher, Valérie A; Harper, Steven J; Hinton, David R; Bates, David O; Ladomery, Michael R

2013-01-01

284

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.  

PubMed

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. PMID:24960161

Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H; Selbach, Matthias; Barton, Paul J R; Cook, Stuart A; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

2014-08-01

285

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing  

PubMed Central

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3? and 5? splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. PMID:24960161

Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H.; Selbach, Matthias; Barton, Paul J.R.; Cook, Stuart A.; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

2014-01-01

286

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

287

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

PubMed Central

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

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

2013-01-01

288

Regulated functional alternative splicing in Drosophila  

PubMed Central

Alternative splicing expands the coding capacity of metazoan genes, and it was largely genetic studies in the fruit-fly Drosophila melanogaster that established the principle that regulated alternative splicing results in tissue- and stage-specific protein isoforms with different functions in development. Alternative splicing is particularly prominent in germ cells, muscle and the central nervous system where it modulates the expression of various proteins including cell-surface molecules and transcription factors. Studies in flies have given us numerous insights into alternative splicing in terms of upstream regulation, the exquisite diversity of their forms and the key differential cellular functions of alternatively spliced gene products. The current inundation of transcriptome sequencing data from Drosophila provides an unprecedented opportunity to gain a comprehensive view of alternative splicing. PMID:21908400

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

2012-01-01

289

RNA Splicing in Plant Mitochondria  

Microsoft Academic Search

\\u000a Abstract\\u000a \\u000a Introns within the mitochondrial genes of land plants belong mostly to the group II category of ribozyme mobile retroelements,\\u000a although a minor number (particularly in nonvascular plants) are members of the group I intron family of “homing” ribozymes.\\u000a In vascular plants, the mitochondrial introns often lack conventional structural features, and their degenerate nature is\\u000a correlated with unusual splicing pathways.

Linda Bonen

290

Photocurrent measurement on donor bound excitons in Si  

NASA Astrophysics Data System (ADS)

Donor bound excitons are formed when free excitons are captured by neutral donor impurities. Due to the spatial localization of exciton at the impurity site, the decay process of donor bound exciton state to neutral donor state features extremely narrow linewidth in energy. Utilizing this inherent feature, it would be feasible to identify nuclear spin states of the donor impurity resulting from the hyperfine interaction between phosphorus nucleus spin and electron spin. We study ensembles of phosphorus donor bound excitons in Si via photocurrent measurements at low temperatures since Auger non-radiative decay process is primarily dominant in an indirect band-gap semiconductor such as Si. We report electric and magnetic field effects on photocurrent signals of phosphorus donor bound excitons.

Kim, Na Young; Sleiter, Darin; Ladd, Thaddeus; Nozawa, Katsuya; Yamamoto, Yoshihisa

2009-03-01

291

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2012 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2012-07-01

292

30 CFR 57.12088 - Splicing trailing cables.  

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2014-07-01

293

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2011 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2011-07-01

294

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2010 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2010-07-01

295

Autoregulation of Fox protein expression to produce dominant negative splicing factors  

PubMed Central

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (Fox?RRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These Fox?RRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform. PMID:20042473

Damianov, Andrey; Black, Douglas L.

2010-01-01

296

Toward predicting self-splicing and protein-facilitated splicing of group I introns.  

PubMed

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence. PMID:18768647

Vicens, Quentin; Paukstelis, Paul J; Westhof, Eric; Lambowitz, Alan M; Cech, Thomas R

2008-10-01

297

The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans.  

PubMed

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted. PMID:11342634

Tchilian, E Z; Wallace, D L; Imami, N; Liao, H X; Burton, C; Gotch, F; Martinson, J; Haynes, B F; Beverley, P C

2001-05-15

298

3'UTR-located ALU elements: donors of potential miRNA target sites and mediators of network miRNA-based regulatory interactions.  

PubMed

Recent research data reveal complex, network-based interactions between mobile elements and regulatory systems of eukaryotic cells. In this article, we focus on regulatory interactions between Alu elements and micro RNAs (miRNAs). Our results show that the majority of the Alu sequences inserted in 3'UTRs of analyzed human genes carry strong potential target sites for at least 53 different miRNAs. Thus, 3'UTR-located Alu elements may play the role of mobile regulatory modules that supply binding sites for miRNA regulation. Their abundance and ability to distribute a set of certain miRNA target sites may have an important role in establishment, extension, network organization, and, as we suppose - in the regulation and environment-dependent activation/inactivation of some elements of the miRNA regulatory system, as well as for a larger scale RNA-based regulatory interactions. The Alu-miRNA connection may be crucial especially for the primate/human evolution. PMID:19455205

Daskalova, Evelina; Baev, Vesselin; Rusinov, Ventsislav; Minkov, Ivan

2006-01-01

299

In Brief: (Mis)splicing in disease  

PubMed Central

Splicing of pre-mRNAs is a crucial step in the gene expression pathway. Disruption of splicing has been linked to the pathogenesis of several human diseases and is particularly widespread in cancer. Recently, a number of mutations affecting genes of the core spliceosome machinery have been identified in haematological malignancies, yet the effect of such mutations on RNA splicing is unclear. A better understanding of how mis-splicing contributes to malignancies may provide diagnostic or prognostic information and new drug targets for therapeutic approaches. PMID:24615176

Pedrotti, Simona; Cooper, Thomas A

2015-01-01

300

Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation  

SciTech Connect

In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

Gesnel, Marie-Claude [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Theoleyre, Sandrine [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Del Gatto-Konczak, Fabienne [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Breathnach, Richard [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France) and Faculte des Sciences, Universite de Nantes, Nantes (France)]. E-mail: breathna@nantes.inserm.fr

2007-07-13

301

Cooperative-Binding and Splicing-Repressive Properties of hnRNP A1? †  

PubMed Central

hnRNP A1 binds to RNA in a cooperative manner. Initial hnRNP A1 binding to an exonic splicing silencer at the 3? end of human immunodeficiency virus type 1 (HIV-1) tat exon 3, which is a high-affinity site, is followed by cooperative spreading in a 3?-to-5? direction. As hnRNP A1 propagates toward the 5? end of the exon, it antagonizes binding of a serine/arginine-rich (SR) protein to an exonic splicing enhancer, thereby inhibiting splicing at that exon's alternative 3? splice site. tat exon 3 and the preceding intron of HIV-1 pre-mRNA can fold into an elaborate RNA secondary structure in solution, which could potentially influence hnRNP A1 binding. We report here that hnRNP A1 binding and splicing repression can occur on an unstructured RNA. Moreover, hnRNP A1 can effectively unwind an RNA hairpin upon binding, displacing a bound protein. We further show that hnRNP A1 can also spread in a 5?-to-3? direction, although when initial binding takes place in the middle of an RNA, spreading preferentially proceeds in a 3?-to-5? direction. Finally, when two distant high-affinity sites are present on the same RNA, they facilitate cooperative spreading of hnRNP A1 between the two sites. PMID:19667073

Okunola, Hazeem L.; Krainer, Adrian R.

2009-01-01

302

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression  

PubMed Central

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3?splice site, and the other involves an exonic splicing silencer (ESS) situated 3? to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator. PMID:23818605

Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L.; Feng, Ying

2013-01-01

303

Imbalanced splicing in MAPK signaling sustains Ras-induced transformation.  

PubMed

Increasingly, evidence suggests that phosphorylation of the mRNA translation initiation factor eIF4E plays an important role in carcinogenesis, downstream of Ras. The eIF4E factor is phosphorylated by MAPK-interacting protein kinases 1 and 2 (MNK1 and MNK2). Due to alternative splicing, two MNK2 proteins exist (MNK2a and MNK2b). While MNK2a possesses a binding site for the stress-induced p38-MAPK, MNK2b does not. Recently, Maimon et al. revealed that a splicing shift towards the MNK2b isoform, in Ras-activated cells, sustains transformation, due to a defect in p38-induced cell death, while the MNK2b-dependent phosphorylation of eIF4E is maintained. PMID:25468550

Müller, David; Martineau, Yvan; Bousquet, Corinne; Pyronnet, Stéphane

2014-11-20

304

Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds  

SciTech Connect

Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.

Doerk, T.; Wulbrand, U.; Tuemmler, B. (Medizinische Hochschule Hannover (Germany))

1993-03-01

305

Finding a Donor  

MedlinePLUS

... business Public engagement Registry & patient services Research & science Information technology Search Open Jobs Employee benefits Career events Job application FAQs E-Verify Financial information Annual report Funding patient assistance Funding donor recruitment ...

306

Tumor microenvironment–associated modifications of alternative splicing  

PubMed Central

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ?20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors. PMID:24335142

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

2014-01-01

307

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

308

A Widespread and Unusual RNA Trans-Splicing Type in Dinoflagellate Mitochondria  

PubMed Central

Cytochrome oxidase subunit 3 (Cox3) is a mitochondrion-encoded core membrane protein of complex IV of the mitochondrial respiratory chain, and consists of seven trans-membrane helices. Here we show that in diverse later-branching dinoflagellates, cox3 is consistently split into two exons in the mitochondrial genome between helices six and seven. Gene exons are transcribed as two discrete oligoadenylated precursor RNAs, and these are subsequently trans-spliced to form a complete coding mRNA. This trans-splicing is highly unusual in that some of the oligoadenylated tail is incorporated at the splice site, such that a short string of adenosines links the two coding exons. This feature is consistently represented in diverse dinoflagellates, however the number of adenosines added varies according to the size of the coding gap between the two exons. Thus we observed between zero (Amphidinium carterae) and 10 (Symbiodinium sp.) adenosines added in different taxa, but the final coding sequence length is identical with the reading frame maintained. Northern analyses show that precursor cox3 transcripts are approximately equally abundant as mature cox3 mRNAs, suggesting a slow or regulated maturation process. These data indicate that the splicing mechanism in dinoflagellate mitochondria is tolerant of variations in the length of the precursor coding sequence, and implicates the use of a splicing template, or guide molecule, during splicing that controls mature mRNA length. PMID:23437234

Jackson, Christopher J.; Waller, Ross F.

2013-01-01

309

Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity  

PubMed Central

SUMMARY Removal of introns from pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we have directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and ?N, we show that beta-globin introns are transcribed and excised in 20-30 s. We further show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin ? (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min-1, and that transcription can be rate limiting for splicing. These results have important implications for mechanistic understanding of co-transcriptional splicing regulation in the live-cell context. PMID:24035393

Martin, Robert M.; Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, Maria

2013-01-01

310

In silico prediction of splice-altering single nucleotide variants in the human genome  

PubMed Central

In silico tools have been developed to predict variants that may have an impact on pre-mRNA splicing. The major limitation of the application of these tools to basic research and clinical practice is the difficulty in interpreting the output. Most tools only predict potential splice sites given a DNA sequence without measuring splicing signal changes caused by a variant. Another limitation is the lack of large-scale evaluation studies of these tools. We compared eight in silico tools on 2959 single nucleotide variants within splicing consensus regions (scSNVs) using receiver operating characteristic analysis. The Position Weight Matrix model and MaxEntScan outperformed other methods. Two ensemble learning methods, adaptive boosting and random forests, were used to construct models that take advantage of individual methods. Both models further improved prediction, with outputs of directly interpretable prediction scores. We applied our ensemble scores to scSNVs from the Catalogue of Somatic Mutations in Cancer database. Analysis showed that predicted splice-altering scSNVs are enriched in recurrent scSNVs and known cancer genes. We pre-computed our ensemble scores for all potential scSNVs across the human genome, providing a whole genome level resource for identifying splice-altering scSNVs discovered from large-scale sequencing studies. PMID:25416802

Jian, Xueqiu; Boerwinkle, Eric; Liu, Xiaoming

2014-01-01

311

Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract.  

PubMed

Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intron. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, we determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. We demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3' and 5' splice sites as are used in C. elegans. The branch point used lies in the inserted sequence. We conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts. PMID:2308820

Ogg, S C; Anderson, P; Wickens, M P

1990-01-11

312

RNA sequencing for the study of splicing  

E-print Network

in splicing patterns in a disease context. By analysing healthy and tumor samples from kidney cancer patients, I show that most of the detected splicing alterations do not lead to big changes in the relative abundance of major transcripts, at least in a...

Gonza?lez-Porta, Mar

2014-10-07

313

The Effect of Donor Age on Corneal Transplantation Outcome: Results of the Cornea Donor Study  

PubMed Central

Objective To determine whether graft survival over a 5-year follow-up period using corneal tissue from donors older than 65 years of age is similar to graft survival using corneas from younger donors. Design Multi-center prospective, double-masked, controlled clinical trial Participants 1090 subjects undergoing corneal transplantation for a moderate risk condition (principally Fuchs’ dystrophy or pseudophakic corneal edema); 11 subjects with ineligible diagnoses were not included Methods 43 participating eye banks provided corneas from donors in the age range of 12 to 75 with endothelial cell densities of 2300 to 3300 cells/mm2, using a random approach without respect to recipient factors. The 105 participating surgeons at 80 sites were masked to information about the donor cornea including donor age. Surgery and post-operative care were performed according to the surgeons’ usual routines. Subjects were followed for five years. Main Outcome Measures Graft failure, defined as a regraft or a cloudy cornea that was sufficiently opaque as to compromise vision for a minimum of three consecutive months. Results The 5-year cumulative probability of graft survival was 86% in both the <66.0 donor age group and the ?66.0 donor age group (difference = 0%, upper limit of one-sided 95% confidence interval = 4%). In a statistical model with donor age as a continuous variable, there was not a significant relationship between donor age and outcome (P=0.11). Three graft failures were due to primary donor failure, 8 to uncorrectable refractive error, 48 to graft rejection, 46 to endothelial decompensation (23 of which had a prior, resolved episode of probable or definite graft rejection), and 30 to other causes. The distribution of the causes of graft failure did not differ between donor age groups. Conclusions Five-year graft survival for cornea transplants at moderate risk for failure is similar using corneas from donors ? 66.0 years and donors < 66.0 years. Surgeons and patients now have evidence that corneas comparable in quality to those used in this study from donors through age 75 years are suitable for transplantation. PMID:18387407

2009-01-01

314

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.  

PubMed

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1?, Nrxn1?, Nrxn2?, Nrxn3?, and Nrxn3? mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1? and Nrxn3? (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-?, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that ?-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing. PMID:24639501

Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

2014-04-01

315

Modulation of alternative splicing by expression of small nuclear ribonucleoprotein polypeptide N.  

PubMed

Alternative splicing of pre-mRNA, catalyzed by small nuclear ribonucleoproteins (snRNPs), plays an important role in proteome complexity and the modulation of cellular functions. snRNP polypeptide N (SmN), is tissue-specifically expressed, where it replaces snRNP polypeptide B (SmB)/B' in the Sm core assembly of snRNPs. Recent studies have demonstrated that perturbation of snRNPs leads to alternative splicing, but whether SmN modulates functions of the splicing machinery remains unclear. In this study, we found that ectopic expression of SmN increased utilization of the proximal 5' splice site on an adenovirus early gene 1A reporter. To evaluate the molecular mechanisms underlying SmN-dependent alternative splicing, we generated a HeLa cell line with an inducible expression system for SmN. Upon SmN induction, SmB/B' expression decreased dramatically, despite only small changes in the level and splicing pattern of SNRPB mRNA. In addition, SmN was incorporated into the U2 snRNP but not into the U1 snRNP after induction. Sedimentation analysis revealed a decrease in the level of mature U2 snRNP. This result suggests that SmN incorporation into the Sm core may impede processing, decreasing the level of functional U2 snRNP. We also found that the inclusion frequencies of alternatively spliced exons in the bridging integrator 1 and exocyst complex component 7 (EXOC7) genes were modulated by SmN expression. An enhanced GFP-EXOC7 reporter was used to confirm that SmN increases the inclusion frequency of EXOC7 exon 7. Taken together, our findings indicate that SmN expression reduces the level of mature U2 snRNP, leading to alternative splicing. PMID:25238490

Lee, Moon-Sing; Lin, Yu-Shan; Deng, Yi-Fang; Hsu, Wan-Ting; Shen, Chiung-Chun; Cheng, Yi-Hsin; Huang, Yao-Ting; Li, Chin

2014-12-01

316

Extensive Alternative Splicing of the Repressor Element Silencing Transcription Factor Linked to Cancer  

PubMed Central

The repressor element silencing transcription factor (REST) is a coordinate transcriptional and epigenetic regulator which functions as a tumor suppressor or an oncogene depending on cellular context, and a truncated splice variant REST4 has been linked to various types of cancer. We performed a comprehensive analysis of alternative splicing (AS) of REST by rapid amplification of cDNA ends and PCR amplification of cDNAs from various tissues and cell lines with specific primers. We identified 8 novel alternative exons including an alternate last exon which doubles the REST gene boundary, along with numerous 5?/3? splice sites and ends in the constitutive exons. With the combination of various splicing patterns (e.g. exon skipping and alternative usage of the first and last exons) that are predictive of altered REST activity, at least 45 alternatively spliced variants of coding and non-coding mRNA were expressed in a species- and cell-type/tissue-specific manner with individual differences. By examining the repertoire of REST pre-mRNA splicing in 27 patients with kidney, liver and lung cancer, we found that all patients without exception showed differential expression of various REST splice variants between paired tumor and adjacent normal tissues, with striking cell-type/tissue and individual differences. Moreover, we revealed that exon 3 skipping, which causes no frame shift but loss of a domain essential for nuclear translocation, was affected by pioglitazone, a highly selective activator of the peroxisome proliferator-activated receptor gamma (PPAR?) which contributes to cell differentiation and tumorigenesis besides its metabolic actions. Accordingly, this study demonstrates an extensive AS of REST pre-mRNA which redefines REST gene boundary and structure, along with a general but differential link between REST pre-mRNA splicing and various types of cancer. These findings advance our understanding of the complex, context-dependent regulation of REST gene expression and function, and provide potential biomarkers and therapeutic targets for cancer. PMID:23614038

Chen, Guo-Lin; Miller, Gregory M.

2013-01-01

317

Integrative analysis revealed the molecular mechanism underlying RBM10-mediated splicing regulation.  

PubMed

RBM10 encodes an RNA binding protein. Mutations in RBM10 are known to cause multiple congenital anomaly syndrome in male humans, the TARP syndrome. However, the molecular function of RBM10 is unknown. Here we used PAR-CLIP to identify thousands of binding sites of RBM10 and observed significant RBM10-RNA interactions in the vicinity of splice sites. Computational analyses of binding sites as well as loss-of-function and gain-of-function experiments provided evidence for the function of RBM10 in regulating exon skipping and suggested an underlying mechanistic model, which could be subsequently validated by minigene experiments. Furthermore, we demonstrated the splicing defects in a patient carrying an RBM10 mutation, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10-mediated splicing regulation and provided a molecular link to understanding a human congenital disorder. PMID:24000153

Wang, Yongbo; Gogol-Döring, Andreas; Hu, Hao; Fröhler, Sebastian; Ma, Yunxia; Jens, Marvin; Maaskola, Jonas; Murakawa, Yasuhiro; Quedenau, Claudia; Landthaler, Markus; Kalscheuer, Vera; Wieczorek, Dagmar; Wang, Yang; Hu, Yuhui; Chen, Wei

2013-09-01

318

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

PubMed Central

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

Corrionero, Anna; Miñana, Belén; Valcárcel, Juan

2011-01-01

319

The Characterizations of Different Splicing Systems  

NASA Astrophysics Data System (ADS)

The concept of splicing system was first introduced by Head in 1987 to model the biological process of DNA recombination mathematically. This model was made on the basis of formal language theory which is a branch of applied discrete mathematics and theoretical computer science. In fact, splicing system treats DNA molecule and the recombinant behavior by restriction enzymes and ligases in the form of words and splicing rules respectively. The notion of splicing systems was taken into account from different points of view by many mathematicians. Several modified definitions have been introduced by many researchers. In this paper, some properties of different kinds of splicing systems are presented and their relationships are investigated. Furthermore, these results are illustrated by some examples.

Karimi, Fariba; Sarmin, Nor Haniza; Heng, Fong Wan

320

Splicing determines the glycosylation state of ameloblastin.  

PubMed

In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro(11) and Ser(17) confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser(86) is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated. PMID:17890672

Kobayashi, K; Yamakoshi, Y; Hu, J C-C; Gomi, K; Arai, T; Fukae, M; Krebsbach, P H; Simmer, J P

2007-10-01

321

U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing  

PubMed Central

To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5? splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5? splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3? splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5? splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations—a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release. PMID:17403782

Hilliker, Angela K.; Mefford, Melissa A.; Staley, Jonathan P.

2007-01-01

322

Residual Strength Analyses of Riveted Lap-Splice Joints  

NASA Technical Reports Server (NTRS)

The objective of this paper was to analyze the crack-linkup behavior in riveted-stiffened lap-splice joint panels with small multiple-site damage (MSD) cracks at several adjacent rivet holes. Analyses are based on the STAGS (STructural Analysis of General Shells) code with the critical crack-tip-opening angle (CTOA) fracture criterion. To account for high constraint around a crack front, the "plane strain core" option in STAGS was used. The importance of modeling rivet flexibility with fastener elements that accurately model load transfer across the joint is discussed. Fastener holes are not modeled but rivet connectivity is accounted for by attaching rivets to the sheet on one side of the cracks that simulated both the rivet diameter and MSD cracks. Residual strength analyses made on 2024-T3 alloy (1.6-mm thick) riveted-lap-splice joints with a lead crack and various size MSD cracks were compared with test data from Boeing Airplane Company. Analyses were conducted for both restrained and unrestrained buckling conditions. Comparison of results from these analyses and results from lap-splice-joint test panels, which were partially restrained against buckling indicate that the test results were bounded by the failure loads predicted by the analyses with restrained and unrestrained conditions.

Seshadri, B. R.; Newman, J. C., Jr.

2000-01-01

323

Behind the Scene Role of Conserved Threonine in Intein Splicing  

NASA Astrophysics Data System (ADS)

Protein splicing is an autocatalytic process where an ``intein'' self-cleaves from a precursor protein and catalyzes ligation of the flanking fragments. Inteins occur in all domains of life and have myriad uses in biotechnology. While reaction steps of intein splicing are known, mechanistic details remain incomplete. Here, we investigate the possible role of a highly conserved active-site Threonine residue in bringing about the initial step of splicing: peptide bond rearrangement at a conserved Glycine-Cysteine motif. We report that although not part of the active transition state in this reaction, Threonine plays an important role in reducing the energy barrier through charge screening of active residues in the transition state. Interestingly, Threonine-Glycine hydrogen bonding makes sulfur of the attacking Cysteine less nucleophilic, thereby minimizing Coulomb repulsion in the transition state. These non-intuitive results are obtained through a combination of crystal structure, quantum mechanical simulations, and mutagenesis data. Our results further predict that the sluggish reaction rates observed with intein mutants harboring Threonine-Alanine substitutions can be accelerated in the presence of non-aqueous solvents.

Dearden, Albert; Callahan, Brian; Belfort, Marlene; Nayak, Saroj

2012-02-01

324

ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing.  

PubMed

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T-->C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G-->A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G-->C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site. PMID:18611979

Burgess, R; MacLaren, R E; Davidson, A E; Urquhart, J E; Holder, G E; Robson, A G; Moore, A T; Keefe, R O'; Black, G C M; Manson, F D C

2009-09-01

325

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

PubMed

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

2013-12-01

326

Temperature-sensitive splicing in the floral homeotic mutant apetala3-1.  

PubMed Central

The floral homeotic gene APETALA3 (AP3) is required for stamen and petal development in Arabidopsis. The previously described ap3-1 allele is temperature sensitive and carries a missense mutation near a 5' splice site. The missense mutation lies within a domain of the AP3 protein that is thought to be important for protein-protein interactions, which suggests that temperature sensitivity of ap3-1 could reflect an unstable interaction with cofactors. Here, we show instead that the ap3-1 mutation causes a temperature-dependent splicing defect and that temperature sensitivity is not a property of the protein products of ap3-1 but of RNA processing, possibly because of unstable base pairing between the transcript and small nuclear RNAs. The unexpected defect of the ap3-1 mutant offers unique opportunities for genetic and molecular studies of splice site recognition in plants. PMID:9724692

Sablowski, R W; Meyerowitz, E M

1998-01-01

327

Nova regulates brain-specific splicing to shape the synapse  

Microsoft Academic Search

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune

Jernej Ule; Aljaž Ule; Joanna Spencer; Alan Williams; Jing-Shan Hu; Melissa Cline; Hui Wang; Tyson Clark; Claire Fraser; Matteo Ruggiu; Barry R Zeeberg; David Kane; John N Weinstein; John Blume; Robert B Darnell

2005-01-01

328

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

329

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system  

PubMed Central

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5? and 3? exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3? splice site (3?SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3?SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing. PMID:23685439

Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

2013-01-01

330

Regular languages, regular grammars and automata in splicing systems  

NASA Astrophysics Data System (ADS)

Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

2013-04-01

331

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating  

PubMed Central

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ?50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development. PMID:22719839

Senatore, Adriano; Spafford, J. David

2012-01-01

332

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

PubMed

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

Li, Sanshu; Breaker, Ronald R

2013-03-01

333

Regulation of Neurexin 1[beta] Tertiary Structure and Ligand Binding through Alternative Splicing  

SciTech Connect

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{beta} carrying an alternative splice insert at site SS{number_sign}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{beta}+SS{number_sign}4 reveals dramatic rearrangements to the 'hypervariable surface', the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop {beta}10-{beta}11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca{sup 2+}-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1{beta} isoforms acquire neuroligin splice isoform selectivity.

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

2008-08-04

334

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells  

PubMed Central

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing. PMID:24872507

Ammon, Tim; Mishra, Shravan Kumar; Kowalska, Kaja; Popowicz, Grzegorz M.; Holak, Tad A.; Jentsch, Stefan

2014-01-01

335

Understanding donors' motivations: a study of unrelated bone marrow donors.  

PubMed

Medical advances in bone marrow transplantation techniques and immunosuppressive medications have dramatically increased the number of such transplants performed each year, and consequently, the demand for bone marrow from unrelated donors. Although physiological aspects of bone marrow donation have been thoroughly investigated, very few studies have examined psychosocial factors that may impact individuals' donation decisions and outcomes. To examine one particular set of donor psychosocial issues, this study investigated motives for bone marrow donation among 343 unrelated bone marrow donors who donated through the National Marrow Donor Program. Six distinct types of donor motives were identified from open-ended questionnaire responses. Donors most frequently reported motives reflecting some awareness of both the costs (to themselves) and potential benefits (to themselves and the recipient) of donation. A desire to act in accordance with social or religious precepts, expected positive feelings about donating, empathy for the recipient, and the simple desire to help another person were also commonly cited reasons for donating. Among a series of donor background characteristics, donors' gender was the variable most strongly associated with motive type; women were most likely to cite expected positive feelings, empathy, and the desire to help someone. Central study findings indicated that donor motives predicted donors reactions to donation even after the effects of donor background characteristics (including gender) were controlled. Donors who reported exchange motives (weighing costs and benefits) and donors who reported simple (or idealized) helping motives experienced the donation as less positive in terms of higher predonation ambivalence and negative postdonation psychological reactions than did remaining donors. Donors who reported positive feeling and empathy motives had the most positive donation reactions in terms of lower ambivalence, and feeling like better persons postdonation. These finding add substantially to the body of work concerning medical volunteerism generally, and also have important practical implications for the recruitment and education of potential bone marrow donors. PMID:9203278

Switzer, G E; Dew, M A; Butterworth, V A; Simmons, R G; Schimmel, M

1997-07-01

336

A Pan-Cancer Analysis of Transcriptome Changes Associated with Somatic Mutations in U2AF1 Reveals Commonly Altered Splicing Events  

PubMed Central

Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35) have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA). Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML), in which U2AF1 is somatically mutated in 3–4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3? splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3? splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types. PMID:24498085

Brooks, Angela N.; Choi, Peter S.; de Waal, Luc; Sharifnia, Tanaz; Imielinski, Marcin; Saksena, Gordon; Pedamallu, Chandra Sekhar; Sivachenko, Andrey; Rosenberg, Mara; Chmielecki, Juliann; Lawrence, Michael S.; DeLuca, David S.; Getz, Gad; Meyerson, Matthew

2014-01-01

337

Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations  

PubMed Central

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts. PMID:23451180

Colombo, Mara; De Vecchi, Giovanna; Caleca, Laura; Foglia, Claudia; Ripamonti, Carla B.; Ficarazzi, Filomena; Barile, Monica; Varesco, Liliana; Peissel, Bernard; Manoukian, Siranoush; Radice, Paolo

2013-01-01

338

ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation  

PubMed Central

The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ?Exon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration. PMID:24204307

Zhou, Yueqin; Liu, Songyan; Liu, Guodong; Öztürk, Arzu; Hicks, Geoffrey G.

2013-01-01

339

Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2? in Development  

PubMed Central

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2? (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2? is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2? binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2? regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2? binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2? protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2?. Versions of Tra2? lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2? protein. PMID:22194695

Best, Andrew; Liu, Yilei; Jakubik, Miriam; Mende, Ylva; Ehrmann, Ingrid; Curk, Tomaz; Rossbach, Kristina; Bourgeois, Cyril F.; Stévenin, James; Grellscheid, David; Jackson, Michael S.; Wirth, Brunhilde; Elliott, David J.

2011-01-01

340

Archaeal introns: splicing, intercellular mobility and evolution  

Microsoft Academic Search

Until recently, it appeared that archaeal introns were spliced by a process specific to the archaeal domain in which an endoribonuclease cuts a ‘bulge-helix-bulge’ motif that forms at exon-intron junctions. Recent results, however, have shown that the endoribonuclease involved in archaeal intron splicing is a homologue of two subunits of the enzyme complex that excises eukaryotic nuclear tRNA introns. Moreover,

Jens Lykke-Andersen; Claus Aagaard; Mikhail Semionenkov; Roger A. Garrett

1997-01-01

341

Using Yeast Genetics to Study Splicing Mechanisms  

PubMed Central

Pre-mRNA splicing is a critical step in eukaryotic gene expression, which involves removal of noncoding intron sequences from pre-mRNA and ligation of the remaining exon sequences to make a mature message. Splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Since the first description of the pre-mRNA splicing reaction in the 1970s, elegant genetic and biochemical studies have revealed that the enzyme that catalyzes the reaction, the spliceosome, is an exquisitely dynamic macromolecular machine, and its RNA and protein components undergo highly ordered, tightly coordinated rearrangements in order to carry out intron recognition and splicing catalysis. Studies using the genetically tractable unicellular eukaryote budding yeast (Saccharomyces cerevisiae) have played an instrumental role in deciphering splicing mechanisms. In this chapter, we discuss how yeast genetics has been used to deepen our understanding of the mechanism of splicing and explore the potential for future mechanistic insights using S. cerevisiae as an experimental tool. PMID:24549672

Hossain, Munshi Azad; Johnson, Tracy L.

2014-01-01

342

Counting on co-transcriptional splicing  

PubMed Central

Splicing is the removal of intron sequences from pre-mRNA by the spliceosome. Researchers working in multiple model organisms – notably yeast, insects and mammalian cells – have shown that pre-mRNA can be spliced during the process of transcription (i.e. co-transcriptionally), as well as after transcription termination (i.e. post-transcriptionally). Co-transcriptional splicing does not assume that transcription and splicing machineries are mechanistically coupled, yet it raises this possibility. Early studies were based on a limited number of genes, which were often chosen because of their experimental accessibility. Since 2010, eight studies have used global datasets as counting tools, in order to quantify co-transcriptional intron removal. The consensus view, based on four organisms, is that the majority of splicing events take place co-transcriptionally in most cells and tissues. Here, we discuss the nature of the various global datasets and how bioinformatic analyses were conducted. Considering the broad differences in experimental approach and analysis, the level of agreement on the prevalence of co-transcriptional splicing is remarkable. PMID:23638305

2013-01-01

343

Missense Mutations in hMLH1 and hMSH2 Are Associated with Exonic Splicing Enhancers  

PubMed Central

There is a critical need to understand why missense mutations are deleterious. The deleterious effects of missense mutations are commonly attributed to their impact on primary amino acid sequence and protein structure. However, several recent studies have shown that some missense mutations are deleterious because they disturb cis-acting splicing elements—so-called “exonic splicing enhancers” (ESEs). It is not clear whether the ESE-related deleterious effects of missense mutations are common. We have evaluated colocalization of pathogenic missense mutations (found in affected individuals) with high-score ESE motifs in the human mismatch-repair genes hMSH2 and hMLH1. We found that pathogenic missense mutations in the hMSH2 and hMLH1 genes are located in ESE sites significantly more frequently than expected. Pathogenic missense mutations also tended to decrease ESE scores, thus leading to a higher propensity for splicing defects. In contrast, nonpathogenic missense mutations (polymorphisms found in unaffected individuals) and nonsense mutations are distributed randomly in relation to ESE sites. Comparison of the observed and expected frequencies of missense mutations in ESE sites shows that pathogenic effects of ?20% of mutations in hMSH2 result from disruption of ESE sites and disturbed splicing. Similarly, pathogenic effects of ?16% of missense mutations in the hMLH1 gene are ESE related. The colocalization of pathogenic missense mutations with ESE sites strongly suggests that their pathogenic effects are splicing related. PMID:14526391

Gorlov, Ivan P.; Gorlova, Olga Y.; Frazier, Marsha L.; Amos, Christopher I.

2003-01-01

344

The exon junction complex: a splicing factor for long intron containing transcripts?  

PubMed

Alternative splicing provides one of the major mechanisms by which eukaryotic genomes can increase the diversity of transcripts and protein products encoded by a limited amount of genes. The spliceosome is involved in recognizing and removing intronic sequences and it is guided in this activity by splicing regulatory factors that determine the particular splice sites that are to be recognized and used for intron removal. Thus, by modulating the spliceosome's activity, these factors can cause different mRNA transcripts to be generated from the same precursor mRNA. In a recent RNAi screen for factors modulating RAS/MAPK signaling, we identified the Drosophila exon junction complex (EJC) as one of the components regulating the splicing of mapk transcripts. We showed that removal of EJC components caused multiple consecutive mapk exons to be skipped. Moreover, we determined that other fly genes that, like mapk, had particularly large introns, were also sensitive to disruption of the EJC. The importance of intron length in determining sensitivity to EJC disruption suggests that the EJC is functioning in exon definition, a splicing mechanism that occurs for long introns in particular. PMID:21478676

Ashton-Beaucage, Dariel; Therrien, Marc

2011-01-01

345

Alternative splice variants of the USH3A gene Clarin 1 (CLRN1)  

PubMed Central

Clarin 1 (CLRN1) is a four-transmembrane protein expressed in cochlear hair cells and neural retina, and when mutated it causes Usher syndrome type 3 (USH3). The main human splice variant of CLRN1 is composed of three exons that code for a 232-aa protein. In this study, we aimed to refine the structure of CLRN1 by an examination of transcript splice variants and promoter regions. Analysis of human retinal cDNA revealed 11 CLRN1 splice variants, of which 5 have not been previously reported. We studied the regulation of gene expression by several promoter domains using a luciferase assay, and identified 1000?nt upstream of the translation start site of the primary CLRN1 splice variant as the principal promoter region. Our results suggest that the CLRN1 gene is significantly more complex than previously described. The complexity of the CLRN1 gene and the identification of multiple splice variants may partially explain why mutations in CLRN1 result in substantial variation in clinical phenotype. PMID:20717163

Västinsalo, Hanna; Jalkanen, Reetta; Dinculescu, Astra; Isosomppi, Juha; Geller, Scott; Flannery, John G; Hauswirth, William W; Sankila, Eeva-Marja

2011-01-01

346

Global impact of RNA splicing on transcriptome remodeling in the heart *  

PubMed Central

In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30 000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart. PMID:22843179

GAO, Chen; Wang, Yibin

2012-01-01

347

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.  

PubMed

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron?165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed. PMID:25220236

Moon, Heegyum; Cho, Sunghee; Loh, Tiing Jen; Oh, Hyun Kyung; Jang, Ha Na; Zhou, Jianhua; Kwon, Young-Soo; Liao, D Joshua; Jun, Youngsoo; Eom, Soohyun; Ghigna, Claudia; Biamonti, Giuseppe; Green, Michael R; Zheng, Xuexiu; Shen, Haihong

2014-11-01

348

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

349

Identifying Potential Kidney Donors Using Social Networking Websites  

PubMed Central

Social networking sites like Facebook may be a powerful tool for increasing rates of live kidney donation. They allow for wide dissemination of information and discussion, and could lessen anxiety associated with a face-to-face request for donation. However, sparse data exist on the use of social media for this purpose. We searched Facebook, the most popular social networking site, for publicly available English-language pages seeking kidney donors for a specific individual, abstracting information on the potential recipient, characteristics of the page itself, and whether potential donors were tested. In the 91 pages meeting inclusion criteria, the mean age of potential recipients was 37 (range: 2–69); 88% were U.S. residents. Other posted information included the individual’s photograph (76%), blood type (64%), cause of kidney disease (43%), and location (71%). Thirty-two percent of pages reported having potential donors tested, and 10% reported receiving a live donor kidney transplant. Those reporting donor testing shared more potential recipient characteristics, provided more information about transplantation, and had higher page traffic. Facebook is already being used to identify potential kidney donors. Future studies should focus on how to safely, ethically, and effectively use social networking sites to inform potential donors and potentially expand live kidney donation. PMID:23600791

Chang, Alexander; Anderson, Emily E.; Turner, Hang T.; Shoham, David; Hou, Susan H.; Grams, Morgan

2013-01-01

350

Genetic Enhancement of RNA-Processing Defects by a Dominant Mutation inB52, theDrosophila Gene for an SR Protein Splicing Factor  

Microsoft Academic Search

B52, the gene for aDrosophilaSR protein. The alleleB52EDwas identified as a dominant second-site enhancer ofwhite-apricot(wa), a retrotransposon insertion in the second intron of the eye pigmentation genewhitewith a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5* splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized

XIANBU PENG; ANDSTEPHEN M. MOUNT

1995-01-01

351

In silico to in vivo splicing analysis using splicing code models  

PubMed Central

With the growing appreciation of RNA splicing’s role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing. PMID:24321485

Gazzara, Matthew R.; Vaquero-Garcia, Jorge; Lynch, Kristen W.; Barash, Yoseph

2015-01-01

352

A biophysical model for identifying splicing regulatory elements and their interactions.  

PubMed

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a crucial step in the expression of most eukaryotic genes. Splicing factors (SFs) play an important role in AS regulation by binding to the cis-regulatory elements on the pre-mRNA. Although many splicing factors (SFs) and their binding sites have been identified, their combinatorial regulatory effects remain to be elucidated. In this paper, we derive a biophysical model for AS regulation that integrates combinatorial signals of cis-acting splicing regulatory elements (SREs) and their interactions. We also develop a systematic framework for model inference. Applying the biophysical model to a human RNA-Seq data set, we demonstrate that our model can explain 49.1%-66.5% variance of the data, which is comparable to the best result achieved by biophysical models for transcription. In total, we identified 119 SRE pairs between different regions of cassette exons that may regulate exon or intron definition in splicing, and 77 SRE pairs from the same region that may arise from a long motif or two different SREs bound by different SFs. Particularly, putative binding sites of polypyrimidine tract-binding protein (PTB), heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and E/K are identified as interacting SRE pairs, and have been shown to be consistent with the interaction models proposed in previous experimental results. These results show that our biophysical model and inference method provide a means of quantitative modeling of splicing regulation and is a useful tool for identifying SREs and their interactions. The software package for model inference is available under an open source license. PMID:23382993

Wen, Ji; Chen, Zhibin; Cai, Xiaodong

2013-01-01

353

Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron.  

PubMed Central

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells. Images PMID:1847501

Chain, A C; Zollman, S; Tseng, J C; Laski, F A

1991-01-01

354

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

PubMed

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

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

2015-01-01

355

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions.  

PubMed

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner. PMID:22829983

Ishigaki, Shinsuke; Masuda, Akio; Fujioka, Yusuke; Iguchi, Yohei; Katsuno, Masahisa; Shibata, Akihide; Urano, Fumihiko; Sobue, Gen; Ohno, Kinji

2012-01-01

356

Blood Donor Management in China  

PubMed Central

Summary Despite a steady increase in total blood collections and voluntary non-remunerated blood donors, China continues to have many challenges with its blood donation system. The country's donation rate remains low at 9%o, with over 60% of donors being first-time donors. Generally there is a lack of adequate public awareness about blood donation. The conservative donor selection criteria, the relatively long donation interval, and the small donation volume have further limited blood supply. To ensure a sufficient and safe blood supply that meets the increasing clinical need for blood products, there is an urgent need to strengthen the country's blood donor management. This comprehensive effort should include educating and motivating more individuals especially from the rural areas to be involved in blood donation, developing rational and evidence-based selection criteria for donor eligibility, designing a donor follow-up mechanism to encourage more future donations, assessing the current donor testing strategy, improving donor service and care, building regional and national shared donor deferral database, and enhancing the transparency of the blood donation system to gain more trust from the general public. The purpose of the review is to provide an overview of the key process of and challenges with the blood donor management system in China. PMID:25254023

Shi, Ling; Wang, Jingxing; Liu, Zhong; Stevens, Lori; Sadler, Andrew; Ness, Paul; Shan, Hua

2014-01-01

357

Differential splicing using whole-transcript microarrays  

PubMed Central

Background The latest generation of Affymetrix microarrays are designed to interrogate expression over the entire length of every locus, thus giving the opportunity to study alternative splicing genome-wide. The Exon 1.0 ST (sense target) platform, with versions for Human, Mouse and Rat, is designed primarily to probe every known or predicted exon. The smaller Gene 1.0 ST array is designed as an expression microarray but still interrogates expression with probes along the full length of each well-characterized transcript. We explore the possibility of using the Gene 1.0 ST platform to identify differential splicing events. Results We propose a strategy to score differential splicing by using the auxiliary information from fitting the statistical model, RMA (robust multichip analysis). RMA partitions the probe-level data into probe effects and expression levels, operating robustly so that if a small number of probes behave differently than the rest, they are downweighted in the fitting step. We argue that adjacent poorly fitting probes for a given sample can be evidence of differential splicing and have designed a statistic to search for this behaviour. Using a public tissue panel dataset, we show many examples of tissue-specific alternative splicing. Furthermore, we show that evidence for putative alternative splicing has a strong correspondence between the Gene 1.0 ST and Exon 1.0 ST platforms. Conclusion We propose a new approach, FIRMAGene, to search for differentially spliced genes using the Gene 1.0 ST platform. Such an analysis complements the search for differential expression. We validate the method by illustrating several known examples and we note some of the challenges in interpreting the probe-level data. Software implementing our methods is freely available as an R package. PMID:19463149

Robinson, Mark D; Speed, Terence P

2009-01-01

358

Splicing therapy for neuromuscular disease?  

PubMed Central

Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. PMID:23631896

Douglas, Andrew G.L.; Wood, Matthew J.A.

2013-01-01

359

Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids  

PubMed Central

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur. PMID:10877811

Hohnstock, A. M.; Stuart-Keil, K. G.; Kull, E. E.; Madsen, E. L.

2000-01-01

360

Discovery of a Splicing Regulator Required for Cell Cycle Progression  

E-print Network

Discovery of a Splicing Regulator Required for Cell Cycle Progression Elena S. Suvorova1 , Matthew) Discovery of a Splicing Regulator Required for Cell Cycle Progression. PLoS Genet 9(2): e1003305. doi:10

Arnold, Jonathan

361

30 CFR 77.504 - Electrical connections or splices; suitability.  

Code of Federal Regulations, 2011 CFR

...2011-07-01 2011-07-01 false Electrical connections or splices; suitability...SURFACE WORK AREAS OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices;...

2011-07-01

362

30 CFR 77.504 - Electrical connections or splices; suitability.  

...2014-07-01 2014-07-01 false Electrical connections or splices; suitability...SURFACE WORK AREAS OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices;...

2014-07-01

363

30 CFR 77.504 - Electrical connections or splices; suitability.  

Code of Federal Regulations, 2012 CFR

...2012-07-01 2012-07-01 false Electrical connections or splices; suitability...SURFACE WORK AREAS OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices;...

2012-07-01

364

30 CFR 77.504 - Electrical connections or splices; suitability.  

Code of Federal Regulations, 2013 CFR

...2013-07-01 2013-07-01 false Electrical connections or splices; suitability...SURFACE WORK AREAS OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices;...

2013-07-01

365

30 CFR 77.504 - Electrical connections or splices; suitability.  

Code of Federal Regulations, 2010 CFR

...2010-07-01 2010-07-01 false Electrical connections or splices; suitability...SURFACE WORK AREAS OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices;...

2010-07-01

366

Photoluminescence study of hydrogen donors in ZnO  

NASA Astrophysics Data System (ADS)

A photoluminescence study of hydrogenated ZnO bulk crystals is presented. Two excitonic recombination lines at 3362.8 and 3360.1 meV are assigned to hydrogen shallow donors. Experimental evidence is presented that the corresponding donor to the line at 3362.8 meV, previously labeled I4, originates from hydrogen trapped within the oxygen vacancy, HO. The line at 3360.1 meV was found to be due to hydrogen located at the bond-centered lattice site, HBC. The corresponding shallow donor has an ionization energy of 53 meV.

Herklotz, F.; Lavrov, E. V.; Weber, J.

2009-12-01

367

In silico to in vivo splicing analysis using splicing code models.  

PubMed

With the growing appreciation of RNA splicing's role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing. PMID:24321485

Gazzara, Matthew R; Vaquero-Garcia, Jorge; Lynch, Kristen W; Barash, Yoseph

2014-05-01

368

The 20S Proteasome Splicing Activity Discovered by SpliceMet  

PubMed Central

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected. PMID:20613855

Textoris-Taube, Kathrin; Janek, Katharina; Keller, Christin; Henklein, Petra; Kloetzel, Peter Michael; Zaikin, Alexey

2010-01-01

369

Neuronal regulation of alternative pre-mRNA splicing  

Microsoft Academic Search

Alternative pre-mRNA splicing has an important role in the control of neuronal gene expression. Many neuronal proteins are structurally diversified through the differential inclusion and exclusion of sequences in the final spliced mRNA. Here, we discuss common mechanisms of splicing regulation and provide examples of how alternative splicing has important roles in neuronal development and mature neuron function. Finally, we

Qin Li; Douglas L. Black

2007-01-01

370

Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches  

PubMed Central

RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations. PMID:23861443

Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Griffith, Graziella; Philippi, Susanne; Précigout, Guillaume; Mamchaoui, Kamel; Jollet, Arnaud; Voit, Thomas; Garcia, Luis

2013-01-01

371

A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase.  

PubMed

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed. PMID:1996139

Jamieson, D J; Rahe, B; Pringle, J; Beggs, J D

1991-02-21

372

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA  

SciTech Connect

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

373

Genome-wide prediction, display and refinement of binding sites with information theory-based models  

PubMed Central

Background We present Delila-genome, a software system for identification, visualization and analysis of protein binding sites in complete genome sequences. Binding sites are predicted by scanning genomic sequences with information theory-based (or user-defined) weight matrices. Matrices are refined by adding experimentally-defined binding sites to published binding sites. Delila-Genome was used to examine the accuracy of individual information contents of binding sites detected with refined matrices as a measure of the strengths of the corresponding protein-nucleic acid interactions. The software can then be used to predict novel sites by rescanning the genome with the refined matrices. Results Parameters for genome scans are entered using a Java-based GUI interface and backend scripts in Perl. Multi-processor CPU load-sharing minimized the average response time for scans of different chromosomes. Scans of human genome assemblies required 4–6 hours for transcription factor binding sites and 10–19 hours for splice sites, respectively, on 24- and 3-node Mosix and Beowulf clusters. Individual binding sites are displayed either as high-resolution sequence walkers or in low-resolution custom tracks in the UCSC genome browser. For large datasets, we applied a data reduction strategy that limited displays of binding sites exceeding a threshold information content to specific chromosomal regions within or adjacent to genes. An HTML document is produced listing binding sites ranked by binding site strength or chromosomal location hyperlinked to the UCSC custom track, other annotation databases and binding site sequences. Post-genome scan tools parse binding site annotations of selected chromosome intervals and compare the results of genome scans using different weight matrices. Comparisons of multiple genome scans can display binding sites that are unique to each scan and identify sites with significantly altered binding strengths. Conclusions Delila-Genome was used to scan the human genome sequence with information weight matrices of transcription factor binding sites, including PXR/RXR?, AHR and NF-?B p50/p65, and matrices for RNA binding sites including splice donor, acceptor, and SC35 recognition sites. Comparisons of genome scans with the original and refined PXR/RXR? information weight matrices indicate that the refined model more accurately predicts the strengths of known binding sites and is more sensitive for detection of novel binding sites. PMID:12962546

Gadiraju, Sashidhar; Vyhlidal, Carrie A; Leeder, J Steven; Rogan, Peter K

2003-01-01

374

Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events.  

PubMed

Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology. PMID:23033341

Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D; Gazdar, Adi F; Stern, Howard M; Hoeflich, Klaus P; Wu, Thomas D; Settleman, Jeff; de Sauvage, Frederic J; Gentleman, Robert C; Neve, Richard M; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S; Zhang, Zemin

2012-12-01

375

Genome-wide identification of spliced introns using a tiling microarray  

PubMed Central

The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA from the debranching mutant to the wild-type parent strain, and thus the identification of lariats on a genome-wide scale. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others. In many instances, signals from the tiling array delineated the 5? splice site and branchpoint site, confirming predicted gene structures. Nearly all introns that went undetected are present in mRNAs expressed at low levels. Overall, 97% of the significant probes could be attributed either to spliced introns or to genes up-regulated by deletion of the debranching enzyme. Because the debranching enzyme is conserved among eukaryotes, this approach could be generally applicable for the annotation of eukaryotic genes and the detection of novel and alternative splice forms. PMID:17351133

Zhang, Zhihong; Hesselberth, Jay R.; Fields, Stanley

2007-01-01

376

Genome-wide identification of spliced introns using a tiling microarray.  

PubMed

The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA from the debranching mutant to the wild-type parent strain, and thus the identification of lariats on a genome-wide scale. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others. In many instances, signals from the tiling array delineated the 5' splice site and branchpoint site, confirming predicted gene structures. Nearly all introns that went undetected are present in mRNAs expressed at low levels. Overall, 97% of the significant probes could be attributed either to spliced introns or to genes up-regulated by deletion of the debranching enzyme. Because the debranching enzyme is conserved among eukaryotes, this approach could be generally applicable for the annotation of eukaryotic genes and the detection of novel and alternative splice forms. PMID:17351133

Zhang, Zhihong; Hesselberth, Jay R; Fields, Stanley

2007-04-01

377

Functional roles of protein splicing factors  

PubMed Central

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system. PMID:22762203

Chen, Hsin-Chou; Cheng, Soo-Chen

2012-01-01

378

Tau Splicing and the Intricacies of Dementia  

PubMed Central

Tau is a microtubule associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its localization, conformation and post-translational modifications and hence its availability and affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases which lead to dementia. In this review, I present our cumulative knowledge of tau splicing regulation in connection with neurodegeneration and also briefly go over the still-extensive list of questions that are connected to tau (dys)function. PMID:21604267

Andreadis, Athena

2011-01-01

379

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.  

PubMed Central

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45. Images PMID:7556075

Screaton, G R; Cáceres, J F; Mayeda, A; Bell, M V; Plebanski, M; Jackson, D G; Bell, J I; Krainer, A R

1995-01-01

380

Analysis of Multiply Spliced Transcripts in Lymphoid Tissue Reservoirs of Rhesus Macaques Infected with RT-SHIV during HAART  

PubMed Central

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4+ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat an