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Sample records for splice donor site

  1. Thermodynamic Modeling of Donor Splice Site Recognition in pre-mRNA

    NASA Astrophysics Data System (ADS)

    Aalberts, Daniel P.; Garland, Jeffrey A.

    2004-03-01

    When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 snRNA with the donor (5') splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our Finding with Binding method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

  2. Thermodynamic modeling of donor splice site recognition in pre-mRNA

    NASA Astrophysics Data System (ADS)

    Garland, Jeffrey A.; Aalberts, Daniel P.

    2004-04-01

    When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5' ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our “finding with binding” method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

  3. A computational approach for prediction of donor splice sites with improved accuracy.

    PubMed

    Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R; Wahi, S D

    2016-09-01

    Identification of splice sites is important due to their key role in predicting the exon-intron structure of protein coding genes. Though several approaches have been developed for the prediction of splice sites, further improvement in the prediction accuracy will help predict gene structure more accurately. This paper presents a computational approach for prediction of donor splice sites with higher accuracy. In this approach, true and false splice sites were first encoded into numeric vectors and then used as input in artificial neural network (ANN), support vector machine (SVM) and random forest (RF) for prediction. ANN and SVM were found to perform equally and better than RF, while tested on HS3D and NN269 datasets. Further, the performance of ANN, SVM and RF were analyzed by using an independent test set of 50 genes and found that the prediction accuracy of ANN was higher than that of SVM and RF. All the predictors achieved higher accuracy while compared with the existing methods like NNsplice, MEM, MDD, WMM, MM1, FSPLICE, GeneID and ASSP, using the independent test set. We have also developed an online prediction server (PreDOSS) available at http://cabgrid.res.in:8080/predoss, for prediction of donor splice sites using the proposed approach. PMID:27302911

  4. A novel donor splice-site mutation of major intrinsic protein gene associated with congenital cataract in a Chinese family

    PubMed Central

    Zeng, Lu; Liu, Wenqiang; Feng, Wenguo; Wang, Xing; Dang, Hui; Gao, Luna; Yao, Jing

    2013-01-01

    Purpose To identify the disease-causing gene in a Chinese family with autosomal dominant congenital cataract. Methods Clinical and ophthalmologic examinations were performed on all members of a Chinese family with congenital cataract. Nine genes associated with congenital cataract were screened using direct DNA sequencing. Mutations were confirmed using restriction fragment length polymorphism (RFLP) analysis. The mutated major intrinsic protein (MIP) minigene, which carries the disease-causing splice-site mutation, and the wild-type (WT) MIP minigene were constructed using the pcDNA3.1 expression vector. Wild-type and mutant MIP minigene constructs were transiently transfected into HeLa cells. After 48 h of incubation at 37 °C, total RNA isolation and reverse transcription (RT)–PCR analysis were performed, and PCR products were separated and confirmed with sequencing. Results Direct DNA sequence analysis identified a novel splice-site mutation in intron 3 (c.606+1 G>A) of the MIP gene. To investigate the manner in which the splice donor mutation could affect mRNA splicing, WT and mutant MIP minigenes were inserted in the pcDNA3.1 (+) vector. Constructs were transfected into HeLa cells. RT–PCR analysis showed that the donor splice site mutation led to deletion of exon 3 in the mRNA encoded by the MIP gene. Conclusions The present study identified a novel donor splice-site mutation (c.606+1G>A) in the MIP gene in a Chinese family with congenital cataract. In vitro RT–PCR analysis showed that this splice-site mutation resulted in the deletion of exon 3 from mRNA encoded by the MIP gene. This is the first report to show that donor splice-site mutation in MIP gene can cause autosomal dominant congenital cataract. PMID:24319327

  5. A novel splice donor site mutation in EPHA2 caused congenital cataract in a Chinese family

    PubMed Central

    Bu, Juan; He, Sijie; Wang, Lejin; Li, Jiankang; Liu, Jing; Zhang, Xiuqing

    2016-01-01

    Background: Congenital cataract is a rare disorder characterized by crystallin denaturation, which becomes a major cause of childhood blindness. Although more than fifty pathogenic genes for congenital cataract have been reported, the genetic causes of many cataract patients remain unknown. In this study, the aim is to identify the genetic cause of a five-generation Chinese autosomal dominant congenital cataract family. Methods: Whole exome sequencing (WES) was performed on three affected and one unaffected member of the family, known causative genes were scanned first. Sanger sequencing was used to validate co-segregation of the candidate variant in the family. The impact on the transcript and amino acid sequences of the variant was further analyzed. Results: We identified a novel splice donor site mutation c. 2825+1G >A in EPHA2 that was absent in public and in-house databases and showed co-segregation in the family. This variant resulted in an altered splice that led to protein truncation. Conclusions: The mutation we identified was responsible for congenital cataract in our studied family. Our findings broaden the spectrum of causative mutations in EPHA2 gene for congenital cataract and suggest that WES is an efficient strategy to scan variants in known causative genes for genetically heterogeneous diseases. PMID:27380975

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

    SciTech Connect

    Poppelreuther, Sven; Iftner, Thomas; Stubenrauch, Frank

    2008-01-05

    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.

  7. Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

    PubMed

    Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

    2015-12-15

    We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. PMID:26671123

  8. Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

    PubMed Central

    Bradley, K; Cavaco, B; Bowl, M; Harding, B; Young, A; Thakker, R

    2005-01-01

    More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g→a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant. PMID:16061557

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

    SciTech Connect

    Willing, M.; Deschenes, S.

    1994-09-01

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

  10. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

    SciTech Connect

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard; Bies, Juraj; Wolff, Linda; Mougel, Marylene . E-mail: mmougel@univ-montp1.fr

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

  11. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

    PubMed

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard; Bies, Juraj; Wolff, Linda; Mougel, Marylène

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism. PMID:15567434

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

    PubMed Central

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

    2014-01-01

    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

  13. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

    PubMed Central

    Solera, J; Magallón, M; Martin-Villar, J; Coloma, A

    1992-01-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. Images Figure 1 PMID:1346483

  14. A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

    PubMed Central

    Broen, Kelly; Levenga, Henriette; Vos, Johanna; van Bergen, Kees; Fredrix, Hanny; Greupink-Draaisma, Annelies; Kester, Michel; Falkenburg, J. H. Frederik; de Witte, Theo; Griffioen, Marieke; Dolstra, Harry

    2011-01-01

    Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT. PMID:21738768

  15. Splicing defects in the COL3A1 gene: marked preference for 5' (donor) spice-site mutations in patients with exon-skipping mutations and Ehlers-Danlos syndrome type IV.

    PubMed Central

    Schwarze, U; Goldstein, J A; Byers, P H

    1997-01-01

    Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis. Images Figure 1 Figure 3 Figure 6 PMID:9399899

  16. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed Central

    Hagiwara, Y.; Nishio, H.; Kitoh, Y.; Takeshima, Y.; Narita, N.; Wada, H.; Yokoyama, M.; Nakamura, H.; Matsuo, M.

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. Images Figure 2 Figure 5 PMID:8279470

  17. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed

    Hagiwara, Y; Nishio, H; Kitoh, Y; Takeshima, Y; Narita, N; Wada, H; Yokoyama, M; Nakamura, H; Matsuo, M

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. PMID:8279470

  18. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    SciTech Connect

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi )

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  19. ERISdb: a database of plant splice sites and splicing signals.

    PubMed

    Szcześniak, Michał Wojciech; Kabza, Michał; Pokrzywa, Rafał; Gudyś, Adam; Makałowska, Izabela

    2013-02-01

    Splicing is one of the major contributors to observed spatiotemporal diversification of transcripts and proteins in metazoans. There are numerous factors that affect the process, but splice sites themselves along with the adjacent splicing signals are critical here. Unfortunately, there is still little known about splicing in plants and, consequently, further research in some fields of plant molecular biology will encounter difficulties. Keeping this in mind, we performed a large-scale analysis of splice sites in eight plant species, using novel algorithms and tools developed by us. The analyses included identification of orthologous splice sites, polypyrimidine tracts and branch sites. Additionally we identified putative intronic and exonic cis-regulatory motifs, U12 introns as well as splice sites in 45 microRNA genes in five plant species. We also provide experimental evidence for plant splice sites in the form of expressed sequence tag and RNA-Seq data. All the data are stored in a novel database called ERISdb and are freely available at http://lemur.amu.edu.pl/share/ERISdb/. PMID:23299413

  20. Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

    SciTech Connect

    Solera, J. ); Magallon, M.; Martin-Villar, J. ); Coloma, A. )

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  1. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

    PubMed Central

    Chillón, M; Dörk, T; Casals, T; Giménez, J; Fonknechten, N; Will, K; Ramos, D; Nunes, V; Estivill, X

    1995-01-01

    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+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->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. Images Figure 3 PMID:7534040

  2. 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

    Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X.; Doerk, T.; Will, K.; Fonknechten, N.

    1995-03-01

    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.

  3. Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs.

    PubMed Central

    Tigges, M A; Raskas, H J

    1984-01-01

    We localized the splice junctions in adenovirus 2 early region 4 (E4) mRNAs. Processing of the E4 precursor RNA positioned the donor splice site of the 5' leader sequence adjacent to acceptor sites near the 5' ends of five of the six open reading regions in the E4 transcription unit. Of particular interest among the E4 mRNAs is an extensively spliced class which includes multiple species with sizes ranging from 1.1 to 0.75 kilobases (kb). Purified 1.1- to 0.75-kb mRNAs specified at least 10 polypeptides in vitro. We detected eight acceptor and two donor splice sites utilized in the deletion of the intron from the 3' portion of these mRNAs. E4 RNAs were isolated from the cytoplasm of infected cells at 5, 9, 12, and 18 h after infection. The E4 mRNAs were present throughout infection, but different members of the 1.1- to 0.7-kb class were predominant at each time assayed. Alternate splicing of the 3.0-kb E4 precursor RNA can generate as many as 25 mRNAs that encode at least 16 polypeptides. Images PMID:6336328

  4. Role of the 3′ Splice Site in U12-Dependent Intron Splicing

    PubMed Central

    Dietrich, Rosemary C.; Peris, Marian J.; Seyboldt, Andrew S.; Padgett, Richard A.

    2001-01-01

    U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site. PMID:11238930

  5. Splice Sites Seldom Slide: Intron Evolution in Oomycetes

    PubMed Central

    Sêton Bocco, Steven; Csűrös, Miklós

    2016-01-01

    We examine exon junctions near apparent amino acid insertions and deletions in alignments of orthologous protein-coding genes. In 1,917 ortholog families across nine oomycete genomes, 10–20% of introns are near an alignment gap, indicating at first sight that splice-site displacements are frequent. We designed a robust algorithmic procedure for the delineation of intron-containing homologous regions, and combined it with a parsimony-based reconstruction of intron loss, gain, and splice-site shift events on a phylogeny. The reconstruction implies that 12% of introns underwent an acceptor-site shift, and 10% underwent a donor-site shift. In order to offset gene annotation problems, we amended the procedure with the reannotation of intron boundaries using alignment evidence. The corresponding reconstruction involves much fewer intron gain and splice-site shift events. The frequency of acceptor- and donor-side shifts drops to 4% and 3%, respectively, which are not much different from what one would expect by random codon insertions and deletions. In other words, gaps near exon junctions are mostly artifacts of gene annotation rather than evidence of sliding intron boundaries. Our study underscores the importance of using well-supported gene structure annotations in comparative studies. When transcription evidence is not available, we propose a robust ancestral reconstruction procedure that corrects misannotated intron boundaries using sequence alignments. The results corroborate the view that boundary shifts and complete intron sliding are only accidental in eukaryotic genome evolution and have a negligible impact on protein diversity. PMID:27412607

  6. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    PubMed

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

    2015-09-01

    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 IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient. PMID:26016411

  7. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  8. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

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

    PubMed

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

    2013-02-01

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

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

    PubMed Central

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

    1995-01-01

    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

  11. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal

    PubMed Central

    Mueller, Nancy; Das, Atze T.; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5′ splice site (5′ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5′ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5′ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  12. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    PubMed

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  13. SeeSite: Characterizing Relationships between Splice Junctions and Splicing Enhancers.

    PubMed

    Lo, Christine; Kakaradov, Boyko; Lokshtanov, Daniel; Boucher, Christina

    2014-01-01

    RNA splicing is a cellular process driven by the interaction between numerous regulatory sequences and binding sites, however, such interactions have been primarily explored by laboratory methods since computational tools largely ignore the relationship between different splicing elements. Current computational methods identify either splice sites or other regulatory sequences, such as enhancers and silencers. We present a novel approach for characterizing co-occurring relationships between splice site motifs and splicing enhancers. Our approach relies on an efficient algorithm for approximately solving Consensus Sequence with Outliers , an NP-complete string clustering problem. In particular, we give an algorithm for this problem that outputs near-optimal solutions in polynomial time. To our knowledge, this is the first formulation and computational attempt for detecting co-occurring sequence elements in RNA sequence data. Further, we demonstrate that SeeSite is capable of showing that certain ESEs are preferentially associated with weaker splice sites, and that there exists a co-occurrence relationship with splice site motifs. PMID:26356335

  14. Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription

    PubMed Central

    Hirochika, Hirohiko; Takatsuji, Hiroshi; Ubasawa, Aiko; Ikeda, Joh-E

    1985-01-01

    A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism. ImagesFig. 1.Fig. 3.Fig. 5.Fig. 7. PMID:16453624

  15. Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

    PubMed Central

    Kameyama, Toshiki; Suzuki, Hitoshi; Mayeda, Akila

    2012-01-01

    Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5′ and 3′ splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events. PMID:22675076

  16. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  17. Nucleotide sequence composition adjacent to intronic splice sites improves splicing efficiency via its effect on pre-mRNA local folding in fungi.

    PubMed

    Zafrir, Zohar; Tuller, Tamir

    2015-10-01

    RNA splicing is the central process of intron removal in eukaryotes known to regulate various cellular functions such as growth, development, and response to external signals. The canonical sequences indicating the splicing sites needed for intronic boundary recognition are well known. However, the roles and evolution of the local folding of intronic and exonic sequence features adjacent to splice sites has yet to be thoroughly studied. Here, focusing on four fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, and Candida albicans), we performed for the first time a comprehensive high-resolution study aimed at characterizing the encoding of intronic splicing efficiency in pre-mRNA transcripts and its effect on intron evolution. Our analysis supports the conjecture that pre-mRNA local folding strength at intronic boundaries is under selective pressure, as it significantly affects splicing efficiency. Specifically, we show that in the immediate region of 12-30 nucleotides (nt) surrounding the intronic donor site there is a preference for weak pre-mRNA folding; similarly, in the region of 15-33 nt surrounding the acceptor and branch sites there is a preference for weak pre-mRNA folding. We also show that in most cases there is a preference for strong pre-mRNA folding further away from intronic splice sites. In addition, we demonstrate that these signals are not associated with gene-specific functions, and they correlate with splicing efficiency measurements (r = 0.77, P = 2.98 × 10(-21)) and with expression levels of the corresponding genes (P = 1.24 × 10(-19)). We suggest that pre-mRNA folding strength in the above-mentioned regions has a direct effect on splicing efficiency by improving the recognition of intronic boundaries. These new discoveries are contributory steps toward a broader understanding of splicing regulation and intronic/transcript evolution. PMID:26246046

  18. Inhibition of Splicing but not Cleavage at the 5' Splice Site by Truncating Human β -globin Pre-mRNA

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

    Human β -globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.

  19. Congenital contractural arachnodactyly due to a novel splice site mutation in the FBN2 gene

    PubMed Central

    Mehar, Virendra; Yadav, Dinesh; Kumar, Ravindra; Yadav, Summi; Singh, Kuldeep; Callewaert, Bert; Pathan, Shahnawaz; De Paepe, Anne; Coucke, Paul J.

    2014-01-01

    Congenital contractural arachnodactyly is a rare autosomal dominant disorder characterized by crumpled ears, congenital contractures, arachnodactyly and scoliosis. Only few cases have been described to date. Here we report a newborn with congenital contractures, crumpled ears and scoliosis. Molecular analysis revealed a novel fibrillin-2 mutation at the donor splice site of intron 28. We discuss the differential diagnosis of neonates with congenital contractures and review the current knowledge on congenital contractural arachnodactyly.

  20. Co-transcriptional commitment to alternative splice site selection.

    PubMed

    Roberts, G C; Gooding, C; Mak, H Y; Proudfoot, N J; Smith, C W

    1998-12-15

    Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription. PMID:9837984

  1. Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

    PubMed

    Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

    2008-01-01

    Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3. PMID:18404972

  2. Method of predicting Splice Sites based on signal interactions

    PubMed Central

    Churbanov, Alexander; Rogozin, Igor B; Deogun, Jitender S; Ali, Hesham

    2006-01-01

    Background Predicting and proper ranking of canonical splice sites (SSs) is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE) and Intronic (ISE) Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand. PMID:16584568

  3. Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

    PubMed Central

    Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human

  4. Characterization of a novel TYMP splice site mutation associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

    PubMed

    Taanman, Jan-Willem; Daras, Mariza; Albrecht, Juliane; Davie, Charles A; Mallam, Elizabeth A; Muddle, John R; Weatherall, Mark; Warner, Thomas T; Schapira, Anthony H V; Ginsberg, Lionel

    2009-02-01

    Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disorder caused by loss-of-function mutations in the thymidine phosphorylase gene (TYMP). We report here a patient compound heterozygous for two TYMP mutations: a novel g.4009G>A transition affecting the consensus splice donor site of intron 9, and a previously reported g.675G>C splice site mutation. The novel mutation causes exon 9 skipping but leaves the reading frame intact; however, TYMP protein was not detected by immunoblot analysis, suggesting that neither mutant allele is expressed as protein. The patient's fibroblasts showed gradual loss of the mitochondrial DNA-encoded subunit I of cytochrome-c oxidase, suggesting a progressive mitochondrial DNA defect in culture. PMID:19056268

  5. Molecular characterization of novel splice site mutation causing protein C deficiency.

    PubMed

    Al-Hamed, Mohamed H; AlBatniji, Fatma; AlDakheel, Ghadah A; El-Faraidi, Huda; Al-Zahrani, Azzah; Al-Abbass, Fahed; Imtiaz, Faiqa

    2016-07-01

    Congenital protein C deficiency is an inherited coagulation disorder associated with an elevated risk of venous thromboembolism. A Saudi Arabian male from a consanguineous family was admitted to neonatal intensive care unit in his first days of life because of transient tachypnea and hematuria. Laboratory investigations determined low platelet and protein C deficiency. Direct sequencing of PROC gene and RNA analysis were performed. Analysis of factor V Leiden (G1691A) and factor II (G20210A) mutations was also done. Novel homozygous splice site mutation c.796+3A>T was detected in the index case and segregation was confirmed in the family. RNA analysis revealed the pathogenicity of the mutation by skipping exon 8 of PROC gene and changing the donor splice site of the exon. Detection of the molecular cause of protein C deficiency reduces life threatening and facilitates inductive carrier testing, prenatal and preimplantation genetic diagnosis for families. PMID:26656900

  6. Case 1: chronic infected donor site.

    PubMed

    2016-03-01

    Following a coronary bypass surgery, a vein donor site became infected and failed to heal despite use of antibiotics and a variety of topical treatments. Octenilin Wound Gel not only helped to promote healing, but also increased the patient's ability to tolerate dressing changes. PMID:26949845

  7. Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

    PubMed

    Semlow, Daniel R; Blanco, Mario R; Walter, Nils G; Staley, Jonathan P

    2016-02-25

    During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3' splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3' to 5' translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome. PMID:26919433

  8. Prp40 and early events in splice site definition.

    PubMed

    Becerra, Soraya; Andrés-León, Eduardo; Prieto-Sánchez, Silvia; Hernández-Munain, Cristina; Suñé, Carlos

    2016-01-01

    The alternative splicing (AS) of precursor messenger RNA (pre-mRNA) is a tightly regulated process through which introns are removed to leave the resulting exons in the mRNA appropriately aligned and ligated. The AS of pre-mRNA is a key mechanism for increasing the complexity of proteins encoded in the genome. In humans, more than 90% of genes undergo AS, underscoring the importance of this process in RNA biogenesis. As such, AS misregulation underlies multiple human diseases. The splicing reaction is catalyzed by the spliceosome, a highly dynamic complex that assembles at or near the intron/exon boundaries and undergoes sequential conformational and compositional changes during splicing. The initial recognition of splice sites defines the exons that are going to be removed, which is a critical step in the highly regulated splicing process. Although the available lines of evidence are increasing, the molecular mechanisms governing AS, including the initial interactions occurring at intron/exon boundaries, and the factors that modulate these critical connections by functioning as a scaffold for active-site RNAs or proteins, remain poorly understood. In this review, we summarize the major hallmarks of the initial steps in the splicing process and the role of auxiliary factors that contribute to the assembly of the spliceosomal complex. We also discuss the role of the essential yeast Prp40 protein and its mammalian homologs in the specificity of this pre-mRNA processing event. In addition, we provide the first exhaustive phylogenetic analysis of the molecular evolution of Prp40 family members. WIREs RNA 2016, 7:17-32. doi: 10.1002/wrna.1312 For further resources related to this article, please visit the WIREs website. PMID:26494226

  9. Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

    PubMed Central

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

    2015-01-01

    Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

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

    PubMed Central

    Sowden, M P; Smith, H C

    2001-01-01

    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

  11. Successful Treatment of Chronic Donor Site Pain

    PubMed Central

    Yanow, Jennifer H; Lorenzo, Luigi Di; Worosilo, Sharon C; Pappagallo, Marco

    2015-01-01

    Introduction: This is a case presentation of a 45-year-old male with chronic donor site pain following autologous iliac crest bone harvest successfully treated with superior cluneal nerve blockade. Donor site pain following autologous bone harvest is a common postoperative complication of lumbar fusion procedures that can cause significant morbidity and diminish quality of life, even in the context of an otherwise successful surgery. Dysfunction of the superior cluneal nerves is an etiology of this chronic pain. The patient’s medical history, attempted treatments, and literature were reviewed. Case Presentation: A 45-year-old male with a six year history of severe pain over the right iliac crest following an otherwise successful lumbar laminectomy and fusion underwent two sets of superior cluneal nerve blocks, with sustained relief of more than 80% at seven months follow up. Conclusions: Donor site pain following autologous iliac crest bone harvest is a common surgical complication that is often resistant to conservative treatments such as physical therapy and oral medications. Blockade of the superior cluneal nerves is a safe and technically simple procedure that may result in long-term pain relief, obviating the need to consider more invasive options. PMID:26587399

  12. Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

    PubMed Central

    Munroe, S H; Duthie, R S

    1986-01-01

    The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing. Images PMID:3024107

  13. Detection of Splice Sites Using Support Vector Machine

    NASA Astrophysics Data System (ADS)

    Varadwaj, Pritish; Purohit, Neetesh; Arora, Bhumika

    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.

  14. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

    PubMed Central

    Thanaraj, T. A.; Clark, Francis

    2001-01-01

    It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms. PMID:11410667

  15. A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

    PubMed Central

    Elrick, Leslie L.; Humphrey, Mary Beth; Cooper, Thomas A.; Berget, Susan M.

    1998-01-01

    Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in

  16. A case of mild CHARGE syndrome associated with a splice site mutation in CHD7.

    PubMed

    Wells, Constance; Loundon, Natalie; Garabedian, Noël; Wiener-Vacher, Sylvette; Cordier-Bouvier, Marie-Dominique; Goudeffroye, Géraldine; Attié-Bitach, Tania; Marlin, Sandrine

    2016-04-01

    CHARGE syndrome (MIM#214800) (Coloboma, Heart defect, Atresia of choanae, Retarded growth and development, Genital hypoplasia, Ear abnormalities/deafness) is caused by heterozygous mutation of CHD7 transmitted in an autosomal dominant manner. In this report, we describe a patient with bilateral hearing impairment, unusually-shaped ears, no intellectual disability and a patent ductus arteriosus. Further investigation showed abnormal semicircular canals and the presence of olfactory bulbs. He does not fulfill the Blake or the Verloes criteria for CHARGE. A de novo mutation at the donor splice site of intron 33 was identified (c.7164 + 1G > A). It is of importance to diagnose mildly affected patients for appropriate genetic counselling and to better understand the mild end of the phenotypic spectrum of CHARGE syndrome. PMID:26921530

  17. DBASS3 and DBASS5: databases of aberrant 3′- and 5′-splice sites

    PubMed Central

    Chivers, Martin; Hwang, Gyulin; Vorechovsky, Igor

    2011-01-01

    DBASS3 and DBASS5 provide comprehensive repositories of new exon boundaries that were induced by pathogenic mutations in human disease genes. Aberrant 5′- and 3′-splice sites were activated either by mutations in the consensus sequences of natural exon–intron junctions (cryptic sites) or elsewhere (‘de novo’ sites). DBASS3 and DBASS5 currently contain approximately 900 records of cryptic and de novo 3′- and 5′-splice sites that were produced by over a thousand different mutations in approximately 360 genes. DBASS3 and DBASS5 data can be searched by disease phenotype, gene, mutation, location of aberrant splice sites in introns and exons and their distance from authentic counterparts, by bibliographic references and by the splice-site strength estimated with several prediction algorithms. The user can also retrieve reference sequences of both aberrant and authentic splice sites with the underlying mutation. These data will facilitate identification of introns or exons frequently involved in aberrant splicing, mutation analysis of human disease genes and study of germline or somatic mutations that impair RNA processing. Finally, this resource will be useful for fine-tuning splice-site prediction algorithms, better definition of auxiliary splicing signals and design of new reporter assays. DBASS3 and DBASS5 are freely available at http://www.dbass.org.uk/. PMID:20929868

  18. Controlled trial of Biocol versus Jelonet on donor sites.

    PubMed

    Ebbehoj, J; Gavrilyuk, B K; Menzul, V A; Paklin, E L; Rochev YuA

    1996-11-01

    A controlled study of healing time in donor sites covered with Biocol, a new latex-based material, compared with donor sites covered with Jelonet showed a shorter healing time after treatment with Biocol (P < 0.001). No significant difference in cosmetic results 3 months post wounding was observed. No difference in discomfort was observed. PMID:8909760

  19. A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism.

    PubMed

    Ieiri, T; Cochaux, P; Targovnik, H M; Suzuki, M; Shimoda, S; Perret, J; Vassart, G

    1991-12-01

    A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient. PMID:1752952

  20. Noncanonical registers and base pairs in human 5′ splice-site selection

    PubMed Central

    Tan, Jiazi; Ho, Jia Xin Jessie; Zhong, Zhensheng; Luo, Shufang; Chen, Gang; Roca, Xavier

    2016-01-01

    Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5′ splice sites are mainly recognized by canonical base-pairing to the 5′ end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5′ splice site/U1 helix contributes to the recognition of certain 5′ splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5′ splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5′ splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences. PMID:26969736

  1. Noncanonical registers and base pairs in human 5' splice-site selection.

    PubMed

    Tan, Jiazi; Ho, Jia Xin Jessie; Zhong, Zhensheng; Luo, Shufang; Chen, Gang; Roca, Xavier

    2016-05-01

    Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences. PMID:26969736

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2006-01-01

    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

  4. The DEAH-box ATPases Prp16 and Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing

    PubMed Central

    Koodathingal, Prakash; Novak, Thaddeus; Piccirilli, Joseph A.; Staley, Jonathan P.

    2010-01-01

    SUMMARY To investigate the mechanism of fidelity in pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5′ splice site cleavage. We inactivated spliceosomes by disrupting a critical metal-ligand interaction at the catalytic center and discovered that when the DEAH-box ATPase Prp16 was disabled these spliceosomes successfully catalyzed 5′ splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5′ splice site cleavage, we found that Prp16 can associate with spliceosomes before 5′ splice site cleavage, consistent with a role for Prp16 in proofreading 5′ splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH-box ATPase Prp43, a spliceosome disassembly factor. These data provide evidence that splicing fidelity mechanisms discriminate against slow substrates and that the mechanisms for establishing the fidelity of 5′ splice site cleavage and exon ligation share a common ATP-dependent framework. PMID:20705241

  5. On splice site prediction using weight array models: a comparison of smoothing techniques

    NASA Astrophysics Data System (ADS)

    Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

    2007-11-01

    In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called "splicing". The positions where introns are cut and exons are spliced together are called "splice sites". Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed.

  6. Healing of Donor-site Buccal Mucosa Urethroplasty

    PubMed Central

    Alwaal, Amjad; Harris, Catherine R.; Enriquez, Anthony; McAninch, Jack W.; Breyer, Benjamin N.

    2016-01-01

    Buccal mucosal graft represents the gold standard graft material for urethroplasty because of its thick epithelium and a thin lamina propria for maximal graft uptake. There is an ongoing debate whether to close the buccal graft donor site. We show a unique look at buccal donor site healing through serial pictures over a 100-day period. In this patient, the anterior half of the buccal donor site was closed at the time of harvest, allowing real-time observation of wound healing from both the closed and open aspects of the wound. PMID:26151892

  7. A role for U2/U6 helix Ib in 5' splice site selection.

    PubMed Central

    Luukkonen, B G; Séraphin, B

    1998-01-01

    Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II. PMID:9701283

  8. Computer-assisted selection of donor sites for autologous grafts

    NASA Astrophysics Data System (ADS)

    Krol, Zdzislaw; Zeilhofer, Hans-Florian U.; Sader, Robert; Hoffmann, Karl-Heinz; Gerhardt, Paul; Horch, Hans-Henning

    1997-05-01

    A new method is proposed for a precise planning of autologous bone grafts in cranio- and maxillofacial surgery. In patients with defects of the facial skeleton, autologous bone transplants can be harvested from various donor sites in the body. The preselection of a donor site depends i.a. on the morphological fit of the available bone mass and the shape of the part that is to be transplanted. A thorough planning and simulation of the surgical intervention based on 3D CT studies leads to a geometrical description and the volumetric characterization of the bone part to be resected and transplanted. Both, an optimal fit and a minimal lesion of the donor site are guidelines in this process. We use surface similarity and voxel similarity measures in order to select the optimal donor region for an individually designed transplant.

  9. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

    PubMed Central

    Vega, Yolanda; Delgado, Elena; de la Barrera, Jorge; Carrera, Cristina; Zaballos, Ángel; Cuesta, Isabel; Mariño, Ana; Ocampo, Antonio; Miralles, Celia; Pérez-Castro, Sonia; Álvarez, Hortensia; López-Miragaya, Isabel; García-Bodas, Elena; Díez-Fuertes, Francisco; Thomson, Michael M.

    2016-01-01

    HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known. PMID:27355361

  10. Secondary structure of splice sites in adenovirus mRNA precursors.

    PubMed Central

    Munroe, S H

    1984-01-01

    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

  11. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  12. Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2.

    PubMed Central

    Bhat, B M; Brady, H A; Wold, W S

    1985-01-01

    Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region. Images PMID:3879768

  13. Structural Features of a 3′ Splice Site in Influenza A

    PubMed Central

    2015-01-01

    Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3′ splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3′ splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3′ splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on 1H chemical shifts. The internal loop containing the 3′ splice site has a dynamic guanosine and a stable imino (cis Watson–Crick/Watson–Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson–Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair. PMID:25909229

  14. Deep intron elements mediate nested splicing events at consecutive AG dinucleotides to regulate alternative 3' splice site choice in vertebrate 4.1 genes.

    PubMed

    Parra, Marilyn K; Gallagher, Thomas L; Amacher, Sharon L; Mohandas, Narla; Conboy, John G

    2012-06-01

    Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3' splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ∼120 kb upstream of E2 an ultradistal intraexon, iE(B), that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iE(B) in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iE(B) function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iE(R)-mediated intrasplicing at the first available exons downstream of iE(R), namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3' splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3' splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity. PMID:22473990

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

    PubMed

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

    2015-04-01

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

  16. A novel splice site mutation in SMARCAL1 results in aberrant exon definition in a child with schimke immunoosseous dysplasia.

    PubMed

    Carroll, Clinton; Hunley, Tracy E; Guo, Yan; Cortez, David

    2015-10-01

    Schimke Immunoosseous Dysplasia (SIOD) is a rare, autosomal recessive disorder of childhood characterized by spondyloepiphyseal dysplasia, focal segmental glomerulosclerosis and renal failure, T-cell immunodeficiency, and cancer in certain instances. Approximately half of patients with SIOD are reported to have biallelic mutations in SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA translocase that localizes to sites of DNA replication and repairs damaged replication forks. We present a novel mutation (NM_014140.3:c.2070+2insT) that results in defective SMARCAL1 mRNA splicing in a child with SIOD. This mutation, within the donor site of intron 12, results in the skipping of exon 12, which encodes part of a critical hinge region connecting the two lobes of the ATPase domain. This mutation was not recognized as deleterious by diagnostic SMARCAL1 sequencing, but discovered through next generation sequencing and found to result in absent SMARCAL1 expression in patient-derived lymphoblasts. The splicing defect caused by this mutation supports the concept of exon definition. Furthermore, it illustrates the need to broaden the search for SMARCAL1 mutations in patients with SIOD lacking coding sequence variants. PMID:25943327

  17. Compensatory signals associated with the activation of human GC 5′ splice sites

    PubMed Central

    Kralovicova, Jana; Hwang, Gyulin; Asplund, A. Charlotta; Churbanov, Alexander; Smith, C. I. Edvard; Vorechovsky, Igor

    2011-01-01

    GC 5′ splice sites (5′ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5′ss activated by a mutation in BTK intron 3. This GC 5′ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5′ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5′ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5′ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5′ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5′ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5′ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites. PMID:21609956

  18. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb. PMID:27368438

  19. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    SciTech Connect

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. Erasmus Univ. of Rotterdam )

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  20. Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

    PubMed

    Mendez, Gregory S; Delwiche, Charles F; Apt, Kirk E; Lippmeier, J Casey

    2015-01-01

    Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR-based contigs. The sequence contains an intron-rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3' splice site are repeated by an identical two to nine base pairs around the 5' splice site. The 5' and 3' splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes. PMID:25963315

  1. Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

    PubMed Central

    Tang, Rongying; Prosser, Debra O.; Love, Donald R.

    2016-01-01

    The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative. PMID:27313609

  2. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  3. Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

    PubMed Central

    Tan, W; Schalling, M; Zhao, C; Luukkonen, M; Nilsson, M; Fenyö, E M; Pavlakis, G N; Schwartz, S

    1996-01-01

    The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site. PMID:8648699

  4. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

    PubMed Central

    Teigelkamp, S; Newman, A J; Beggs, J D

    1995-01-01

    Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP. Images PMID:7781612

  5. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

    PubMed Central

    Teraoka, S N; Telatar, M; Becker-Catania, S; Liang, T; Onengüt, S; Tolun, A; Chessa, L; Sanal, O; Bernatowska, E; Gatti, R A; Concannon, P

    1999-01-01

    Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations. PMID:10330348

  6. ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

    PubMed Central

    Alvi, R K; Lund, M; Okeefe, R T

    2001-01-01

    Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site. PMID:11453062

  7. Splicing fidelity

    PubMed Central

    Koodathingal, Prakash; Staley, Jonathan P.

    2013-01-01

    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

  8. [An optimum donor site for venous grafting for microsurgery].

    PubMed

    Grimaud, O; Delpit, X; Hardy, P

    2011-06-01

    We report the results of an anatomic study based on 10 cadavers. The aim of this work is to find an optimum donor site for venous grafts which is safe, reproducible, and suitable for microsurgery stitches especially in finger reimplantation, when a long and small calibre graft is needed. This study describes the deep venous network of the radial artery, an original donor site for microsurgical venous grafts. The second aim is to describe our technique of harvesting. Dissections always show two satellite veins, that can be harvested with optimal average diameter of 1.8mm constant over the whole length. The maximum length available is about 126.5mm for the radial satellite vein, and 125 mm for the ulnar one, with a few number of collateral ligatures needed. No tying is required in 60% cases for radial satellite vein, and one ligature for the other 40%, whereas in the ulnar satellite vein, no tying is needed in 80% and just one in the other 20%. This original site is advantageous in microsurgery of the upper limb, offering an easy, quick, safe and reproducible option in an emergency situation. PMID:21621446

  9. The Consensus 5' Splice Site Motif Inhibits mRNA Nuclear Export

    PubMed Central

    Lee, Eliza S.; Akef, Abdalla; Mahadevan, Kohila; Palazzo, Alexander F.

    2015-01-01

    In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3’terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5’splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3’cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3’terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5’splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise. PMID:25826302

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

    PubMed

    Piasecka, Alicja; Brzuzan, Paweł; Woźny, Maciej; Ciesielski, Sławomir; Kaczmarczyk, Dariusz

    2015-02-01

    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

  11. Achieving ideal donor site aesthetics with autologous breast reconstruction

    PubMed Central

    2015-01-01

    The appearance of the donor site following breast reconstruction with abdominal flaps has become an important topic for study. Given the variety of flaps that are derived from the abdomen, decisions are often based on how much muscle and fascia will be harvested. Comparisons between muscle sparing and non-muscle sparing techniques have been performed with outcomes related to function and contour. Closure techniques will vary and include primary fascial closure, mesh reinforcement and additional fascial plication all of which can produce natural and sometimes improved abdominal contours. Proper patient selection however is important. This manuscript will describe various techniques in order to achieve ideal abdominal contour following autologous reconstruction. PMID:26005646

  12. Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

    SciTech Connect

    Zhou, Z.; Olsen, J.C.; Silverman, L.M.

    1994-09-01

    Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. The remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.

  13. Association of Carney Complex with an Intronic Splice Site Mutation in the PRKAR1A Gene.

    PubMed

    Guo, H; Xiong, H; Li, Z; Xu, J; Zhang, H; Chen, X; Hu, S

    2016-06-01

    This study was aimed to investigate the clinical features and mutations in the PRKAR1A gene of a multigenerational kindred including 17 individuals at risk for Carney complex. Eight patients were diagnosed with Carney complex among the 17 individuals (47.1%). Among the 8 affected patients, 4 had cardiac myxomas, 8 had skin pigmentation, and 3 had diabetes. Genomic DNA sequencing in 14 surviving patients showed 6 had the same germline mutation in the sixth intron and affected the splice site. cDNA sequencing and DNAMAN software showed 159 bases were absent, resulting in the absence of the amino acids 249 to 301 from the protein. All 6 patients with this PRKAR1A gene mutation had skin pigmentation. In conclusion, the present study reported for the first time an intronic splice site mutation in the PRKAR1A gene of a Chinese family with Carney complex, which probably caused skin pigmentation observed in affected family members. PMID:26788925

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

    PubMed Central

    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

    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

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

    PubMed Central

    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

    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

  16. Feature selection for splice site prediction: A new method using EDA-based feature ranking

    PubMed Central

    Saeys, Yvan; Degroeve, Sven; Aeyels, Dirk; Rouzé, Pierre; Van de Peer, Yves

    2004-01-01

    Background The identification of relevant biological features in large and complex datasets is an important step towards gaining insight in the processes underlying the data. Other advantages of feature selection include the ability of the classification system to attain good or even better solutions using a restricted subset of features, and a faster classification. Thus, robust methods for fast feature selection are of key importance in extracting knowledge from complex biological data. Results In this paper we present a novel method for feature subset selection applied to splice site prediction, based on estimation of distribution algorithms, a more general framework of genetic algorithms. From the estimated distribution of the algorithm, a feature ranking is derived. Afterwards this ranking is used to iteratively discard features. We apply this technique to the problem of splice site prediction, and show how it can be used to gain insight into the underlying biological process of splicing. Conclusion We show that this technique proves to be more robust than the traditional use of estimation of distribution algorithms for feature selection: instead of returning a single best subset of features (as they normally do) this method provides a dynamical view of the feature selection process, like the traditional sequential wrapper methods. However, the method is faster than the traditional techniques, and scales better to datasets described by a large number of features. PMID:15154966

  17. Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome.

    PubMed

    Lee, Byeonghyeon; Duz, Mehmet Bugrahan; Sagong, Borum; Koparir, Asuman; Lee, Kyu-Yup; Choi, Jae Young; Seven, Mehmet; Yuksel, Adnan; Kim, Un-Kyung; Ozen, Mustafa

    2016-02-01

    Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome. PMID:26551301

  18. Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

    PubMed Central

    Kiss-László, Z; Blanc, S; Hohn, T

    1995-01-01

    A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV. Images PMID:7628455

  19. A 5' splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated pro alpha 1(I) chains with a non-collagenous insertion destabilizing the triple helix.

    PubMed Central

    Bateman, J F; Chan, D; Moeller, I; Hannagan, M; Cole, W G

    1994-01-01

    A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3' limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7945197

  20. Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.

    PubMed Central

    Montell, C; Fisher, E F; Caruthers, M H; Berk, A J

    1984-01-01

    The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells. Images PMID:6727875

  1. Alternative splicing of HLA-DQB transcripts and secretion of HLA-DQ beta-chain proteins: allelic polymorphism in splicing and polyadenylylation sites.

    PubMed Central

    Briata, P; Radka, S F; Sartoris, S; Lee, J S

    1989-01-01

    HLA class II antigens are highly polymorphic cell-surface proteins involved in initiation and regulation of the immune response. Allelic sequence variation primarily affects the structure of the first external domains of alpha and beta component chains. Here we provide evidence for other types of allelic polymorphism for the genes encoding these chains. Sequences of two cDNA clones corresponding to HLA-DQB mRNAs from an HLA-homozygous cell line exhibit both alternative splicing and read-through of polyadenylylation. Furthermore, alternative splicing that deletes the transmembrane exon is associated with only a subset of HLA-DQB alleles, while the polyadenylylation-site read-through is found in a larger subset. This suggest that polymorphic cis-acting elements within the HLA-DQB gene control both processing steps. Proteins, presumably encoded by alternatively spliced mRNAs lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against HLA-DQ. These proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines that do not contain alternatively spliced mRNAs. Images PMID:2464826

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

    PubMed Central

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

    2001-01-01

    Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA. PMID:11266544

  3. Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

    PubMed

    Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

    2015-07-15

    The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. PMID:25904108

  4. A prospective comparison of a new, synthetic donor site dressing versus an impregnated gauze dressing.

    PubMed

    Hickerson, W L; Kealey, G P; Smith, D J; Thomson, P D

    1994-01-01

    Three institutions enrolled 38 patients who required bilateral skin graft donor sites into a safety and efficacy study of a new synthetic donor site dressing. Bilateral donor sites were randomized to receive either a new, synthetic donor site dressing or an impregnated gauze dressing. Wounds were assessed by time to healing, pain, and patient preference. Synthetic dressing wounds were treated 7.9 days compared with 10.2 days for gauze dressing wounds (p < 0.001), and synthetic dressing wounds were more completely epithelialized. Visual analogue pain analysis revealed significantly less donor site pain with synthetic dressing (2.94) versus gauze dressing (4.64) (p < 0.001). Synthetic dressing had fewer treatment-related adverse experiences than gauze dressing (2 vs 7) and was judged by recipients to be superior to gauze dressing in comfort, pain relief, cosmetic appeal, ease of ambulation, and overall acceptance. PMID:7929519

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

    SciTech Connect

    Hunter, T.C.; Albertini, R.J.; O`Neill, J.P.

    1996-03-01

    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.

  6. Ice application to minimize pain in the split-thickness skin graft donor site.

    PubMed

    Akan, Mithat; Misirlioğlu, Aykut; Yildirim, Serkan; Cakir, Bariş; Taylan, Gaye; Aköz, Tayfun

    2003-01-01

    Reconstruction of tissue defects with skin grafts is one of the most used processes in soft tissue defects. While any part of the body can be used as a donor site of split-thickness skin grafts, the posterolateral thigh is the most used one. Pain in the graft donor site may be the primary concern of patients in the postoperative period. Various kinds of donor site dressings and procedures have been described for this purpose. The main goal of this practice is the fast recovery of the donor site. Nevertheless, avoiding infection, decreasing the pain in the donor site, and minimizing the cost should be considered. To minimize pain in the graft donor site, ice pack application, used for its local anesthetic effect, was utilized for patients postoperatively in our clinic. Thirty-six patients were included in this study between June 2001 and May 2002. Patients were divided into two groups, with 18 patients in-group I, to whom ice was applied, and 18 patients in group II, which was specified as the control group. The patients were evaluated according to the pain in the graft donor site. The visual analog scale (VAS) was used to evaluate the pain of the patients. The data were statistically evaluated with the Mann-Whitney U test procedure. In addition to this, infections, recovery periods, and cost benefit effects were also determined. The mean pain scores in the graft donor site were found to be quite low in patients in the group with ice application (p < 0.05). No significant difference was determined between the two groups when their pain scales were compared on the fourth and the fifth days (p > 0.05). Ice can be safely used in patients for whom donor site pain is the primary concern, with advantages such as ease of application, low cost (almost free), and a significant decrease in pain in the donor site. PMID:15058555

  7. Biased exon/intron distribution of cryptic and de novo 3′ splice sites

    PubMed Central

    Královičová, Jana; Christensen, Mikkel B.; Vořechovský, Igor

    2005-01-01

    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

  8. A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher.

    PubMed

    Meurs, Kathryn M; Lahmers, Sunshine; Keene, Bruce W; White, Stephen N; Oyama, Mark A; Mauceli, Evan; Lindblad-Toh, Kerstin

    2012-08-01

    Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (p(raw) = 9.999e-05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190-23,781,919 (p = 0.001) and DNA sequencing identified a 16-base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (p < 0.0001). Electron microscopy of myocardium from affected dogs demonstrated disorganization of the Z line, mild to moderate T tubule and sarcoplasmic reticulum dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog. PMID:22447147

  9. 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

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-03-30

    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.

  10. Constant splice-isoform ratios in human lymphoblastoid cells support the concept of a splico-stat.

    PubMed

    Kramer, Marcel; Huse, Klaus; Menzel, Uwe; Backhaus, Oliver; Rosenstiel, Philip; Schreiber, Stefan; Hampe, Jochen; Platzer, Matthias

    2011-03-01

    Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. PMID:21220357

  11. A novel splice site mutation in EYA4 causes DFNA10 hearing loss.

    PubMed

    Hildebrand, Michael S; Coman, David; Yang, Tao; Gardner, R J McKinlay; Rose, Elizabeth; Smith, Richard J H; Bahlo, Melanie; Dahl, Hans-Henrik M

    2007-07-15

    Nonsyndromic autosomal dominant sensorineural hearing loss (SNHL) at the DFNA10 locus was described in two families in 2001. Causative mutations that affect the EyaHR domain of the 'Eyes absent 4' (EYA4) protein were identified. We report on the clinical and genetic analyses of an Australian family with nonsyndromic SNHL. Screening of the EYA4 gene showed the novel polypyrimidine tract variation ca. 1,282-12T > A that introduces a new 3' splice acceptor site. This is the first report of a point mutation in EYA4 that is hypothesized to lead to aberrant pre-mRNA splicing and human disease. The DFNA10 family described is only the fourth to be identified. One individual presented with apparently the same phenotype as other affected members of the family. However, genotyping illustrated that he did not share the DFNA10 disease haplotype. Detailed clinical investigation showed differences in the onset and severity of his hearing loss and thus he is presumed to represent a phenocopy, perhaps resulting from long-term exposure to loud noise. PMID:17568404

  12. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    PubMed Central

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Geneviève; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; Østergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I

    2015-01-01

    Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing. PMID:26125040

  13. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

    PubMed Central

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-01-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations. PMID:19773425

  14. A novel 3′ splice site recognition by the two zinc fingers in the U2AF small subunit

    PubMed Central

    Yoshida, Hisashi; Park, Sam-Yong; Oda, Takashi; Akiyoshi, Taeko; Sato, Mamoru; Shirouzu, Mikako; Tsuda, Kengo; Kuwasako, Kanako; Unzai, Satoru; Muto, Yutaka; Urano, Takeshi; Obayashi, Eiji

    2015-01-01

    The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3′ splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the β sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein. PMID:26215567

  15. Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene

    PubMed Central

    Gastaldo, Elena; Harries, Lorna W.; Rubio-Cabezas, Oscar; Castaño, Luis

    2012-01-01

    Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. PMID:22235272

  16. MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation.

    PubMed

    Szigeti, K; Wong, L-J C; Perng, C-L; Saifi, G M; Eldin, K; Adesina, A M; Cass, D L; Hirano, M; Lupski, J R; Scaglia, F

    2004-02-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder caused by thymidine phosphorylase (TP) deficiency, resulting in severe gastrointestinal dysmotility and skeletal muscle abnormalities. A patient is reported with a classical MNGIE clinical presentation but without skeletal muscle involvement at morphological, enzymatic, or mitochondrial DNA level, though gastrointestinal myopathy was present. MNGIE was diagnosed by markedly raised plasma thymidine and reduced thymidine phosphorylase activity. Molecular genetic analysis showed a homozygous novel splice site mutation in TP. On immunohistochemical studies there was marked TP expression in the CNS, in contrast to what has been observed in rodents. It is important to examine the most significantly affected tissue and to measure TP activity and plasma thymidine in order to arrive at an accurate diagnosis in this condition. PMID:14757860

  17. PCR differentiation of commercial yeast strains using intron splice site primers.

    PubMed Central

    de Barros Lopes, M; Soden, A; Henschke, P A; Langridge, P

    1996-01-01

    The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms in commercial yeast strains. The speed of the method and the ability to analyze many samples in a single day permit the monitoring of specific yeast strains during fermentations. Furthermore, the simplicity of the technique, which does not require the isolation of DNA, makes it accessible to industrial laboratories that have limited molecular expertise and resources. PMID:8953723

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

    PubMed Central

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

    2014-01-01

    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

  19. Dominant Splice Site Mutations in PIK3R1 Cause Hyper IgM Syndrome, Lymphadenopathy and Short Stature.

    PubMed

    Petrovski, Slavé; Parrott, Roberta E; Roberts, Joseph L; Huang, Hongxiang; Yang, Jialong; Gorentla, Balachandra; Mousallem, Talal; Wang, Endi; Armstrong, Martin; McHale, Duncan; MacIver, Nancie J; Goldstein, David B; Zhong, Xiao-Ping; Buckley, Rebecca H

    2016-07-01

    The purpose of this research was to use next generation sequencing to identify mutations in patients with primary immunodeficiency diseases whose pathogenic gene mutations had not been identified. Remarkably, four unrelated patients were found by next generation sequencing to have the same heterozygous mutation in an essential donor splice site of PIK3R1 (NM_181523.2:c.1425 + 1G > A) found in three prior reports. All four had the Hyper IgM syndrome, lymphadenopathy and short stature, and one also had SHORT syndrome. They were investigated with in vitro immune studies, RT-PCR, and immunoblotting studies of the mutation's effect on mTOR pathway signaling. All patients had very low percentages of memory B cells and class-switched memory B cells and reduced numbers of naïve CD4+ and CD8+ T cells. RT-PCR confirmed the presence of both an abnormal 273 base-pair (bp) size and a normal 399 bp size band in the patient and only the normal band was present in the parents. Following anti-CD40 stimulation, patient's EBV-B cells displayed higher levels of S6 phosphorylation (mTOR complex 1 dependent event), Akt phosphorylation at serine 473 (mTOR complex 2 dependent event), and Akt phosphorylation at threonine 308 (PI3K/PDK1 dependent event) than controls, suggesting elevated mTOR signaling downstream of CD40. These observations suggest that amino acids 435-474 in PIK3R1 are important for its stability and also its ability to restrain PI3K activity. Deletion of Exon 11 leads to constitutive activation of PI3K signaling. This is the first report of this mutation and immunologic abnormalities in SHORT syndrome. PMID:27076228

  20. A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis

    PubMed Central

    Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y.; Distl, Ottmar

    2015-01-01

    Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57–58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future. PMID:26506231

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

    PubMed Central

    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

    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

  2. The ideal split-thickness skin graft donor site dressing: rediscovery of polyurethane film.

    PubMed

    Dornseifer, Ulf; Fichter, Andreas M; Herter, Frank; Sturtz, Gustavo; Ninkovic, Milomir

    2009-08-01

    The almost single disadvantage of polyurethane film dressings, an uncontrolled leakage, is probably as often described as its numerous advantages for split-thickness skin graft donor sites. We solved this problem by perforating the polyurethane film, which permits a controlled leakage into a secondary absorbent dressing. The study included 30 adult patients. Skin graft donor sites at the proximal thigh were dressed with the modified film dressing. Our results indicate that this dressing concept is associated with a reliable, rapid rate of epithelization. Both, controlled leakage and minimal pain caused particular comfort for patients and ward staff. Furthermore, this dressing was also suited for differently shaped and large donor sites. We conclude that the modification results in a more practicable, comfortable, and cost-effective film dressing, which requalifies the polyurethane film as an ideal dressing material for split-thickness skin graft donor sites. PMID:19571740

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

    PubMed

    Finley, Jahahreeh

    2014-11-01

    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

  4. Founder Effect of a c.828+3A>T Splice Site Mutation in Peripherin 2 (PRPH2) Causing Autosomal Dominant Retinal Dystrophies

    PubMed Central

    Shankar, Suma P.; Birch, David G.; Ruiz, Richard S.; Hughbanks-Wheaton, Dianna K.; Sullivan, Lori S.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

    2015-01-01

    Importance Screening for splice site mutation c.828+3A>T in the peripherin 2 (PRPH2) gene should be a high priority in families with highly variable retinal dystrophies. The correction of missplicing is a potential therapeutic target. Objective To determine the prevalence, genetic origin, and molecular mechanism of a donor c.828+3A>T mutation in the PRPH2 (peripherin 2, retinal degeneration slow) gene in individuals with retinal dystrophies. Design, Setting, and Participants Case-control study that took place at the University of Texas Health Science Center, the University of Iowa, and the Retina Foundation of the Southwest, from January 1, 1987, to August 1, 2014, including affected individuals from 200 families with a diagnosis of autosomal dominant retinitis pigmentosa, 35 families with unspecified macular dystrophies, and 116 families with pattern dystrophy. Participants were screened for the c.828+3A>T mutation by restriction-enzyme digest, single-strand conformational polymorphism screening, or bidirectional sequencing. Haplotypes of polymorphic markers flanking the PRPH2 locus and sequence variants within the gene were determined by denaturing gel electrophoresis or automated capillary-based cycle sequencing. The effect of the splice site mutation on the PRPH2 transcript was analyzed using NetGene2, a splice prediction program and by the reverse transcription polymerase chain reaction of illegitimate transcripts from peripheral white blood cells. Main Outcomes and Measures Results of testing for splice site mutation, haplotypes, and alternate transcripts. Results The PRPH2 mutation was found in 97 individuals of 19 independently ascertained families with a clinical diagnosis of retinitis pigmentosa, macular dystrophy, and/or pattern dystrophy. All affected individuals also shared a rare haplotype of approximately 644 kilobase pairs containing the c.828+3A>T mutation, which extends from the short tandem repeat polymorphism D6S282 to c.1013G>A (rs434102, a

  5. Stabilizing Morbidity and Predicting the Aesthetic Results of Radial Forearm Free Flap Donor Sites

    PubMed Central

    Yun, Tae Kyoung; Ahn, Duck Sun; Park, Seung Ha; Lee, Byung Il; Kim, Hyon Surk; You, Hi Jin

    2015-01-01

    Background The radial forearm flap is a versatile, widely used flap. However, the possibility of donor site complications has led to concern over its use. Some surgeons prefer using other flaps whose donor sites can be closed primarily with less morbidity, including avoiding unpleasant scarring. However, in our experience, donor site stability of the radial forearm flap can be reliably achieved by using well-implemented specific procedures. Here, we present a collection of donor site cases of the radial forearm flap and investigate factors that affect the aesthetic results as the basis for a reference for selecting a radial forearm flap. Methods In this retrospective study, we reviewed 171 cases in which a radial forearm flap was used for free tissue transfer after resecting head and neck cancer. We focused on donor site morbidity rates. Each operation involved a detailed procedure designed to minimize donor site morbidity. Moreover, statistical investigations were conducted for 22 cases to determine factors affecting the scar appearance. Results Only one case developed total skin graft necrosis as a major complication. Scar-related aesthetic results were acceptable, and the body-mass index, body weight, diabetes, and cardiac problems were significant factors related to the appearance of scars. Conclusions Performing the radial forearm flap using a well-implemented detailed technique helps achieve acceptable donor site morbidity results. The aesthetic results were more promising for patients without excess body weight, diabetes, or cardiac problems. Therefore, anxiety about donor site morbidity should not be a reason to avoid selecting the radial forearm flap in suitable patients. PMID:26618126

  6. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

    PubMed

    Itani, Omar A; Flibotte, Stephane; Dumas, Kathleen J; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  7. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo

    PubMed Central

    Itani, Omar A.; Flibotte, Stephane; Dumas, Kathleen J.; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J.

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  8. Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families

    PubMed Central

    Khan, Shahid Y.; Ali, Shahbaz; Naeem, Muhammad Asif; Khan, Shaheen N.; Husnain, Tayyab; Butt, Nadeem H.; Qazi, Zaheeruddin A.; Akram, Javed; Riazuddin, Sheikh; Ayyagari, Radha; Hejtmancik, J. Fielding

    2015-01-01

    Purpose This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin. Methods Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1. Results A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028–1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408–2A>G. In silico analysis suggested that these

  9. The Peculiarities of Large Intron Splicing in Animals

    PubMed Central

    Fedorov, Alexei

    2009-01-01

    In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These “large introns” must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5′ and 3′ acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing—a consecutive splicing from the 5′-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites. PMID:19924226

  10. Probabilistic simple splicing systems

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

    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.

  11. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal

    PubMed Central

    Agrawal, Anant A.; Salsi, Enea; Chatrikhi, Rakesh; Henderson, Steven; Jenkins, Jermaine L.; Green, Michael R.; Ermolenko, Dmitri N.; Kielkopf, Clara L.

    2016-01-01

    How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals. PMID:26952537

  12. Functional and esthetic assessment of radial forearm flap donor site repaired with split thickness skin graft.

    PubMed

    Lee, Jong-Ho; Alrashdan, Mohammad S; Kim, Su-Gon; Rim, Jae-Seok; Jabaiti, Samir; Kim, Myung-Jin; Kim, Soung-Min

    2011-01-01

    The purpose of this study was to evaluate the long-term functional and esthetic outcomes of radial forearm flap (RFF) donor site repaired with split thickness skin graft (STSG). Nineteen patients underwent surgical reconstruction of oro-facial defects by the use of RFF and their donor sites were reconstructed with STSG. The patients were followed up at least for 12 months postoperatively and the left hand was the non-dominant hand in all of them. Objective methods including pinch strength, grip strength, range of motion, current perception threshold (CPT) and two-point discrimination, and subjective methods including patients interview, visual analogue score (VAS) about function, sensitivity, pain and color match, were collectively employed for donor site assessment. Our data revealed some degree of reduction in motor function and sensation compared to the non-donor hand. The difference of pinch strength means was 9.81% and of the grip strength was 12.6%. The difference of wrist flexion means was 17.6% and of wrist extension was 13.4%. However, none of the patients had functional defects of forearm supination and pronation, wrist ulnar deviation or wrist radial deviation. Subjective evaluation showed that the donor site repaired with STSG was well accepted by the patients particularly from a functional point of view. These results demonstrate that STSG represents a favorable choice for RFF donor site repair. PMID:20589506

  13. Intensive insulin treatment increases donor site wound protein synthesis in burn patients

    PubMed Central

    Tuvdendorj, Demidmaa; Zhang, Xiao-Jun; Chinkes, David L.; Aarsland, Asle; Kulp, Gabriela A.; Jeschke, Marc G.; Herndon, David N.

    2013-01-01

    Background In the treatment of burns, patients’ own skin is the preferred material to cover burn wounds, resulting in the need to create a donor site wound. Enhancement of healing of the donor site wound would be beneficial in burn patients. Insulin, an anabolic agent, is routinely used to treat hyperglycemia after injury. We investigated whether intensive insulin treatment (INS) increases fractional synthesis rate (FSR) of the donor site wound protein and decreases the length of hospitalization normalized for total body surface area burned (LOS/TBSA). Methods FSR of the donor site wound protein was measured in pediatric patients randomized to control (CNT) (n = 13) and INS (n = 10) treatments. Depending on the postoperative day when the tracer study was done studies were divided into “Early” (days < 5) and “Late” (days >=5) periods. Results FSR of the donor site wound protein was greater in the INS group at the “Early” period of wound healing (CNT vs. INS, 8.2±3.8 vs. 13.1±6.9 %/day, p: < 0.05); but not at the “Late” (CNT vs. INS, 19.7±4.6 vs. 16.6±4.0 %/day, p > 0.05). Despite these differences LOS/TBSA was not decreased in the INS group. Correlation analyses demonstrated that independently of the treatment regimen FSR positively correlated (p < 0.05) with time post creation of the donor site and negatively correlated (p < 0.05) with LOS/TBSA. Conclusions Insulin treatment increased FSR of the donor site wound protein in the early period of wound healing; FSR correlated with LOS/TBSA independently of the treatment regimen. PMID:21236451

  14. The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

    1995-07-01

    The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

  15. An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

    PubMed

    Williams, Christopher; Hoppe, Hans-Jürgen; Rezgui, Dellel; Strickland, Madeleine; Forbes, Briony E; Grutzner, Frank; Frago, Susana; Ellis, Rosamund Z; Wattana-Amorn, Pakorn; Prince, Stuart N; Zaccheo, Oliver J; Nolan, Catherine M; Mungall, Andrew J; Jones, E Yvonne; Crump, Matthew P; Hassan, A Bassim

    2012-11-30

    Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation. PMID:23197533

  16. The USH1C 216G-->A splice-site mutation results in a 35-base-pair deletion.

    PubMed

    Lentz, Jennifer; Savas, Sevtap; Ng, San-San; Athas, Grace; Deininger, Prescott; Keats, Bronya

    2005-02-01

    Usher syndrome is characterized by profound hearing loss and retinal degeneration. A splice-site mutation, 216G-->A, in exon 3 of USH1C is associated with Acadian Usher type IC. This mutation was reported to create an in-frame deletion of 39 base pairs (bp), resulting in an unstable transcript. By RT-PCR analysis of 216A and 216G constructs transfected into HeLa cells and also of patient cell lines, we have demonstrated a frame-shift deletion of 35 bp, not 39 bp. Thus, the instability of the USH1C mRNA is explained by the 216G-->A out-of-frame splice site mutation. PMID:15578223

  17. An empirical study of ensemble-based semi-supervised learning approaches for imbalanced splice site datasets

    PubMed Central

    2015-01-01

    Background Recent biochemical advances have led to inexpensive, time-efficient production of massive volumes of raw genomic data. Traditional machine learning approaches to genome annotation typically rely on large amounts of labeled data. The process of labeling data can be expensive, as it requires domain knowledge and expert involvement. Semi-supervised learning approaches that can make use of unlabeled data, in addition to small amounts of labeled data, can help reduce the costs associated with labeling. In this context, we focus on the problem of predicting splice sites in a genome using semi-supervised learning approaches. This is a challenging problem, due to the highly imbalanced distribution of the data, i.e., small number of splice sites as compared to the number of non-splice sites. To address this challenge, we propose to use ensembles of semi-supervised classifiers, specifically self-training and co-training classifiers. Results Our experiments on five highly imbalanced splice site datasets, with positive to negative ratios of 1-to-99, showed that the ensemble-based semi-supervised approaches represent a good choice, even when the amount of labeled data consists of less than 1% of all training data. In particular, we found that ensembles of co-training and self-training classifiers that dynamically balance the set of labeled instances during the semi-supervised iterations show improvements over the corresponding supervised ensemble baselines. Conclusions In the presence of limited amounts of labeled data, ensemble-based semi-supervised approaches can successfully leverage the unlabeled data to enhance supervised ensembles learned from highly imbalanced data distributions. Given that such distributions are common for many biological sequence classification problems, our work can be seen as a stepping stone towards more sophisticated ensemble-based approaches to biological sequence annotation in a semi-supervised framework. PMID:26356316

  18. Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3′-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

    2013-01-01

    The most commonly used 3′-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

  19. Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

    PubMed

    Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

    2013-01-01

    The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

  20. Splice-site mutation of the p53 gene in a family with hereditary breast-ovarian cancer.

    PubMed

    Jolly, K W; Malkin, D; Douglass, E C; Brown, T F; Sinclair, A E; Look, A T

    1994-01-01

    Germline mutations within evolutionary conserved exons of the p53 gene predispose to tumor development in several familial cancer syndromes. We now report identification of a novel p53 mutation affecting the splice acceptor site of exon 6 in the germline DNA of a family with hereditary breast-ovarian cancer. This splice-site mutation, which results in omission of exon 6 and creates a frame-shift and premature stop codon in transcripts from the mutant allele, was found in seven family members--four of whom have developed breast, ovarian or choroid plexus tumors before age 35. Our finding suggests the need to examine the entire p53 gene for splice-site, frame-shift, and nonsense (as well as missense) mutations in families with early-onset hereditary breast and breast-ovarian cancers not linked to the BRCA1 gene on chromosome 17q. We propose that the term 'p53 familial cancer syndrome' be applied to clusters of tumors in families with documented germline p53 mutations, regardless of the histopathologic findings or pattern of tumor development. PMID:8302608

  1. Reconstruction of complex defects of the parotid region using a lateral thoracic wall donor site.

    PubMed

    Biglioli, Federico; Pedrazzoli, Marco; Rabbiosi, Dimitri; Colletti, Giacomo; Colombo, Valeria; Frigerio, Alice; Autelitano, Luca

    2013-04-01

    Radical treatment of parotid neoplasms may lead to complex parotid defects that present functional and aesthetic reconstructive challenges. We report our experience using the lateral thoracic wall as a single donor site. Between 2003 and 2009, four patients with malignant tumours in the parotid gland underwent radical parotidectomy and simultaneous reconstruction using a perforator latissimus dorsi cutaneous free flap (de-epithelialized and entire skin paddle in two cases each). A thoracodorsal nerve graft was used in all cases to replace the intraglandular branches of the facial nerve. Costal grafts were used for mandibular reconstruction in two patients. All patients underwent postoperative physiotherapy. No donor-site complication occurred and all treatments achieved good aesthetic results. All patients recovered nearly complete symmetry at rest and partial facial mimetic function. The lateral thoracic wall is a good donor site for the reconstruction of complex parotid defects. PMID:23245945

  2. Cryptic splice site usage in exon 7 of the human fibrinogen Bβ-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon

    PubMed Central

    Spena, Silvia; Tenchini, Maria Luisa; Buratti, Emanuele

    2006-01-01

    In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bβ-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T—initially described in a patient suffering from congenital afibrinogenemia—is present, this SF2/ASF binding site is critical for cryptic 5′ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations. PMID:16611940

  3. Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia

    PubMed Central

    Kar, Anjana; Munjal, Sachin Dev; Sarangi, Aditya N.; Dalal, Ashwin; Aggarwal, Rakesh

    2015-01-01

    Background Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India. Methods DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1. Results Of 71 subjects with UH (68 male; median age [range], 26 [16–63] years; serum bilirubin 56 [26–219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein. Conclusion Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA

  4. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

    PubMed

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J; Setó-Salvia, Núria; Ryan, Natalie S; Morris, Huw R; Arber, Charles; Devine, Michael J; Houlden, Henry; Warner, Thomas T; Bushell, Trevor J; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J; Fox, Nick C; Rossor, Martin N; Hardy, John; Wray, Selina

    2015-09-15

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. PMID:26136155

  5. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT

    PubMed Central

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J.; Setó-Salvia, Núria; Ryan, Natalie S.; Morris, Huw R.; Arber, Charles; Devine, Michael J.; Houlden, Henry; Warner, Thomas T.; Bushell, Trevor J.; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J.; Fox, Nick C.; Rossor, Martin N.; Hardy, John; Wray, Selina

    2015-01-01

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. PMID:26136155

  6. Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

    PubMed Central

    Buratti, Emanuele; Chivers, Martin; Královičová, Jana; Romano, Maurizio; Baralle, Marco; Krainer, Adrian R.; Vořechovský, Igor

    2007-01-01

    Despite a growing number of splicing mutations found in hereditary diseases, utilization of aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences of 346 aberrant 5′splice sites (5′ss) that were activated by mutations in 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic 5′ss and mutations elsewhere activated 92 de novo 5′ss. Point mutations leading to cryptic 5′ss activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substitutions at position +5 were exclusively G>A transitions, which was largely attributable to high mutability rates of C/G>T/A. However, the frequency of point mutations at position +5 was significantly higher than that observed in the Human Gene Mutation Database, suggesting that alterations of this position are particularly prone to aberrant splicing, possibly due to a requirement for sequential interactions with U1 and U6 snRNAs. Cryptic 5′ss were best predicted by computational algorithms that accommodate nucleotide dependencies and not by weight-matrix models. Discrimination of intronic 5′ss from their authentic counterparts was less effective than for exonic sites, as the former were intrinsically stronger than the latter. Computational prediction of exonic de novo 5′ss was poor, suggesting that their activation critically depends on exonic splicing enhancers or silencers. The authentic counterparts of aberrant 5′ss were significantly weaker than the average human 5′ss. The development of an online database of aberrant 5′ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms. PMID:17576681

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

    PubMed Central

    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

    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

  8. A Splice-Site Mutation in a Retina-Specific Exon of BBS8 Causes Nonsyndromic Retinitis Pigmentosa

    PubMed Central

    Riazuddin, S. Amer; Iqbal, Muhammad; Wang, Yue; Masuda, Tomohiro; Chen, Yuhng; Bowne, Sara; Sullivan, Lori S.; Waseem, Naushin H.; Bhattacharya, Shomi; Daiger, Stephen P.; Zhang, Kang; Khan, Shaheen N.; Riazuddin, Sheikh; Hejtmancik, J. Fielding; Sieving, Paul A.; Zack, Donald J.; Katsanis, Nicholas

    2010-01-01

    Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies. PMID:20451172

  9. Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis.

    PubMed

    Belforte, Fiorella S; Citterio, Cintia E; Testa, Graciela; Olcese, María Cecilia; Sobrero, Gabriela; Miras, Mirta B; Targovnik, Héctor M; Rivolta, Carina M

    2016-01-01

    Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of

  10. Donor site healing dynamics: molecular, histological, and noninvasive imaging assessment in a porcine model.

    PubMed

    Mauskar, Neil A; Sood, Subeena; Travis, Taryn E; Matt, Sarah E; Mino, Matthew J; Burnett, Mary-Susan; Moffatt, Lauren T; Fidler, Philip; Epstein, Stephen E; Jordan, Marion H; Shupp, Jeffrey W

    2013-01-01

    Understanding the physiology of donor site healing will lead to advances in how these wounds are treated and may ultimately allow faster healing, more frequent autografting, and more effective care of the burn-injured patient. Unfortunately, a paucity of data exists regarding perfusion metrics over the course of donor site healing. Furthermore, there are no studies that interrelate indices of perfusion with the molecular and cellular processes of donor site healing. Male Duroc pigs were anesthetized and donor site wounds were created using a Zimmer dermatome at a depth of 0.060 inch (1.52 mm). Digital photographs, laser Doppler images, and punch biopsies were obtained before and after excision and on days 2, 4, 7, 9, 11, 14, and 16 until wounds were healed. RNA isolation was performed and quantitative polymerase chain reaction was used to examine differential gene expression over the time course. Formalin-fixed biopsies were embedded in paraffin, sectioned, stained, and examined. Wound surfaces were 83% re-epithelialized by day 16. Perfusion peaked on day 2 then declined, but it remained significantly elevated compared to before excision (P < .05). From day 9 onward, mean perfusion units were not significantly different from baseline (P < .05). Twenty-two representative genes were selected for examination. RNA expression of collagen, tenascin-cytoactin, inflammatory cytokines, remodeling enzymes, growth factors, and Wnt was increased. Inflammatory cells and cytokines were demonstrated histologically. Nuclei per high powered field peaked at day 7 and neodermal thickness increased daily to day 14. A novel porcine model for donor site wound healing that interrelates re-epithelilaizationand perfusion with molecular and cellular indices has been demonstrated. PMID:23511287

  11. An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element.

    PubMed Central

    Amarasinghe, A K; MacDiarmid, R; Adams, M D; Rio, D C

    2001-01-01

    P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro. PMID:11565747

  12. Tumour Transfer to Bone Graft Donor Site: A Case Report and Review of the Literature of the Mechanism of Seeding

    PubMed Central

    Dias, Richard G.; Carter, Simon R.; Grimer, Robert J.; Tillman, Roger M.

    2000-01-01

    Purpose. Transmission of malignant tumour cells to a bone graft donor site is a rare complication of bone grafting.We report a case of seeding of malignant fibrous histiocytoma from the femur to a pelvic bone graft donor site. Discussion. We review the literature, discuss the possible mechanism of tumour transfer and offer advice aimed at avoiding this complication. PMID:18521435

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

    PubMed Central

    Hamid, Fursham M.; Makeyev, Eugene V.

    2014-01-01

    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

  14. Structure, Dynamics, and Interaction of p54(nrb)/NonO RRM1 with 5' Splice Site RNA Sequence.

    PubMed

    Duvignaud, Jean-Baptiste; Bédard, Mikaël; Nagata, Takashi; Muto, Yutaka; Yokoyama, Shigeyuki; Gagné, Stéphane M; Vincent, Michel

    2016-05-10

    p54(nrb)/NonO is a nuclear RNA-binding protein involved in many cellular events such as pre-mRNA processing, transcription, and nuclear retention of hyper-edited RNAs. In particular, it participates in the splicing process by directly binding the 5' splice site of pre-mRNAs. The protein also concentrates in a nuclear body called paraspeckle by binding a G-rich segment of the ncRNA NEAT1. The N-terminal section of p54(nrb)/NonO contains tandem RNA recognition motifs (RRMs) preceded by an HQ-rich region including a threonine residue (Thr15) whose phosphorylation inhibits its RNA binding ability, except for G-rich RNAs. In this work, our goal was to understand the rules that characterize the binding of the p54(nrb)/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54(nrb)/NonO binds to the 5' splice site RNA. We have then determined the structure of the p54(nrb)/NonO RRM1 by liquid-state NMR which revealed the presence of a canonical fold (β1α1β2β3α2β4) and the conservation of aromatic amino acids at the protein surface. We also investigated the dynamics of this domain by NMR. The p54(nrb)/NonO RRM1 displays some motional properties that are typical of a well-folded protein with some regions exhibiting more flexibility (loops and β-strands). Furthermore, we determined the affinity of p54(nrb)/NonO RRM1 interaction to the 5' splice site RNA by NMR and fluorescence quenching and mapped its binding interface by NMR, concluding in a classical nucleic acid interaction. This study provides an improved understanding of the molecular basis (structure and dynamics) that governs the binding of the p54(nrb)/NonO RRM1 to one of its target RNAs. PMID:27064654

  15. The properties of the "ideal" donor site dressing: results of a worldwide online survey.

    PubMed

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

    2013-09-30

    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

  16. Which dressing do donor site wounds need?: study protocol for a randomized controlled trial

    PubMed Central

    2011-01-01

    Background Donor site wounds after split-skin grafting are rather 'standard' wounds. At present, lots of dressings and topical agents for donor site wounds are commercially available. This causes large variation in the local care of these wounds, while the optimum 'standard' dressing for local wound care is unclear. This protocol describes a trial in which we investigate the effectiveness of various treatment options for these donor site wounds. Methods A 14-center, six-armed randomized clinical trial is being carried out in the Netherlands. An a-priori power analysis and an anticipated dropout rate of 15% indicates that 50 patients per group are necessary, totaling 300 patients, to be able to detect a 25% quicker mean time to complete wound healing. Randomization has been computerized to ensure allocation concealment. Adult patients who need a split-skin grafting operation for any reason, leaving a donor site wound of at least 10 cm2 are included and receive one of the following dressings: hydrocolloid, alginate, film, hydrofiber, silicone dressing, or paraffin gauze. No combinations of products from other intervention groups in this trial are allowed. Optimum application and changes of these dressings are pursued according to the protocol as supplied by the dressing manufacturers. Primary outcomes are days to complete wound healing and pain (using a Visual Analogue Scale). Secondary outcomes are adverse effects, scarring, patient satisfaction, and costs. Outcome assessors unaware of the treatment allocation will assess whether or not an outcome has occurred. Results will be analyzed according to the intention to treat principle. The first patient was randomized October 1, 2009. Discussion This study will provide comprehensive data on the effectiveness of different treatment options for donor site wounds. The dressing(s) that will prevail in effectiveness, satisfaction and costs will be promoted among clinicians dealing with such patients. Thus, we aim to

  17. Alternative Splicing in CKD.

    PubMed

    Stevens, Megan; Oltean, Sebastian

    2016-06-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. PMID:26763787

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

    PubMed Central

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

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

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

    PubMed

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

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

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

    PubMed

    Du, Qiang; Zhang, Tianyu

    2014-08-01

    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

  1. Use of continuous local anesthetic infusion in the management of postoperative split-thickness skin graft donor site pain.

    PubMed

    Hernandez, Jorge L Reguero; Savetamal, Alisa; Crombie, Roselle E; Cholewczynski, Walter; Atweh, Nabil; Possenti, Paul; Schulz, John T

    2013-01-01

    Donor sites from split-thickness skin grafts (STSG) impose significant pain on patients in the early postoperative period. We report the use of continuous local anesthetic infusion as a method for the management of postoperative STSG donor site pain. Patients undergoing single or dual, adjacent STSG harvest from the thigh (eight patients) or back (one patient) were included in this study. Immediately after STSG harvest, subcutaneous catheters were placed for continuous infusion of local anesthetic. Daily donor site-specific pain severity scores were prospectively recorded in nine patients receiving local anesthetic infusion. Patient characteristics, technical aspects, and postoperative complications were identified in the study. The thigh was the anatomic location chosen for most donor sites. A single catheter was placed for donor sites limited to 4 inches in width or less. A dual catheter system was used for those wider than 4 inches. An elastomeric pump delivered continuously a total of 4 ml/hr of a solution of 0.5% bupivacaine. The average anesthetic infusion duration was 3.1 days. A substantial decrease in worst, least, and average donor site pain scores was found from the first 24 hours to the second postoperative day in our patients, a treatment trend that continued through postoperative day 3. One patient developed minor anesthetic leakage from the catheter insertion site; and in three cases, accidental dislodgement of the catheters occurred. There were no cases of donor site secondary infection. All donor sites were completely epithelialized at 1-month follow-up. Continuous local anesthetic infusion is technically feasible and may represent an option for postoperative donor site pain control after STSG harvesting. Relative cost-benefit of the technique remains to be determined. PMID:23271060

  2. Prospective biomechanical evaluation of donor site morbidity after radial forearm free flap.

    PubMed

    Riecke, Björn; Kohlmeier, Carsten; Assaf, Alexandre T; Wikner, Johannes; Drabik, Anna; Catalá-Lehnen, Philip; Heiland, Max; Rendenbach, Carsten

    2016-02-01

    Although the radial forearm free flap (RFF) is a commonly-used microvascular flap for orofacial reconstruction, we are aware of few prospective biomechanical studies of the donor site. We have therefore evaluated the donor site morbidity biomechanically of 30 consecutive RFF for orofacial reconstruction preoperatively and three months postoperatively. This included the Mayo wrist score, the Disabilities of the Arm, Shoulder and Hand (DASH) score, grip strength, followed by tip pinch, key pinch, palmar pinch, and range of movement of the wrist. Primary defects were all closed with local full-thickness skin grafts from the donor site forearm, thereby circumventing the need for a second defect. Postoperative functional results showed that there was a reduction in hand strength measured by (grip strength: -24.1%, in tip pinch: -23.3%, in key pinch: -16.5, and in palmar pinch: -19.3%); and wrist movement measured by extension (active=14.3% / passive= -11.5%) and flexion = -14.8% / -8.9%), and radial (-9.8% / -9.8%) and ulnar (-11.0% / -9.3%) abduction. The Mayo wrist score was reduced by 9.4 points (-12.9%) and the DASH score increased by 16.1 points (+35.5%) compared with the same forearm preoperatively. The local skin graft resulted in a robust wound cover with a good functional result. Our results show that the reduction in hand strength and wrist movement after harvest of a RFF is objectively evaluable, and did not reflect the subjectively noticed extent and restrictions in activities of daily living. Use of a local skin graft avoids a second donor site and the disadvantages of a split-thickness skin graft. PMID:26708799

  3. Free Flap Donor Site Reconstruction: A Prospective Case Series Using an Optimized Polyurethane Biodegradable Temporizing Matrix

    PubMed Central

    Wagstaff, Marcus J. D.; Schmitt, Bradley J.; Caplash, Yugesh

    2015-01-01

    Introduction: We recently published a 10-patient case series where free flap donor site reconstruction was performed as a 2-stage procedure using an integrating biodegradable polyurethane matrix (to form a neodermis), followed by definitive closure with an autologous split-skin graft. Two issues were revealed by this pilot study that led to further modification of the biodegradable temporizing matrix. This involved alterations to the seal thickness and bonding to the foam matrix and the introduction of fenestrations to the seal. Objective: This article documents a second cohort of patients requiring free flap (fibular and radial forearm) donor site reconstruction with this optimized material. Methods: The biodegradable temporizing matrix was implanted when the free flap was detached from its donor site. Subsequent integration was monitored closely. Five weeks was the usual time of integration before delamination (seal removal), dermabrasion, and definitive closure with autograft. Results: Integration was complete and uncomplicated in every case, delamination occurred in 1 piece in 1 action, and subsequent graft take was 100% for every patient. Long-term scar outcomes improved compared with the pilot group. Degradation is complete by 12 months, other than occasional microscopic remnants undergoing phagocytosis. Conclusion: This study has reiterated that the biodegradable temporizing matrix can be implanted into humans, followed by neovascularization and integration. No infection was observed, and split-skin overgrafting was successful and uncomplicated. PMID:26171099

  4. Donor site wound protein synthesis correlates with length of acute hospitalization in severely burned children: a pilot study

    PubMed Central

    Tuvdendorj, Demidmaa; Chinkes, David L.; Zhang, Xiao-Jun; Aarsland, Asle; Herndon, David N.

    2014-01-01

    Autografting of burn wounds results in generation of donor site wounds. Here we measured donor site wound protein Fractional Synthesis Rate (FSR) in a burn pediatric population and showed that FSR increases over time postsurgery and correlates with the length of hospital stay (LOS) normalized for total body surface area (TBSA) burn size. 3.9±1.1 days after the grafting surgery patients participated in a metabolic study consisting of continuous infusion of L-[ring-2H5]-phenylalanine and donor site wound punch biopsies. Donor site wound protein FSR was 10.4±7.5 %/day. Wound FSR demonstrated linear correlation with the time postsurgery (p < 0.05). Multiple regression analysis showed that LOS/TBSA correlated with donor site wound protein FSR and time postsurgery (p < 0.001) and the following equation describes the relationship: Estimated LOS/TBSA = (FSR - 12.95 – 1.414 × Postsurgery day)/(−17.8). This equation predicted that FSR corrected for the postsurgery day when the metabolic study was conducted accounted for 67 % of the variability (r2 = 0.673) in the LOS/TBSA. Donor site wound protein FSR correlated to LOS/TBSA of burn patients admitted to the intensive care unit. Measurement of protein deposition in regenerating donor site wound using stable isotope technique provides a quantitative measure of wound healing. PMID:20412556

  5. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    PubMed Central

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-01-01

    Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy. PMID:26325627

  6. Characterization of growth hormone enhanced donor site healing in patients with large cutaneous burns.

    PubMed Central

    Herndon, D N; Hawkins, H K; Nguyen, T T; Pierre, E; Cox, R; Barrow, R E

    1995-01-01

    BACKGROUND: Human growth hormone is an anabolic agent that attenuates injury-induced catabolism and stimulates protein synthesis. Recombinant human growth hormone (rhGH) administered therapeutically to patients with massive burns has been shown to increase the rate of skin graft donor site healing. It has been postulated that growth hormone affects wound healing and tissue repair by stimulating the production of insulin-like growth factor-1 (IGF-1) by the liver to increase circulating IGF-1 concentrations. The mechanism by which it improves wound healing, however, remains in question. The authors hypothesize that rhGH up-regulates IGF-1 receptors and IGF-1 levels both systemically and locally in the wound site to stimulate cell mitosis and increase synthesis of laminin, collagen types IV and VII, and cytokeratin. This hypothesis was tested in nine patients with burns covering > 40% of total body surface area. OBJECTIVE: The authors assessed the efficacy of rhGH in promoting several major building materials in the donor site of patients with massive burns. METHODS: Ten massively burned patients with full-thickness burns covering more than 40% of total body surface area were participants in a placebo-controlled prospective study to determine the efficacy of 0.2 mg/kg/day rhGH on donor site wound healing and to identify some of the major components involved in wound healing and its integrity. RESULTS: Donor sites in burn patients receiving rhGH showed an increased coverage by the basal lamina of 26% for placebo to 68% coverage of the dermal-epidermal junction. Insulin-like growth factor-1 receptors and laminin, types IV and VII collagen, and cytokeratin-14 all increased significantly. Healing times of the donor sites were significantly decreased compared with patients receiving placebo. CONCLUSION: Results indicate that growth hormone or its secondary mediators may directly stimulate the cells of the epidermis and dermis during wound healing to produce the structural

  7. Solid phase electron donors control denitrification in groundwater at agricultural sites

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

    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

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

    PubMed

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

    2015-01-01

    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

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

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.04986.001 PMID:25555158

  10. Identification of conserved RNA secondary structures at influenza B and C splice sites reveals similarities and differences between influenza A, B, and C

    PubMed Central

    2014-01-01

    Background Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection. Findings All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure. Conclusions When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3′ splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation. PMID:24405943

  11. A splice site mutation in HERC1 leads to syndromic intellectual disability with macrocephaly and facial dysmorphism: Further delineation of the phenotypic spectrum.

    PubMed

    Aggarwal, Shagun; Bhowmik, Aneek Das; Ramprasad, Vedam L; Murugan, Sakthivel; Dalal, Ashwin

    2016-07-01

    We report on a sib pair of Indian origin presenting with intellectual disability, dysmorphism, and macrocephaly. Exome sequencing revealed a homozygous splice site HERC1 mutation in both probands. Functional analysis revealed use of an alternate splice site resulting in formation of a downstream stop codon and nonsense mediated decay. In the light of recent reports of HERC1 mutations in two families with a similar phenotypic presentation, this report reiterates the pathogenic nature and clinical consequences of HERC1 disruption. © 2016 Wiley Periodicals, Inc. PMID:27108999

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

    SciTech Connect

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

    2008-11-07

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

  13. A Novel Local Autologous Bone Graft Donor Site After Scalp Tissue Expansion in Aplasia Cutis Congenita.

    PubMed

    Hadad, Ivan; Meara, John G; Rogers-Vizena, Carolyn R

    2016-06-01

    Aplasia cutis congenita (ACC) is a rare condition often presenting as an absent area of cutaneous scalp. The calvarium and dura may also be affected. Scalp reconstruction with tissue expansion is often needed for large defects. Patients involving deficient calvarial bone present a dilemma for the reconstructive surgeon, because bone graft donor sites are limited in young children.A thick, bony rim has been noted to form around the periphery of scalp tissue expanders. The authors present a series of 3 patients with ACC for whom this bony hyperostosis was used as donor particulate bone graft at the time of scalp tissue expansion. There was 85 to 100% graft ossification on postoperative computed tomography scan. There were no bone graft-related complications.In conclusion, the hyperostotic rim that forms after scalp tissue expansion can be successfully used as particulate bone graft, decreasing the number of procedures needed for patient with ACC and obviating the need for other donor sites. PMID:27192637

  14. U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs.

    PubMed

    Jenkins, Jermaine L; Agrawal, Anant A; Gupta, Ankit; Green, Michael R; Kielkopf, Clara L

    2013-04-01

    Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65). PMID:23376934

  15. Iliac Crest Donor Site for Children With Cleft Lip and Palate Undergoing Alveolar Bone Grafting: A Long-term Assessment.

    PubMed

    Wheeler, Jonathan; Sanders, Megan; Loo, Stanley; Moaveni, Zac; Bartlett, Glenn; Keall, Heather; Pinkerton, Mark

    2016-05-01

    The authors aimed to accurately assess the donor site morbidity from iliac crest bone grafts for secondary bone grafting in patients with cleft lip and palate alveolar defects. Fifty patients between 3 months and 10 years following alveolar bone grafting for cleft lip and palate were entered into the study. Two-thirds of patients had no significant concerns about the donor site. The remaining third had some concerns about the appearance of their hips and less than 10% of patients expressing strong agreement with statements about concerns with shape, appearance, and self-consciousness about the iliac crest donor site. Examination findings showed the average length of scar being 5.4 cm and a third of patients having some minor palpable boney irregularities of the iliac crest. The authors found that the alveolar crest donor site is well tolerated by patients long term but has a measurable morbidity long term. PMID:27035602

  16. The missing puzzle piece: splicing mutations

    PubMed Central

    Lewandowska, Marzena A

    2013-01-01

    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

  17. A novel BTK gene mutation creates a de-novo splice site in an X-linked agammaglobulinemia patient.

    PubMed

    Chear, Chai Teng; Ripen, Adiratna Mat; Mohamed, Sharifah Adlena Syed; Dhaliwal, Jasbir Singh

    2015-04-15

    Bruton's tyrosine kinase (BTK), encoded by the BTK gene, is a cytoplasmic protein critical in B cell development. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA), a primary immunodeficiency with characteristically low or absent B cells and antibodies. This report describes a five year-old boy who presented with otitis externa, arthritis, reduced immunoglobulins and no B cells. Flow cytometry showed undetectable monocyte BTK expression. Sequencing revealed a novel mutation at exon 13 of the BTK gene which created a de novo splice site with a proximal 5 nucleotide loss resulting in a truncated BTK protein. The patient still suffered from ear infection despite intravenous immunoglobulin replacement therapy. In this study, mosaicism was seen only in the mother's genomic DNA. These results suggest that a combination of flow cytometry and BTK gene analysis is important for XLA diagnosis and carrier screening. PMID:25680287

  18. Rhodopsin mutations in a Scottish retinitis pigmentosa population, including a novel splice site mutation in intron four.

    PubMed Central

    Bell, C; Converse, C A; Hammer, H M; Osborne, A; Haites, N E

    1994-01-01

    Retinitis pigmentosa (RP) is the name given to a group of disorders, both clinically and genetically heterogeneous, that primarily affect the photoreceptor function of the eye. Mutations in the genes encoding for rhodopsin, RDS-peripherin, or the beta subunit of the cGMP phosphodiesterase enzyme can be responsible for the phenotype. In this study the rhodopsin gene has been screened for mutations in a panel of RP individuals and five different sequence changes have been detected to date in three dominantly inherited and two unclassified families. One of these, a base substitution in the 3'UTR, has not yet been confirmed as disease specific, while three missense substitutions have previously been reported and are likely to be responsible for the phenotype. The fifth change, a base substitution at the intron 4 acceptor splice site, represents a novel mutation and is assumed to be the causative mutation. Images PMID:7819178

  19. Activation of a cryptic splice site in the growth hormone receptor associated with growth hormone insensitivity syndrome in a genetic isolate of Laron Syndrome

    SciTech Connect

    Schiavi, A.; Bartlett, R.; Brown, M.

    1994-09-01

    Laron syndrome (LS) is a rare, autosomal recessive disease found worldwide. Despite various ethnic differences, all patients with LS described display classic dysmorphic features and extreme short stature due to defects in the growth hormone receptor (GHR). The vast majority of these patients are sporadic occurrences resulting from consanguineous matings; however, an Ecuadorian genetic isolate of LS has been reported. Our investigations have identified a genetic isolate of LS of Anglo Saxon origin. Seven individuals, by all clinical and biochemical criteria, have LS. As a result of extensive review of family and medical histories we have constructed a pedigree tracing the lineage of our affected patients through the 17th century. No GHR gross deletions were detected using an exon-specific PCR assay developed in our laboratory. Previous molecular analyses have identified mutations in exons 2-7 in numerous patients with classical LS. Single strand conformational polymorphism (SSCP) analysis was performed on GHR exons 2-7, and a marked conformational shift was noted in exon 7. Cycle sequencing of exon 7 from three affected individuals, and from four first-degree relatives, revealed a C{r_arrow}T transition at position 766 of the cDNA, and a heterozygous C{r_arrow}T transition at the identical position in the obligate carriers studied. This mutation is predicted to activate a cryptic donor splice site 63 base pairs upstream from the 3{prime} end of exon 7, effectively truncating the GHR cDNA without changing the reading frame. The resultant GHR protein is shortened by a proposed 21 amino acids. The identification and conformation of this mutation not only identifies a novel mutation in the GHR, and the first to be described in LS patients of English descent, but also allows for comparisons between genotypes and phenotypes in an inbred population.

  20. Identification of novel splice site mutation IVS9 + 1(G > A) and novel complex allele G355R/R359X in Type 1 Gaucher patients heterozygous for mutation N370S.

    PubMed

    Hoitsema, Kourtnee; Amato, Dominick; Khan, Aneal; Sirrs, Sandra; Choy, Francis Y M

    2016-09-01

    Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. More than 350 mutations that cause Gaucher disease have been described to date. Novel mutations can potentially provide insight into the glucocerebrosidase structure-function relationship and biochemical basis of the disease. Here, we report the identification of two novel mutations in two unrelated patients with type I (non-neuronopathic) Gaucher disease: 1) a splice site mutation IVS9 + 1G > A; and (2) a complex allele (cis) G355R/R359X. Both patients have a common N370S mutation in the other allele. The splice site mutation results from an intronic base substitution (G to A, c.1328 + 1, g.5005) at the donor splice site of exon and intron 9. The complex allele results from two point mutations in exon 8 of glucocerebrosidase (G to C at c.1180, g.4396, and T to C at c. 1192, g.4408) substituting glycine by arginine (G355R) and arginine by a premature termination (R359X), respectively. In order to demonstrate that G355R/R359X are in cis arrangement, PCR-amplified glucocerebrosidase exon 8 genomic DNA from the patient was cloned into the vector pJET1.2 in Escherichia coli TOP10® strain. Out of the 15 clones that were sequence analyzed, 10 contained the normal allele sequence and 5 contained the complex allele G355R/R359X sequence showing both mutations in cis arrangement. Restriction fragment length polymorphism analysis using Hph1 restriction endonuclease digest was established for the IVS9 + 1G > A mutation for confirmation and efficient identification of this mutation in future patients. Past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in Gaucher complex alleles are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings. PMID:27222815

  1. Identification of novel splice site mutation IVS9 + 1(G > A) and novel complex allele G355R/R359X in Type 1 Gaucher patients heterozygous for mutation N370S☆

    PubMed Central

    Hoitsema, Kourtnee; Amato, Dominick; Khan, Aneal; Sirrs, Sandra; Choy, Francis Y.M.

    2016-01-01

    Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. More than 350 mutations that cause Gaucher disease have been described to date. Novel mutations can potentially provide insight into the glucocerebrosidase structure–function relationship and biochemical basis of the disease. Here, we report the identification of two novel mutations in two unrelated patients with type I (non-neuronopathic) Gaucher disease: 1) a splice site mutation IVS9 + 1G > A; and (2) a complex allele (cis) G355R/R359X. Both patients have a common N370S mutation in the other allele. The splice site mutation results from an intronic base substitution (G to A, c.1328 + 1, g.5005) at the donor splice site of exon and intron 9. The complex allele results from two point mutations in exon 8 of glucocerebrosidase (G to C at c.1180, g.4396, and T to C at c. 1192, g.4408) substituting glycine by arginine (G355R) and arginine by a premature termination (R359X), respectively. In order to demonstrate that G355R/R359X are in cis arrangement, PCR-amplified glucocerebrosidase exon 8 genomic DNA from the patient was cloned into the vector pJET1.2 in Escherichia coli TOP10® strain. Out of the 15 clones that were sequence analyzed, 10 contained the normal allele sequence and 5 contained the complex allele G355R/R359X sequence showing both mutations in cis arrangement. Restriction fragment length polymorphism analysis using Hph1 restriction endonuclease digest was established for the IVS9 + 1G > A mutation for confirmation and efficient identification of this mutation in future patients. Past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in Gaucher complex alleles are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings. PMID:27222815

  2. Novel compound heterozygous Thyroglobulin mutations c.745+1G>A/c.7036+2T>A associated with congenital goiter and hypothyroidism in a Vietnamese family. Identification of a new cryptic 5' splice site in the exon 6.

    PubMed

    Citterio, Cintia E; Morales, Cecilia M; Bouhours-Nouet, Natacha; Machiavelli, Gloria A; Bueno, Elena; Gatelais, Frédérique; Coutant, Regis; González-Sarmiento, Rogelio; Rivolta, Carina M; Targovnik, Héctor M

    2015-03-15

    Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein

  3. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    PubMed

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  4. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

    PubMed

    O'Neill, J P; Rogan, P K; Cariello, N; Nicklas, J A

    1998-11-01

    The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are

  5. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  6. Analysis of a splice-site mutation in the sap-precursor gene of a patient with metachromatic leukodystrophy

    SciTech Connect

    Henseler, M.; Klein, A.; Reber, M.

    1996-01-01

    Sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins required for the lysosomal degradation of various sphingolipids with a short oligosaccharide chain by their exohydrolases. Four of the five known activator proteins (sap-A-sap-D), also called {open_quotes}saposins,{close_quotes} are derived from a common precursor by proteolytic processing. sap-B stimulates hydrolysis of sulfatides by arylsulfatase A in vivo. Its recessively inherited deficiency results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is caused by a defect of arylsulfatase A. Here we report on a patient with sap-B deficiency. Reverse-transcription-PCR studies on the patient`s mRNA revealed the occurrence of two distinct mutant species: one with an in-frame deletion of the first 21 bases of exon 6, the other with a complete in-frame deletion of this exon. The patient was homozygous for the underlying mutation, which was found to be a G-{yields}T transversion within the acceptor splice site between intron e and exon 6, abolishing normal RNA splicing. Allele-specific oligonucleotide hybridization revealed that the parents and both grandfathers of the patient were carriers of this mutation. In order to analyze the fate of the mutant precursor proteins, both abnormal cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments showed that the deletion of 21 bp had no effect on the transport and the maturation of the encoded precursor. All sap forms except sap-B were detectable by immunochemical methods. The cDNA bearing a complete deletion of exon 6 encoded a shortened precursor of only 60 kD, and no mature SAPs were detectable. The carbohydrate chains of this polypeptide were of the high-mannose and hybrid type, indicating no transport of the mutant precursor beyond early Golgi apparatus. An endoplasmic-reticulum localization of this polypeptide was supported by indirect immunofluorescence analysis. 31 refs., 8 figs.

  7. Rapid generation of splicing reporters with pSpliceExpress

    PubMed Central

    Kishore, Shivendra; Khanna, Amit; Stamm, Stefan

    2008-01-01

    Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo. PMID:18930792

  8. 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

    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

    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

  9. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.

    PubMed

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-09-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

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

    PubMed

    Slavov, D; Gardiner, K

    2002-10-16

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

  11. An anatomic comparison of the skin of five donor sites for dermal fat graft.

    PubMed

    Hwang, K; Kim, D J; Lee, I J

    2001-03-01

    Kim performed more than 3,000 augmentation rhinoplasties using the dermal fat graft. He preferred the sacral area as the donor site over other areas because the dermis is thick and the fat is more compact. The authors conducted a comparative study of the thickness of the epidermis and dermis, and the numbers of fibroblasts and fibrocytes in the dermis of the abdominal wall, groin, lateral gluteal area, gluteal fold, and sacrum of 7 adult cadavers. The sacrum had the thickest epidermis (86.1 +/- 7.8 microm) and dermis (1,510.7 +/- 201.7 microm), and the groin had the thinnest epidermis (57.3 +/- 22.9 microm) and dermis (783.3 +/- 244.5 microm). The dermal thickness of the abdomen, lateral gluteal area, and gluteal fold was 913.3 +/- 271.7 microm, 1,018.7 +/- 305.6 microm, and 1,107.0 +/- 272.6 microm respectively. The sacral dermis was significantly thicker than the other four sites (p < 0.008), and the groin dermis was the thinnest (p < 0.039). The number of fibroblasts and fibrocytes in the sacral area and the gluteal skin folds was significantly higher than the other areas (p < 0.05). The sacral area, gluteal fold, and lateral gluteal region had relatively thicker panniculus adiposus than the abdomen and groin. The panniculus adiposus of the sacral skin was especially well developed and was comprised of several compact layers that were connected by parallel, thick collagen fibers. The authors conclude that the sacral skin is a suitable donor site for dermal grafting because its dermis has more fibroblasts and fibrocytes than the other areas studied, and its dermis is more viable and durable. PMID:11293528

  12. MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery

    PubMed Central

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J.; Huang, Yan; Savich, Gleb L.; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A.; Perou, Charles M.; MacLeod, James N.; Chiang, Derek Y.; Prins, Jan F.; Liu, Jinze

    2010-01-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  13. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.

    PubMed

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J; Huang, Yan; Savich, Gleb L; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A; Perou, Charles M; MacLeod, James N; Chiang, Derek Y; Prins, Jan F; Liu, Jinze

    2010-10-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥ 75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  14. Lipoaugmentation of the vocal folds: a survey on alternative donor sites for graft harvesting.

    PubMed

    Prodinger, Peter Michael; Windisch, Gunther; Hammer, Georg Philipp; Anderhuber, Friedrich; Friedrich, Gerhard

    2009-09-01

    Lipoaugmentation is a treatment option for patients suffering from glottic insufficiency. Autologous fat is a nearly ideal material for vocal-fold augmentation from the view of biocompatibility and viscoelasticity, but there is still the problem of high graft resorption. As distribution and biological behavior of fatty tissue is very different in the human body, the aim of the study was to elucidate possible donor sites with respect to the quantity of harvested fat, the surgical accessibility to the region, the donor site morbidity and possibility of aesthetic defects and the quality of harvested tissue. Possible donor sites for harvesting were examined by magnetic resonance imaging in thirty-five patients with special emphasis to the buccal fat pad, the neck, the dorsolateral side of the proximal upper extremity, the subcutaneous layer of the abdominal wall, the superficial trochanteric region, the medial thigh, and the infrapatellar fat pad. Identified regions that failed to be chosen into consideration because of an elaborate surgical approach (superficial axillary's space, ischio-anal fossa, subcutaneous layer of buttock, popliteal fossa) were not taken into consideration. The mean volume of the buccal fat was 3.994 cm(3); the average thickness of the fat at the level of C7 was 1.721 cm, the mean value in the upper extremities was 1.913 cm laterally and 1.275 cm dorsally. The subcutaneous fat of the abdominal wall was divided into a superficial compartment (mean: 1.527 cm) and a deep one (average: 3.545 cm). In the superficial trochanteric region, the mean thickness was 2.536 cm, in the medial thigh 2.127 cm; the mean volume of the infrapatellar fat pad was 20.198 cm(3). All regions of interest showed reproducible and sufficient amounts of harvestable tissue, we found significant intersexual differences in dorsolateral side of the upper arm, subcutaneous layer of the abdominal wall and superficial trochanteric region. When harvesting subcutaneous tissue of the

  15. Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

    PubMed Central

    Alexandersen, S; Carpenter, S; Christensen, J; Storgaard, T; Viuff, B; Wannemuehler, Y; Belousov, J; Roth, J A

    1993-01-01

    The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown. Images PMID:8380084

  16. [Cosmesis and Body Image after Laparo-Endoscopic Single Site Donor Nephrectomy].

    PubMed

    Yanishi, Masaaki; Kinoshita, Hidefumi; Yoshida, Takashi; Takayasu, Kenta; Mishima, Takao; Yoshida, Kenji; Sugi, Motohiko; Kawa, Gen; Matsuda, Tadashi

    2015-07-01

    Using a questionnaire, we objectively assessed the body image of donors who underwent conventional laparoscopic donor nephrectomy (L-DN) or laparoscopic single-site donor nephrectomy (LESS-DN). Subjects were 15 patients who underwent an L-DN and 15 who underwent an LESS-DN. The questionnaire consisted of the Body Image Questionnaire (BIQ), including a Body Image Scale (BIS) and Cosmetic Scale (CS), and a Photo-Series Questionnaire (PSQ). A higher score indicated a more favorable assessment, and patient scores were compared. Subjects were also asked which procedure they preferred if they had to undergo donor nephrectomy again. Pain was assessed by comparing the number of times an analgesic was administered during hospitalization. The average BIS score was 18.7 points (out of 20) for patients who underwent an L-DN and 19.5 points for patients who underwent an LESS-DN ; those who underwent an LESS-DN had a significantly higher score (p=0.03). Patients who underwent an L-DN had a median CS score of 17.5 points (out of 24) while patients who underwent an LESS-DN had a median CS score of 19.1 points ; those who underwent an LESS-DN had a higher score, but the difference in average CS scores was not significant (p=0.123). The average PSQ score was 7.1 points for patients who underwent an L-DN and 8.8 points for patients who underwent an LESS-DN ; the higher score for LESS-patients was statistically significant (p=0.01). Patients who underwent an L-DN were administered an analgesic a median of 4 times during hospitalization (range : 3-10 times) while patients who underwent an LESS-DN were administered an analgesic a median of 2 times (range : 0-4 times), which was significantly less (p=0.01). Patients who underwent LESS-DN had a better body image and better cosmetic appearance than those who underwent LDN, thus indicating the usefulness of LESS-DN. However, a more prospective larger study needs to be performed. PMID:26278210

  17. Novel method of laparoendoscopic single-site and natural orifice specimen extraction for live donor nephrectomy: single-port laparoscopic donor nephrectomy and transvaginal graft extraction

    PubMed Central

    Jeong, Won Jun; Choi, Byung Jo; Hwang, Jeong Kye; Yuk, Seung Mo; Song, Min Jong

    2016-01-01

    Laparoscopic live donor nephrectomy (DN) has been established as a useful alternative to the traditional open methods of procuring kidneys. To maximize the advantages of the laparoendoscopic single-site (LESS) method, we applied natural orifice specimen extraction to LESS-DN. A 46-year-old woman with no previous abdominal surgery history volunteered to donate her left kidney to her husband and underwent single-port laparoscopic DN with transvaginal extraction. The procedure was completed without intraoperative complications. The kidney functioned well immediately after transplantation, and the donor and recipient were respectively discharged 2 days and 2 weeks postoperatively. Single-port laparoscopic DN and transvaginal graft extraction is feasible and safe. PMID:26878020

  18. Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

    PubMed Central

    Fukao, T; Yamaguchi, S; Wakazono, A; Orii, T; Hoganson, G; Hashimoto, T

    1994-01-01

    We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. Images PMID:7907600

  19. No-drain DIEP Flap Donor-site Closure Using Barbed Progressive Tension Sutures

    PubMed Central

    Nagarkar, Purushottam; Lakhiani, Chrisovalantis; Cheng, Angela; Lee, Michael; Teotia, Sumeet

    2016-01-01

    Background: The use of progressive tension sutures has been shown to be comparable to the use of abdominal drains in abdominoplasty. However, the use of barbed progressive tension sutures (B-PTSs) in deep inferior epigastric artery perforator (DIEP) flap donor-site closure has not been investigated. Methods: A retrospective chart review was performed on patients with DIEP flap reconstruction in a 3-year period at 2 institutions by 2 surgeons. Patients were compared by method of DIEP donor-site closure. Group 1 had barbed running progressive tension sutures without drain placement. Group 2 had interrupted progressive tension closure with abdominal drain placement (PTS-AD). Group 3 had closure with only abdominal drain placement (AD). Data collected included demographics, perioperative data, and postoperative outcomes. Results: Seventy-five patients underwent DIEP reconstruction (25 B-PTS, 25 PTS-AD, and 25 AD). Patient characteristics—age, body mass index, comorbidities, smoking status, and chemotherapy—were not significantly different between groups. Rate of seroma was 1.3% (B-PTS = 0%, PTS-AD = 4%, AD = 0%), wound dehiscence 16% (B-PTS = 8%, PTS-AD = 16%, AD = 24%), and umbilical necrosis 5.3% (B-PTS = 0%, PTS-AD = 0%, AD = 16%). No hematomas were observed in any patients. No statistically significant difference was found between complication rates across groups. Conclusions: Use of B-PTSs for abdominal closure after DIEP flap harvest can obviate the need for abdominal drains. Complication rates following this technique are not significantly different from closure using progressive tension suture and abdominal drain placement. This practice can prevent the use of abdominal drains, which can promote patient mobility, increase independence upon discharge, and contribute to patient satisfaction. PMID:27200234

  20. A Biodegradable Polyurethane Dermal Matrix in Reconstruction of Free Flap Donor Sites: A Pilot Study

    PubMed Central

    Wagstaff, Marcus J.D.; Schmitt, Bradley J.; Coghlan, Patrick; Finkemeyer, James P.; Caplash, Yugesh

    2015-01-01

    We have developed a biodegradable temporizing matrix (BTM) capable of supporting secondary split-skin graft-take in animal studies. We report its first long-term implantation and use as a dermal scaffold in humans. This preliminary study assesses its ability to integrate, its ease of delamination, its ability to sustain split-skin graft in complex wounds, the degree of wound contraction, and ultimately the quality of the scar at 1 year postimplantation. Ten patients were recruited, each requiring elective free flap reconstruction. Free flap donor sites created were anterolateral thigh flaps, fibular osseocutaneous flaps, or radial/ulnar forearm (RF/UF) flaps. The BTM was implanted when the flap was detached from its donor site. Dressing changes were performed twice weekly. The time elapsed between implantation and delamination depended on the type of flap and thus the wound bed left. Once integrated, the BTMs were delaminated in theatre, and the surface of the “neodermis” was refreshed by dermabrasion, prior to application of a split-skin graft. The BTM integration occurred in all patients (100% in 6 patients, with 90%, 84%, 76%, and 60% integration in the remainder). Integrated BTM sustained successful graft-take in all patients. Complete take was marred in 2 patients, over areas of BTM that had not integrated and graft application was performed too early. The BTM can be applied into wounds in humans and can integrate, persist in the presence of infection, and sustain split-skin overgrafting, despite the trial group presenting with significant comorbidities. PMID:25987938

  1. A biodegradable polyurethane dermal matrix in reconstruction of free flap donor sites: a pilot study.

    PubMed

    Wagstaff, Marcus J D; Schmitt, Bradley J; Coghlan, Patrick; Finkemeyer, James P; Caplash, Yugesh; Greenwood, John E

    2015-01-01

    We have developed a biodegradable temporizing matrix (BTM) capable of supporting secondary split-skin graft-take in animal studies. We report its first long-term implantation and use as a dermal scaffold in humans. This preliminary study assesses its ability to integrate, its ease of delamination, its ability to sustain split-skin graft in complex wounds, the degree of wound contraction, and ultimately the quality of the scar at 1 year postimplantation. Ten patients were recruited, each requiring elective free flap reconstruction. Free flap donor sites created were anterolateral thigh flaps, fibular osseocutaneous flaps, or radial/ulnar forearm (RF/UF) flaps. The BTM was implanted when the flap was detached from its donor site. Dressing changes were performed twice weekly. The time elapsed between implantation and delamination depended on the type of flap and thus the wound bed left. Once integrated, the BTMs were delaminated in theatre, and the surface of the "neodermis" was refreshed by dermabrasion, prior to application of a split-skin graft. The BTM integration occurred in all patients (100% in 6 patients, with 90%, 84%, 76%, and 60% integration in the remainder). Integrated BTM sustained successful graft-take in all patients. Complete take was marred in 2 patients, over areas of BTM that had not integrated and graft application was performed too early. The BTM can be applied into wounds in humans and can integrate, persist in the presence of infection, and sustain split-skin overgrafting, despite the trial group presenting with significant comorbidities. PMID:25987938

  2. Erythema persists longer than one year in split-thickness skin graft donor sites.

    PubMed

    Danielsen, Patricia L; Jorgensen, Lars N; Jørgensen, Bo; Karlsmark, Tonny; Agren, Magnus S

    2013-05-01

    The recovery of skin function and appearance after harvest of split-thickness skin autografts is incompletely described. We followed the kinetics of skin restoration after a partial-thickness skin excision relative to adjacent normal skin over 12 months. Standardized donor site wounds were made on the thigh using a pneumatic dermatome in 19 consecutive Caucasian patients, median age 70 years, age range 44-86 years, who were undergoing skin graft surgery for leg ulcers. Transepidermal water loss (TEWL), erythema and pigmentation were measured quantitatively using non-invasive devices. The macroscopically healed wound was compared with adjacent normal skin at 1, 3 and 12 months. At 1 month postoperatively, TEWL was 108% (p = 0.003), erythema 145% (p < 0.0005) and pigmentation 24% (p < 0.001) higher in the wounds compared with adjacent uninjured skin. The corresponding values at 3 months were 48% (p = 0.015), 89% (p < 0.0005) and 15% (p < 0.0005). After 12 months, erythema was elevated by 36% (p < 0.0005), while TEWL (p = 0.246) and pigmentation (p = 0.211) had returned to same levels as in the surrounding normal skin. Diabetes mellitus (p = 0.024) and smoking (p = 0.017) were associated with increased TEWL of normal skin, and erythema decreased with age (rs = -0.53, p = 0.020). In conclusion, erythema appears to be the significant component contributing to long-term postoperative donor site appearance. We hypothesize that this is due to increased microvasculature. PMID:22987230

  3. Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence.

    PubMed

    Hancock, D B; Reginsson, G W; Gaddis, N C; Chen, X; Saccone, N L; Lutz, S M; Qaiser, B; Sherva, R; Steinberg, S; Zink, F; Stacey, S N; Glasheen, C; Chen, J; Gu, F; Frederiksen, B N; Loukola, A; Gudbjartsson, D F; Brüske, I; Landi, M T; Bickeböller, H; Madden, P; Farrer, L; Kaprio, J; Kranzler, H R; Gelernter, J; Baker, T B; Kraft, P; Amos, C I; Caporaso, N E; Hokanson, J E; Bierut, L J; Thorgeirsson, T E; Johnson, E O; Stefansson, K

    2015-01-01

    We conducted a 1000 Genomes-imputed genome-wide association study (GWAS) meta-analysis for nicotine dependence, defined by the Fagerström Test for Nicotine Dependence in 17 074 ever smokers from five European-ancestry samples. We followed up novel variants in 7469 ever smokers from five independent European-ancestry samples. We identified genome-wide significant association in the alpha-4 nicotinic receptor subunit (CHRNA4) gene on chromosome 20q13: lowest P=8.0 × 10(-9) across all the samples for rs2273500-C (frequency=0.15; odds ratio=1.12 and 95% confidence interval=1.08-1.17 for severe vs mild dependence). rs2273500-C, a splice site acceptor variant resulting in an alternate CHRNA4 transcript predicted to be targeted for nonsense-mediated decay, was associated with decreased CHRNA4 expression in physiologically normal human brains (lowest P=7.3 × 10(-4)). Importantly, rs2273500-C was associated with increased lung cancer risk (N=28 998, odds ratio=1.06 and 95% confidence interval=1.00-1.12), likely through its effect on smoking, as rs2273500-C was no longer associated with lung cancer after adjustment for smoking. Using criteria for smoking behavior that encompass more than the single 'cigarettes per day' item, we identified a common CHRNA4 variant with important regulatory properties that contributes to nicotine dependence and smoking-related consequences. PMID:26440539

  4. A novel splice site mutation in the GNPTAB gene in an Iranian patient with mucolipidosis II α/β.

    PubMed

    Hashemi-Gorji, Feyzollah; Ghafouri-Fard, Soudeh; Salehpour, Shadab; Yassaee, Vahid Reza; Miryounesi, Mohammad

    2016-08-01

    Mucolipidosis type II α/β (ML II α/β) and mucolipidosis type III α/β (ML III α/β) have been shown to be caused by an absence or reduced level of uridine diphosphate (UDP)-N-acetylglucosamine-1-phosphotransferase enzyme (EC 2.7.8.17) activity, respectively. Both disorders are caused by mutations in the GNPTAB gene and are inherited in an autosomal recessive manner. Here we report a 2-year-old female patient being diagnosed as a case of ML II α/β due to coarse face, severe developmental delay, multiple dysostosis, noticeable increase of multiple lysosomal enzymes activity in plasma and normal acid mucopolysaccharides in urine. Mutational analysis of the GNPTAB gene has revealed a novel homozygous mutation in the patient (c.3250-2A>G) with both parents being heterozygote. Transcript analyses showed that this novel splice site mutation leads to exon 17 skipping and a frameshift afterwards (p.P1084_R1112del F1113Vfs*1). Consequently, we confirmed the association of this mutation with ML II α/β. Our finding expands the number of reported cases of this rare metabolic disorder and adds to the GNPTAB mutation database. PMID:27180337

  5. Donor-Site Morbidity Following Free Tissue Harvest from the Thigh: A Systematic Review and Pooled Analysis of Complications.

    PubMed

    Lakhiani, Chrisovalantis; DeFazio, Michael V; Han, Kevin; Falola, Reuben; Evans, Karen

    2016-06-01

    Background Donor-site morbidity significantly influences patient satisfaction and quality of life following reconstructive surgery. The relevant donor-site morbidities associated with more commonly utilized thigh-based flaps are reviewed. Methods A systematic search of the MEDLINE and Cochrane databases from 1994 to 2014 was conducted to identify all reports of "anterolateral thigh (ALT)," "vastus lateralis (VL)," "anteromedial thigh (AMT)," "transverse upper gracilis (TUG)," tensor fascia latae (TFL)," "gracilis," and "rectus femoris (RF)," flaps. Only studies that investigated donor-site outcomes related to pain, paresthesia, wound dehiscence, infection, hematoma, seroma, contour deformity, and/or objective functional performance were included. Case series or anecdotal reports with less than five flaps, non-English, and animal studies were excluded. Results A total of 116 articles representing 4,554 flaps were reviewed, including 2,922 ALT/VL, 148 AMT, 436 TUG, 278 TFL, 527 gracilis, and 243 RF flaps. The most frequently cited donor-site complication was paresthesia (range: 0-36.4%), followed by wound dehiscence (range: 0.9-8.3%), contour deformity (range: 0-5.2%), pain (range: 0-6.3%), and seroma (range: 0.4-2.0%). Despite mixed results regarding functional performance, pooled-analysis of dynamometric studies demonstrated a significant reduction in strength only after RF flap harvest (21%). Conclusions Donor-site morbidity for thigh-flaps is minimal and appears to be well-tolerated by the majority of patients. Nevertheless, the appropriate flap selection is highly individualized, and patients must be informed of potential complications and morbidities specific to each flap. We have established the most current review of donor-site morbidity for thigh-based flaps to aid the surgeon in this important discussion. PMID:27144952

  6. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  7. In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3' splice site in the intron excision reaction.

    PubMed Central

    Bufardeci, E; Fabbri, S; Baldi, M I; Mattoccia, E; Tocchini-Valentini, G P

    1993-01-01

    During processing of intron-containing pre-tRNAs, the Xenopus laevis splicing endonuclease binds the precursor and cleaves it at both the 5' and 3' splice sites. In vitro selection was used to determine structural features characteristic of precursor tRNA molecules that are active in this reaction. We performed two types of selection, one for molecules that are not cut, the other for molecules that are cut at only one site. The results shed light on various aspects of the intron excision reaction, including the importance of the three-dimensional structure of the mature domain for recognition and binding of the enzyme, the active role played by the single-stranded region of the intron, and the importance of the cardinal positions which, although not necessarily occupied by the same base in all precursors, nevertheless play a fundamental role in the splicing reaction. A precursor can be cut at the 3' site if a base in the single-stranded loop of the intron is allowed to pair (A-I pair) with the base of the 5' exon situated at the position immediately following the anticodon stem [first cardinal position (CP1)]. The nature of the bases involved in the A-I pair is important, as is the position of the base in the single-stranded loop of the intron. We discuss the role of the cardinal positions in the reaction. Images PMID:8223479

  8. HS3D, A Dataset of Homo Sapiens Splice Regions, and its Extraction Procedure from a Major Public Database

    NASA Astrophysics Data System (ADS)

    Pollastro, Pasquale; Rampone, Salvatore

    The aim of this work is to describe a cleaning procedure of GenBank data, producing material to train and to assess the prediction accuracy of computational approaches for gene characterization. A procedure (GenBank2HS3D) has been defined, producing a dataset (HS3D - Homo Sapiens Splice Sites Dataset) of Homo Sapiens Splice regions extracted from GenBank (Rel.123 at this time). It selects, from the complete GenBank Primate Division, entries of Human Nuclear DNA according with several assessed criteria; then it extracts exons and introns from these entries (actually 4523 + 3802). Donor and acceptor sites are then extracted as windows of 140 nucleotides around each splice site (3799 + 3799). After discarding windows not including canonical GT-AG junctions (65 + 74), including insufficient data (not enough material for a 140 nucleotide window) (686 + 589), including not AGCT bases (29 + 30), and redundant (218 + 226), the remaining windows (2796 + 2880) are reported in the dataset. Finally, windows of false splice sites are selected by searching canonical GT-AG pairs in not splicing positions (271 937 + 332 296). The false sites in a range +/- 60 from a true splice site are marked as proximal. HS3D, release 1.2 at this time, is available at the Web server of the University of Sannio: http://www.sci.unisannio.it/docenti/rampone/.

  9. A Randomized, Prospective, Parallel Group Study of Laparoscopic vs. Laparoendoscopic Single Site Donor Nephrectomy for Kidney Donation

    PubMed Central

    Aull, Meredith J.; Afaneh, Cheguevara; Charlton, Marian; Serur, David; Douglas, Melissa; Christos, Paul J.; Kapur, Sandip; Del Pizzo, Joseph J.

    2014-01-01

    Few prospective, randomized studies have assessed 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 vs. LDN from surgery through 5 years post-donation. Subjects complete recovery/satisfaction questionnaires at 2, 6, and 12 months post-donation; transplant recipient outcomes are also recorded. 100 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%) vs. 2 LDN (3.9%; P=0.67). Questionnaires revealed 97.2% of LESS-DN vs. 79.5% of LDN (P=0.03) were 100% recovered by two 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

  10. Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo

    SciTech Connect

    Rossi, A.M. Pisa Univ. ); Tates, A.D.; van Zeeland, A.A.; Vrieling, H. )

    1992-01-01

    Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, the authors observed (1) complete loss of one or more exons, (2) partial loss of one exon, or (3) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns.

  11. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

    PubMed Central

    Erkelenz, Steffen; Mueller, William F.; Evans, Melanie S.; Busch, Anke; Schöneweis, Katrin; Hertel, Klemens J.; Schaal, Heiner

    2013-01-01

    Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. PMID:23175589

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

    PubMed Central

    2014-01-01

    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

  13. Does distance matter? Variations in alternative 3' splicing regulation.

    PubMed

    Akerman, Martin; Mandel-Gutfreund, Yael

    2007-01-01

    Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5' or 3' splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3' splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3' splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3' splice sites from constitutive 3' splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites. PMID:17704130

  14. Acticoat versus Allevyn as a split-thickness skin graft donor-site dressing: a prospective comparative study.

    PubMed

    Argirova, Maya; Hadjiski, Ognjan; Victorova, Anastasija

    2007-10-01

    The study comprises 27 operated patients with similar burns. Fifteen donor sites treated with Acticoat (Smith & Nephew) and 12 donor sites treated with Allevyn (Smith & Nephew) have been analyzed with respect to epithelization time, antibacterial effect, ease of dressing change, pain, and pharmacologic and cost-effective characteristics. All donor sites after the reepithelization were evaluated using the Vancouver Scar Scale for the assessment of scars at the fourth, eighth, and 12th weeks. The obtained results demonstrate statistically significant faster epithelization (P = 0.012 on the eighth day and P = 0.0081 on the 10th day) and better comfort for the patient with the Acticoat dressing (P < 0.05). With regard to bacterial growth (P > 0.05) there is no statistically significant difference in the application of Acticoat and Allevyn. The Vancouver Scar Scale assessment shows no statistically significant difference (P > 0.05) in the application of both Acticoat and Allevyn. There is no considerable difference in the cost of treatment between both dressings. The results obtained determine both dressings as suitable for application on donor sites. If there is a possibility of choice, the Acticoat dressing is preferable. PMID:17901734

  15. The scalp as a donor site for split-thickness skin graft: a rare complication case report.

    PubMed

    Robert, N; May, P; Binder, J P; Revol, M; Servant, J M

    2011-05-01

    The scalp is a useful and reliable donor site for the paediatric burn population that can be harvested several times with minimal morbidity. However, the scalp cannot be used as skin graft donor site with impunity. Scalp alopecia and chronic folliculitis can be observed among the complications. In these cases, the reconstruction phase offers different surgical procedures such as primary closure, staged excision or tissue expansion. We report the case of a patient (29-years-old), treated 20 years ago for second-degree burns covering up to 20% total body surface area (TBSA) by using thin split-thickness skin grafts of his scalp. As a teenager, he developed multiple episodes of folliculitis at the donor site of the scalp and then of recurrent abscesses, resistant to all existing medical treatments. Surgical treatment consisted in the skin excision of his scalp donor site which was immediately covered by a thin split-thickness skin graft. Four months after surgery, the patient was satisfied with the functional and aesthetic result. PMID:21300581

  16. A noncoding melanophilin gene (MLPH) SNP at the splice donor of exon 1 represents a candidate causal mutation for coat color dilution in dogs.

    PubMed

    Drögemüller, Cord; Philipp, Ute; Haase, Bianca; Günzel-Apel, Anne-Rose; Leeb, Tosso

    2007-01-01

    Coat color dilution in several breeds of dog is characterized by a specific pigmentation phenotype and sometimes accompanied by hair loss and recurrent skin inflammation, the so-called color dilution alopecia or black hair follicular dysplasia. Coat color dilution (d) is inherited as a Mendelian autosomal recessive trait. In a previous study, MLPH polymorphisms showed perfect cosegregation with the dilute phenotype within breeds. However, different dilute haplotypes were found in different breeds, and no single polymorphism was identified in the coding sequence that was likely to be causative for the dilute phenotype. We resequenced the 5'-region of the canine MLPH gene and identified a strong candidate single nucleotide polymorphism within the nontranslated exon 1, which showed perfect association to the dilute phenotype in 65 dilute dogs from 7 different breeds. The A/G polymorphism is located at the last nucleotide of exon 1 and the mutant A-allele is predicted to reduce splicing efficiency 8-fold. An MLPH mRNA expression study using quantitative reverse transcriptase-polymerase chain reaction confirmed that dd animals had only about approximately 25% of the MLPH transcript compared with DD animals. These results provide preliminary evidence that the reported regulatory MLPH mutation might represent a causal mutation for coat color dilution in dogs. PMID:17519392

  17. Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence

    PubMed Central

    Hancock, D B; Reginsson, G W; Gaddis, N C; Chen, X; Saccone, N L; Lutz, S M; Qaiser, B; Sherva, R; Steinberg, S; Zink, F; Stacey, S N; Glasheen, C; Chen, J; Gu, F; Frederiksen, B N; Loukola, A; Gudbjartsson, D F; Brüske, I; Landi, M T; Bickeböller, H; Madden, P; Farrer, L; Kaprio, J; Kranzler, H R; Gelernter, J; Baker, T B; Kraft, P; Amos, C I; Caporaso, N E; Hokanson, J E; Bierut, L J; Thorgeirsson, T E; Johnson, E O; Stefansson, K

    2015-01-01

    We conducted a 1000 Genomes–imputed genome-wide association study (GWAS) meta-analysis for nicotine dependence, defined by the Fagerström Test for Nicotine Dependence in 17 074 ever smokers from five European-ancestry samples. We followed up novel variants in 7469 ever smokers from five independent European-ancestry samples. We identified genome-wide significant association in the alpha-4 nicotinic receptor subunit (CHRNA4) gene on chromosome 20q13: lowest P=8.0 × 10−9 across all the samples for rs2273500-C (frequency=0.15; odds ratio=1.12 and 95% confidence interval=1.08–1.17 for severe vs mild dependence). rs2273500-C, a splice site acceptor variant resulting in an alternate CHRNA4 transcript predicted to be targeted for nonsense-mediated decay, was associated with decreased CHRNA4 expression in physiologically normal human brains (lowest P=7.3 × 10−4). Importantly, rs2273500-C was associated with increased lung cancer risk (N=28 998, odds ratio=1.06 and 95% confidence interval=1.00–1.12), likely through its effect on smoking, as rs2273500-C was no longer associated with lung cancer after adjustment for smoking. Using criteria for smoking behavior that encompass more than the single ‘cigarettes per day' item, we identified a common CHRNA4 variant with important regulatory properties that contributes to nicotine dependence and smoking-related consequences. PMID:26440539

  18. Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution

    PubMed Central

    Parkinson, Oliver T.; Roellecke, Katharina; Freund, Marcel; Gombert, Michael; Lottmann, Nadine; Steward, Charles A.; Kramm, Christof M.; Yarov-Yarovoy, Vladimir; Rettie, Allan E.; Hanenberg, Helmut

    2015-01-01

    CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5–exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution. PMID:26355749

  19. The Maturation of Synthetic Scaffolds for Osteochondral Donor Sites of the Knee

    PubMed Central

    Bedi, Asheesh; Foo, Li Foong; Williams, Riley J.; Potter, Hollis G.

    2010-01-01

    Objective: The purpose of this study was to analyze the morphological imaging characteristics and incorporation of TruFit bone graft substitute (BGS) plugs using cartilage-sensitive magnetic resonance imaging (MRI) and quantitative T2 mapping. Design: Twenty-six patients (mean age, 28.72 years; range, 11-56 years) underwent osteochondral autologous transplantation (OATS) for chondral defects with filling of the knee joint donor sites using Trufit BGS plugs. The mean follow-up interval between implantation and MRI analysis was 21.3 months (range, 6-39 months). During this period, 43 cartilage-sensitive and 25 quantitative T2-mapping MRI studies were performed. The donor sites were assessed for plug and interface morphology, displacement, hypertrophy, subchondral edema, presence of bony overgrowth, percentage fill, and degree of incorporation. T2 relaxation times were measured for the superficial and deep layers of the repair tissue. A linear regression and correlational analysis was performed with Bonferroni correction, and P < 0.05 was defined as significant. Results: Longitudinal analysis revealed favorable plug appearance at early follow-up (≤6 months), with 75% of plugs demonstrating flush morphology and 78% demonstrating near complete to complete fill. Plug appearance deteriorated at intermediate follow-up (~12 months), with only 26% of plugs demonstrating flush morphology and 52% with near complete or complete fill. Plug appearance substantially improved with longer follow-up (≥16 months), with 70% of plugs demonstrating flush morphology and 90% demonstrating near complete or complete fill. Interface resorption was common at ~12 months (P < 0.0001) and was associated with older age (P = 0.01) or a single-plug configuration (P = 0.04). T2 values for the repair cartilage approached that of normal cartilage with increasing duration after surgery (P < 0.004), more so for single- compared with multiple-plug configurations (P = 0.03). Conclusions: The Trufit BGS

  20. Traceless protein splicing utilizing evolved split inteins

    PubMed Central

    Lockless, Steve W.; Muir, Tom W.

    2009-01-01

    Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. PMID:19541616

  1. Recursive splicing in long vertebrate genes

    PubMed Central

    Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-01-01

    It is generally believed that splicing removes introns as single units from pre-mRNA transcripts. However, some long D. melanogaster introns contain a cryptic site, called a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing1,2. The extent to which recursive splicing occurs in other species and its mechanistic basis remain unclear. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of a “RS-exon” that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform due to competition with a reconstituted 5′ splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic exons or promoters that are prevalent in long introns, but which fail to reconstitute an efficient 5′ splice site. Most RS-exons contain a premature stop codon such that their inclusion may decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling inclusion of cryptic elements with RS-exons. PMID:25970246

  2. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons. PMID:25970246

  3. Donor Site Evaluation: Anterior Iliac Crest Following Secondary Alveolar Bone Grafting

    PubMed Central

    Vura, Nandagopal; Reddy K., Rajiv; R., Sudhir; G., Rajasekhar; Kaluvala, Varun Raja

    2013-01-01

    Introduction: The use of autogenous bone graft for Secondary alveolar bone grafting is well established in the treatment of cleft lip and palate patients. Aims and Objectives: To evaluate post-operative morbidity of anterior iliac crest graft after secondary alveolar bone grafting in cleft patients. Material and Methods: Forty patients during the period from July 2008 to March 2013, who underwent secondary alveolar bone grafting by harvesting graft from anterior iliac crest in Mamata Dental Hospital, Khammam, Andhra Pradesh, India are included in the present study. Unilateral and bilateral cleft patients who had undergone secondary alveolar bone grafting (SABG) with anterior iliac crest as their donor site have been selected and post- operative complications from the surgery were evaluated with the help of a questionnaire which included pain, gait disturbances, numbness and scar problems (infection, irritation). Results: Patients who were operated gave maximum score for pain as 8 on visual analogue scale. No pain was observed in any of the cases after 8 days, gait disturbances were seen in all patients (limping) for 2-6 days, there was no post-operative numbness with all the patients returning to their routine in 6- 15 days and 90% of the patients gave a satisfied response towards scar. Conclusion: From the results in our study the morbidity after harvesting bone from iliac crest was found to be moderate to low, which had minimal complications and were well tolerated and greater acceptance from the patient. PMID:24392424

  4. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations.

    PubMed

    Diederichs, Sven; Bartsch, Lorenz; Berkmann, Julia C; Fröse, Karin; Heitmann, Jana; Hoppe, Caroline; Iggena, Deetje; Jazmati, Danny; Karschnia, Philipp; Linsenmeier, Miriam; Maulhardt, Thomas; Möhrmann, Lino; Morstein, Johannes; Paffenholz, Stella V; Röpenack, Paula; Rückert, Timo; Sandig, Ludger; Schell, Maximilian; Steinmann, Anna; Voss, Gjendine; Wasmuth, Jacqueline; Weinberger, Maria E; Wullenkord, Ramona

    2016-01-01

    Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis. PMID:26992833

  5. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing.

    PubMed Central

    Hodges, P E; Rosenberg, L E

    1989-01-01

    Ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is a mitochondrial matrix enzyme of the mammalian urea cycle. The X chromosome-linked spfash mutation in the mouse causes partial ornithine transcarbamylase deficiency and has served as a model for the human disease. We show here that the spfash mutation is a guanine to adenine transition of the last nucleotide of the fourth exon of the ornithine transcarbamylase gene. This nucleotide change produces two remarkably different effects. First, this transition causes ornithine transcarbamylase mRNA deficiency because the involved exon nucleotide plays a part in the recognition of the adjacent splice donor site. As a result of the mutation, ornithine transcarbamylase pre-mRNA is spliced inefficiently both at this site and at a cryptic splice donor site 48 bases into the adjacent intron. Second, two mutant proteins are translated from these mRNAs. From the correctly spliced mRNA, the transition results in a change of amino acid 129 from arginine to histidine. This missense substitution has no discernable effect on mitochondrial import, subunit assembly, or enzyme activity. On the other hand, the elongated mRNA resulting from mis-splicing is translated into a dysfunctional ornithine transcarbamylase subunit elongated by the insertion of 16 amino acid residues. Images PMID:2471197

  6. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  7. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  8. Hemostatic effect of Vivostat patient-derived fibrin sealant on split-thickness skin graft donor sites.

    PubMed

    Drake, David B; Wong, Lesley G

    2003-04-01

    Topical hemostatic agents are used frequently to control bleeding of skin graft donor sites. In this study, the hemostatic properties of Vivostat (Vivolution A/S, Birkerød, Denmark) patient-derived fibrin sealant were compared with a control group of spray thrombin solution, which is considered an industry standard for topical hemostasis. Treatments were applied simultaneously to two randomly chosen halves of a single split-thickness single donor site in patients in five United States surgical centers. The time to achieve satisfactory hemostasis (< or =10 min) was estimated on each half of the wound as the time at which active bleeding had stopped and the wound was suitable for application of a surgical dressing. The time to hemostasis of wounds treated with Vivostat (Vivolution A/S) patient-derived sealant was significantly shorter in comparison with wounds treated with thrombin solution (medians: Vivostat, 31 seconds; thrombin, 58 seconds; p=0.0012). No abnormalities in wound healing were reported for either treatment site 1 week after the operation. Vivostat (Vivolution A/S) sealant is a more rapidly effective topical hemostatic agent than thrombin on split-thickness skin graft donor sites. PMID:12671377

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

    PubMed

    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

    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

  10. Evaluation of Six Split-thickness Skin Graft Donor-site Dressing Materials in a Swine Model

    PubMed Central

    Masella, Pamela C.; Balent, Eric M.; Carlson, Terri L.; Lee, Karen W.

    2013-01-01

    Background: Numerous dressings for split-thickness skin graft donor sites are commercially available with no conclusive evidence-based consensus regarding the optimal dressing choice. This study was conducted to identify which of 5 commonly used materials promotes wound healing most effectively for use on split-thickness donor sites in comparison with our standard dressing, Xeroform (petrolatum gauze). Methods: Twenty-four partial-thickness wounds were created on the backs of 4 pigs using a dermatome. Wounds (n = 4 per dressing type per pig) were treated with Xeroform, Opsite (polyurethane film), Kaltostat ( calcium sodium alginate), DuoDERM (hydrocolloid), Aquacel (hydrofiber), and Mepilex (silicone foam). Full-thickness skin samples were excised at 3 or 5 days and evaluated histologically for reepithelialization and inflammation. Comparisons also included incidence of infection, ease of use, and cost analyses. Results: DuoDERM elicited the greatest percent reepithelialization (81%) and Mepilex the lowest (33%) after 3 days (P = 0.004). All dressings demonstrated complete reepithelialization except Mepilex (85%) at 5 days. There were no infections and inflammation was mild among all treatments. Mepilex was easiest to use, whereas Aquacel, Kaltostat, and Opsite were most difficult (P = 0.03). Xeroform was most cost-effective and Aquacel most expensive. Combined scoring revealed DuoDERM = Xeroform > Opsite = Mepilex > Kaltostat > Aquacel. Conclusions: DuoDERM and Xeroform were most effective overall. DuoDERM tended to outperform all dressings in reepithelialization at 3 days, while Xeroform was least expensive, easy to use, and demonstrated rapid reepithelialization. These findings suggest that Xeroform may be preferred for use on large donor-site areas. DuoDERM may be more appropriate for small donor sites when healing time is a priority. PMID:25289278

  11. Donor site morbidity following iliac crest bone harvesting for cervical fusion: a comparison between minimally invasive and open techniques

    PubMed Central

    Pollock, Raymond; Bhatia, Chandra; Chuter, Graham; Lingutla, Kiran; Budithi, Chakravarty; Krishna, Manoj

    2008-01-01

    We have studied the occurrence of donor site morbidity, cosmesis and overall satisfaction with graft procedure in 76 patients who had undergone iliac crest bone harvesting for anterior cervical discectomy and fusion (ACDF). Totally 24 patients underwent an open procedure and 52 a minimally invasive trephine harvesting method. Although our study demonstrated substantial donor site pain and its effect on ambulation in both groups, this was of limited duration. Two patients, one in each group, suffered long-term pain that was eventually resolved. Totally 8.3% of patients in the open group suffered minor complications and 11.5% in the trephine group. There were two cases of meralgia parasthetica. There were no major complications in either group. There was no statistically significant difference in morbidity between the open and trephine groups. There was a trend towards significance (P = 0.076) for pain at the donor site, with less pain reported by patients who underwent the trephine procedure for harvesting. PMID:18389294

  12. Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites.

    PubMed

    Loeffelbein, Denys J; Rohleder, Nils H; Eddicks, Matthias; Baumann, Claudia M; Stoeckelhuber, Mechthild; Wolff, Klaus-D; Drecoll, Enken; Steinstraesser, Lars; Hennerbichler, Simone; Kesting, Marco R

    2014-01-01

    Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative. PMID:25003117

  13. Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site

    PubMed Central

    Smalle, Jan A

    2013-01-01

    Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 ( sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth. PMID:24358894

  14. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  15. The genetics of splicing in neuroblastoma

    PubMed Central

    Chen, Justin; Hackett, Christopher S.; Zhang, Shile; Song, Young K.; Bell, Robert J.A.; Molinaro, Annette M.; Quigley, David A.; Balmain, Allan; Song, Jun S.; Costello, Joseph F.; Gustafson, W. Clay; Dyke, Terry Van; Kwok, Pui-Yan; Khan, Javed; Weiss, William A.

    2015-01-01

    Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest. Here we describe splicing quantitative trait loci (sQTL) associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. PMID:25637275

  16. Investigation of the low-affinity oxidation site for exogenous electron donors in the Mn-depleted photosystem II complexes.

    PubMed

    Kurashov, V N; Lovyagina, E R; Shkolnikov, D Yu; Solntsev, M K; Mamedov, M D; Semin, B K

    2009-12-01

    In the manganese-depleted photosystem II (PSII[-Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(-Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L. Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to Y(Z)(*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light, Biochemistry 41 (2002) 5854-5864.]. In blocked (PSII[-Mn,+Fe]) preparations electron donation by Mn(II) cations to Y(Z)(*) was not detected at Mn(II) concentration 10 microM (corresponds to K(m) for Mn(II) oxidation at the HA site), but detected at Mn concentration 100 microM (corresponds to K(m) for the LA site) by fluorescence measurements. Comparison of pH-dependencies of electron donation by Mn(II) through the HA and the LA sites revealed the similar pK(a) equal to 6.8. Comparison of K(m) for diphenylcarbazide (DPC) oxidation at the LA site and K(d) for A(T) thermoluminescence band suppression by DPC in PSII(-Mn,+Fe) samples suggests that there is relationship between the LA site and A(T) band formation. The role of D1-His190 as an oxidant of exogenous electron donors at the LA site is discussed. In contrast to electrogenic electron transfer from Mn(II) at the HA site to Y(Z)(*), photovoltage due to Mn(II) oxidation in iron-blocked PSII(-Mn) core particles was not detected. PMID:19616503

  17. Long-term vascular, motor, and sensory donor site outcomes after ulnar forearm flap harvest.

    PubMed

    Brown, Emile N; Chaudhry, Arif; Mithani, Suhail K; Bluebond-Langner, Rachel O; Feiner, Jeffrey M; Shaffer, Cynthia K; Call, Diana; Rodriguez, Eduardo D

    2014-02-01

    Use of the ulnar forearm flap (UFF) is limited by concerns for ulnar nerve injury and impaired perfusion in the donor extremity. Twenty UFFs were performed over a 6-year period. All patients underwent postoperative bilateral upper extremity arterial duplex studies. A subset of postoperative patients (n = 10) also had bilateral upper extremity sensory and motor evaluations, and functional evaluation via the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH). Motor function was tested by digital and key grip dynamometry. Ulnar nerve sensation was tested by evaluation of one- and two-point perceived pressure thresholds and two-point discrimination using the Pressure-Specified Sensory Device (Sensory Management Services, LLC, Baltimore, MD). All UFFs were viable postoperatively. Mean follow-up was 28.8 months for vascular studies and 45.3 months for motor, sensory, and QuickDASH evaluations. Although mid and distal radial artery flow velocities were significantly higher in donor versus control extremities evaluated at less than 1 year postoperatively, there was no significant difference in extremities evaluated at later time points. Digital pressures, grip strength, key pinch strength, and ulnar sensation were equivalent between donor and control extremities. The mean QuickDASH score was 17.4 ± 23.8. The UFF can be harvested reliably and long-term follow-up shows no evidence of impaired vascular, motor, or sensory function in the donor extremity. PMID:24163222

  18. PRKN, DJ-1, and PINK1 screening identifies novel splice site mutation in PRKN and two novel DJ-1 mutations.

    PubMed

    Ghazavi, Farzaneh; Fazlali, Zeinab; Banihosseini, Setareh Sadat; Hosseini, Sayed-Rzgar; Kazemi, Mohammad Hossein; Shojaee, Seyedmehdi; Parsa, Khosro; Sadeghi, Homa; Sina, Farzad; Rohani, Mohammad; Shahidi, Gholam-Ali; Ghaemi, Nasser; Ronaghi, Mostafa; Elahi, Elahe

    2011-01-01

    We present results of mutation screening of PRKN gene in 93 Iranian Parkinson's disease (PD) patients with average age at onset (AAO) of 42.2 years. The gene was screened by direct sequencing and by a semi-quantitative PCR protocol for detection of sequence rearrangements. Heterozygous rearrangements were tested by reverse transcription-polymerase chain reaction (RT-PCR). Nine different PRKN mutations were found. One of these, IVS9+1G>A, affects splicing and is novel. Two mutated PRKN alleles were observed in each of 6 patients whose average AAO was 25.7 years. Only 1 patient carried a single mutated allele and his AAO was 41 years. Among patients with AAO of <30 years, 31.3% had two mutated alleles, while only 2.6% with AAO of >30 years carried a PRKN mutation. Analysis of PRKN by RT-PCR led to identification of a novel exon expressed in leukocytes of control and PD individuals. The alternatively spliced transcript if translated would code a protein without a RING Finger 2 domain. Its functional relevance remains to be shown. DJ-I and PINK1 were also screened. Two novel DJ-1 mutations, c.91-2A>G affecting splicing and c.319G>C causing Ala107Pro, were observed among patients with AAO of <31 years, suggesting that PD in a high fraction (>12%) of this group of Iranian patients may be due to mutations in DJ-1. Mutations in PINK1 were not observed. Our results complement previous findings on LRRK2 mutations among Iranian PD patients. PMID:21322020

  19. Novel splice site mutation in the caveolin-3 gene leading to autosomal recessive limb girdle muscular dystrophy.

    PubMed

    Müller, Juliane S; Piko, Henriett; Schoser, Benedikt G H; Schlotter-Weigel, Beate; Reilich, Peter; Gürster, Stefanie; Born, Christine; Karcagi, Veronika; Pongratz, Dieter; Lochmüller, Hanns; Walter, Maggie C

    2006-07-01

    Mutations in CAV3 gene encoding the protein caveolin-3 are associated with autosomal dominant limb girdle muscular dystrophy 1C, rippling muscle disease, hyperCKemia, distal myopathy, hypertrophic cardiomyopathy and rare autosomal recessive limb girdle muscular dystrophy phenotypes. In a 57-year-old patient with asymmetric limb girdle weakness, we detected a novel homozygous intronic mutation (IVS1 + 2T > C) of the CAV3 gene. This is the first splicing mutation reported for CAV3. These findings add to the clinical and genetic variability of CAV3 mutations. PMID:16730439

  20. Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate

    PubMed Central

    Mishler, Dennis M.; Christ, Alexander B.; Steitz, Joan A.

    2008-01-01

    The exon junction complex (EJC) is critical for mammalian nonsense-mediated mRNA decay and translational regulation, but the mechanism of its stable deposition on mRNA is unknown. To examine requirements for EJC deposition, we created splicing substrates containing either DNA nucleotides or RNA secondary structure in the 5′ exon. Using RNase H protection, toeprinting, and coimmunoprecipitation assays, we found that EJC location shifts upstream when a stretch of DNA or RNA secondary structure appears at the canonical deposition site. These upstream shifts occur prior to exon ligation and are often accompanied by decreases in deposition efficiency. Although the EJC core protein eIF4AIII contacts four ribose 2′OH groups in crystal structures, we demonstrate that three 2′OH groups are sufficient for deposition. Thus, the site of EJC deposition is more flexible than previously appreciated and efficient deposition appears spatially limited. PMID:18952819

  1. An ENU Mutagenesis Screen in Zebrafish for Visual System Mutants Identifies a Novel Splice-Acceptor Site Mutation in patched2 that Results in Colobomas

    PubMed Central

    Lee, Jiwoon; Cox, Ben D.; Daly, Christina M. S.; Lee, Chanjae; Nuckels, Richard J.; Tittle, Rachel K.; Uribe, Rosa A.; Gross, Jeffrey M.

    2012-01-01

    Purpose. To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. Methods. A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. Results. A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta1, identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2uta1 mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2uta1 mutant embryos. Conclusions. We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas. PMID:23150614

  2. Split-thickness skin graft donor site management: a randomized controlled trial comparing polyurethane with calcium alginate dressings.

    PubMed

    Higgins, Louise; Wasiak, Jason; Spinks, Anneliese; Cleland, Heather

    2012-04-01

    Split-thickness skin grafting (SSG) is a common reconstructive technique for the treatment of patients with deep burns and other traumatic injuries. The management of the donor site after harvesting an SSG remains controversial because of a variety of dressings available for use. The aim of this randomized controlled trial was to compare the effectiveness of a polyurethane dressing, Allevyn™, to a calcium alginate, Kaltostat®. From August 2009 to April 2010, 36 patients were randomized to Allevyn™ or Kaltostat® for donor site management following split skin graft surgery. Pain intensity and adverse events were the primary outcomes assessed. Secondary outcome measures included time for wound healing, ease of application and removal and overall patient satisfaction. Time to first dressing change was earlier in those randomized to Allevyn™ compared with Kaltostat® (5·5 days versus 8·11 days, P = 0·014). In patients randomized to Allevyn™, excessive exudate lead to a significantly increased number of dressing changes before day 10 (14 days versus 7 days, P = 0·018). The total number of dressing changes applied was also greater in those with Allevyn™ compared with Kaltstat® (P = 0·007). There were no significant differences between the two treatment groups with respect to time to wound healing, level of pain intensity, length of stay, staff and patient satisfaction levels. This trial showed Allevyn™ to be associated with increase demands on nursing time, increased cost of dressing products, medical consumables and wastes. Kaltostat® remains the dressing of choice for initial donor site dressing in this burns unit. PMID:22051247

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

    PubMed

    Brenner, Maria; Hilliard, Carol; Peel, Glynis; Crispino, Gloria; Geraghty, Ruth; OʼCallaghan, Gill

    2015-01-01

    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

  4. Splicing of Retrotransposon Insertions from Transcripts of the Drosophila Melanogaster Vermilion Gene in a Revertant

    PubMed Central

    Pret, A. M.; Searles, L. L.

    1991-01-01

    A mutation of the Drosophila melanogaster vermilion (v) gene known as v(1) is caused by the insertion of a 412 retrotransposon into the 5' untranslated region of the first exon. Mutants carrying this insertion accumulate a low level of mRNA from which most of the transposon sequences have been eliminated by splicing at cryptic sites within transposon sequences. Here, we demonstrate that a revertant of the v(1) allele called v(+37) is caused by the insertion of a second retrotransposon, the B104/roo element, into a site near one end of the 412 element. The revertant strain accumulates a higher level of mRNA from which most of both transposons have been removed by splicing at new donor sites introduced by the B104/roo insertion and the same acceptor site within 412. Mutations at suppressor of sable [su(s)], which increase the accumulation of v(1) transcripts, slightly elevate the level of v(+37) RNA. In addition, we show that the first v intron downstream of the 412 insertion is not efficiently removed in the v(1) mutant, and suppressor and reversion mutations increase the proportion of transcripts that are properly spliced at that downstream intron. Thus, it appears that both the suppressor and reversion mutations exert an effect at the level of pre-mRNA splicing. PMID:1664404

  5. dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice-Site SNVs.

    PubMed

    Liu, Xiaoming; Wu, Chunlei; Li, Chang; Boerwinkle, Eric

    2016-03-01

    The purpose of the dbNSFP is to provide a one-stop resource for functional predictions and annotations for human nonsynonymous single-nucleotide variants (nsSNVs) and splice-site variants (ssSNVs), and to facilitate the steps of filtering and prioritizing SNVs from a large list of SNVs discovered in an exome-sequencing study. A list of all potential nsSNVs and ssSNVs based on the human reference sequence were created and functional predictions and annotations were curated and compiled for each SNV. Here, we report a recent major update of the database to version 3.0. The SNV list has been rebuilt based on GENCODE 22 and currently the database includes 82,832,027 nsSNVs and ssSNVs. An attached database dbscSNV, which compiled all potential human SNVs within splicing consensus regions and their deleteriousness predictions, add another 15,030,459 potentially functional SNVs. Eleven prediction scores (MetaSVM, MetaLR, CADD, VEST3, PROVEAN, 4× fitCons, fathmm-MKL, and DANN) and allele frequencies from the UK10K cohorts and the Exome Aggregation Consortium (ExAC), among others, have been added. The original seven prediction scores in v2.0 (SIFT, 2× Polyphen2, LRT, MutationTaster, MutationAssessor, and FATHMM) as well as many SNV and gene functional annotations have been updated. dbNSFP v3.0 is freely available at http://sites.google.com/site/jpopgen/dbNSFP. PMID:26555599

  6. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  7. Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

    PubMed Central

    Boukis, L A; Bruzik, J P

    2001-01-01

    The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction. PMID:11421358

  8. Splicing Programs and Cancer

    PubMed Central

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

    2012-01-01

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

  9. A Splice Site Variant in the Bovine RNF11 Gene Compromises Growth and Regulation of the Inflammatory Response

    PubMed Central

    Sartelet, Arnaud; Druet, Tom; Michaux, Charles; Fasquelle, Corinne; Géron, Sarah; Tamma, Nico; Zhang, Zhiyan; Coppieters, Wouter; Georges, Michel; Charlier, Carole

    2012-01-01

    We report association mapping of a locus on bovine chromosome 3 that underlies a Mendelian form of stunted growth in Belgian Blue Cattle (BBC). By resequencing positional candidates, we identify the causative c124-2A>G splice variant in intron 1 of the RNF11 gene, for which all affected animals are homozygous. We make the remarkable observation that 26% of healthy Belgian Blue animals carry the corresponding variant. We demonstrate in a prospective study design that approximately one third of homozygous mutants die prematurely with major inflammatory lesions, hence explaining the rarity of growth-stunted animals despite the high frequency of carriers. We provide preliminary evidence that heterozygous advantage for an as of yet unidentified phenotype may have caused a selective sweep accounting for the high frequency of the RNF11 c124-2A>G mutation in Belgian Blue Cattle. PMID:22438830

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

    PubMed Central

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

    2012-01-01

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

  11. The fat-fascia paddle only with a composite fibula flap: marked reduction in donor site morbidity.

    PubMed

    Mohindra, A; Parmar, S; Praveen, P; Martin, T

    2016-08-01

    Fibula free flaps are used widely in head and neck reconstruction, primarily for their versatility and contribution to aesthetic and functional outcomes. The literature suggests that early complications such as wound dehiscence and skin graft loss can occur in up to a third of patients. The healing of these donor sites can be prolonged, and in certain cases may require an operative intervention. A method to overcome this problem is described herein. In raising the skin paddle, a standard lateral approach to the fibula harvest is used. The skin paddle is not isolated and the posterior margin of the paddle is maintained. The skin paddle epithelium is separated with a small cuff of adipose tissue from the underlying fat-fascia layer. This fat-fascia paddle is then raised with the fibula as normal and tacked to the margins of the recipient soft tissue defect. The fat-fascia paddle heals, resulting in a thin mucosal covering for prosthetic dental rehabilitation. This technique can reduce the incidence of donor site wound complications when raising a composite fibula flap. PMID:27061479

  12. Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing

    PubMed Central

    Mazloomian, Alborz; Meyer, Irmtraud M

    2015-01-01

    RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10−15). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure. PMID:26512413

  13. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

    Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions. PMID:18369186

  14. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  15. Autosomal Dominant Retinal Dystrophies Caused by a Founder Splice Site Mutation, c.828+3A>T, in PRPH2 and Protein Haplotypes in trans as Modifiers

    PubMed Central

    Shankar, Suma P.; Hughbanks-Wheaton, Dianna K.; Birch, David G.; Sullivan, Lori S.; Conneely, Karen N.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

    2016-01-01

    Purpose We determined the phenotypic variation, disease progression, and potential modifiers of autosomal dominant retinal dystrophies caused by a splice site founder mutation, c.828+3A>T, in the PRPH2 gene. Methods A total of 62 individuals (19 families) harboring the PRPH2 c.828+3A>T mutation, had phenotype analysis by fundus appearance, electrophysiology, and visual fields. The PRPH2 haplotypes in trans were sequenced for potential modifying variants and generalized estimating equations (GEE) used for statistical analysis. Results Several distinct phenotypes caused by the PRPH2 c.828+3A>T mutation were observed and fell into two clinical categories: Group I (N = 44) with mild pattern dystrophies (PD) and Group II (N = 18) with more severe cone-rod dystrophy (CRD), retinitis pigmentosa (RP), and central areolar chorioretinal dystrophy (CACD). The PRPH2 Gln304-Lys310-Asp338 protein haplotype in trans was found in Group I only (29.6% vs. 0%), whereas the Glu304-Lys310-Gly338 haplotype was predominant in Group II (94.4% vs. 70.4%). Generalized estimating equations analysis for PD versus the CRD/CACD/RP phenotypes in individuals over 43 years alone with the PRPH2 haplotypes in trans and age as predictors, adjusted for correlation within families, confirmed a significant effect of haplotype on severity (P = 0.03) with an estimated odds ratio of 7.16 (95% confidence interval [CI] = [2.8, 18.4]). Conclusions The PRPH2 c.828+3A>T mutation results in multiple distinct phenotypes likely modified by protein haplotypes in trans; the odds of having the CACD/RP-like phenotype (versus the PD phenotype) are 7.16 times greater with a Glu304-Lys310-Gly338 haplotype in trans. Further functional studies of the modifying haplotypes in trans and PRPH2 splice variants may offer therapeutic targets. PMID:26842753

  16. Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

    SciTech Connect

    Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de; Brinkmann, A.O.; Degenhart, H.J.; Trapman, J. )

    1994-04-01

    The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.

  17. A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.

    PubMed Central

    Villanueva, M S; Williams, S P; Beard, C B; Richards, F F; Aksoy, S

    1991-01-01

    A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons. Images PMID:1719380

  18. Tuning of Stepwise Neutral-Ionic Transitions by Acceptor Site Doping in Alternating Donor/Acceptor Chains.

    PubMed

    Nakabayashi, Keita; Nishio, Masaki; Miyasaka, Hitoshi

    2016-03-01

    The stepwise neutral-ionic (N-I) phase transition found in the alternating donor/acceptor (DA) chain [Ru2(2,3,5,6-F4PhCO2)4(DMDCNQI)]·2(p-xylene) (0; 2,3,5,6-F4PhCO2(-) = 2,3,5,6-tetrafluorobenzoate; DMDCNQI = 2,5-dimethyl-N,N'-dicyanoquinonediimine) was tuned by partly substituting the acceptor DMDCNQI with 2,5-dimethoxy-N,N'-dicyanoquinonediimine (DMeODCNQI), which displays a poorer electron affinity in an isostructural series. The site-doped series comprised [Ru2(2,3,5,6-F4PhCO2)4(DMDCNQI)1-x(DMeODCNQI)x]·2(p-xylene) for doping rates (x) = 0.05 (0.05-MeO), 0.10 (0.10-MeO), 0.15 (0.15-MeO), and 0.20 (0.20-MeO). The neutral chain [Ru2(2,3,5,6-F4PhCO2)4(DMeODCNQI)]·4(p-xylene) (1), which only contained DMeODCNQI, was also characterized. All site-doped compounds were isostructural to 0 except 1 despite their identical DA chain motif. Except at an x value of 0.20, they displayed a two-step N-I transition involving an intermediate phase. This transition occurred at high temperatures in 0 but shifted to lower temperatures in a parallel manner with increasing doping rate. Simultaneously, each transition broadened with increasing doping rate, leading to a convergence of two transitions at an x value approximating 0.2. Donor/acceptor-site-doping techniques present somewhat different impacts in terms of interchain Coulomb effects. PMID:26878151

  19. Radiographic evaluation of the symphysis menti as a donor site for an autologous bone graft in pre-implant surgery

    PubMed Central

    Di Bari, Roberto; Coronelli, Roberto

    2013-01-01

    Purpose This study was performed to obtain a quantitative evaluation of the cortical and cancellous bone graft harvestable from the mental and canine regions, and to evaluate the cortical vestibular thickness. Materials and Methods This study collected cone-beam computed tomographic (CBCT) images of 100 Italian patients. The limits of the mental region were established: 5 mm in front of the medial margin of each mental foramen, 5 mm under the apex of each tooth present, and above the inferior mandibular cortex. Cortical and cancellous bone volumes were evaluated using SimPlant software (SimPlant 3-D Pro, Materialize, Leuven, Belgium) tools. In addition, the cortical vestibular thickness (minimal and maximal values) was evaluated in 3 cross-sections corresponding to the right canine tooth (3R), the median section (M), and the left canine tooth (3L). Results The cortical volume was 0.71±0.23 mL (0.27-1.96 mL) and the cancellous volume was 2.16±0.76 mL (0.86-6.28 mL). The minimal cortical vestibular thickness was 1.54±0.41 mm (0.61-3.25 mm), and the maximal cortical vestibular thickness was 3.14±0.75mm(1.01-5.83 mm). Conclusion The use of the imaging software allowed a patient-specific assessment of mental and canine region bone availability. The proposed evaluation method might help the surgeon in the selection of the donor site by the comparison between bone availability in the donor site and the reconstructive exigency of the recipient site. PMID:24083206

  20. Long-term donor-site morbidity after vascularized free fibula flap harvesting: Clinical and gait analysis.

    PubMed

    Feuvrier, Damien; Sagawa, Yoshimasa; Béliard, Samuel; Pauchot, Julien; Decavel, Pierre

    2016-02-01

    The aim of this study was to determine the clinical morbidity and changes in gait temporal spatial parameters after harvesting of a vascularized free fibula flap. This study included 11 patients (mean age: 52 ± 17 years) and 11 healthy controls (mean age: 50 ± 14 years). The patients were assessed between 5 and 104 months post surgery. The study consisted of a subjective functional evaluation with two validated clinical scores (Kitaoka Score and Point Evaluation System (PES) score), clinical and neurological examination of the legs, and evaluation of gait temporal spatial parameters while walking at a comfortable speed. The mean functional Kitaoka score was 78/100, and the mean PES score of 12.18 was considered average. At the time of the review, five patients had sensory disorders, two had toe deformities, and eight had pain at the donor site. The gait analysis showed that the patient's comfortable walking speed was significantly lower in comparison to that of the controls, and that stride length and cadence were reduced. In addition, most of the gait-specific parameters were significantly different. The donor leg displayed greater variability during walking. To reduce the risk of falling, this study revealed that the patients' gait pattern had changed as they took a more cautious approach during walking. Early rehabilitation is expected to help improve and/or restore the physical abilities of patients after harvesting of the vascularized free fibula flap. PMID:26602741

  1. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  2. Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

    PubMed

    Dal Mas, Andrea; Fortugno, Paola; Donadon, Irving; Levati, Lauretta; Castiglia, Daniele; Pagani, Franco

    2015-05-01

    The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes. PMID:25665175

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

    PubMed Central

    van Santen, V L; Spritz, R A

    1986-01-01

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

  4. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-β -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing

    NASA Astrophysics Data System (ADS)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

    In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human β -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and β -globin sequences, we show that the NS1 5' splice site was effectively utilized by the β -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the β -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from β -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

  5. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides.

    PubMed

    Bergsma, Atze J; In 't Groen, Stijn Lm; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, Wwm Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  6. Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.

    PubMed

    Vallée, Maxime P; Di Sera, Tonya L; Nix, David A; Paquette, Andrew M; Parsons, Michael T; Bell, Russel; Hoffman, Andrea; Hogervorst, Frans B L; Goldgar, David E; Spurdle, Amanda B; Tavtigian, Sean V

    2016-07-01

    Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants. PMID:26913838

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

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

    2014-09-01

    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

  9. RNA splicing factors as oncoproteins and tumour suppressors.

    PubMed

    Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K

    2016-07-01

    The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations. PMID:27282250

  10. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

    PubMed

    Hwang, Daw-Yang; Hung, Chi-Chih; Riepe, Felix G; Auchus, Richard J; Kulle, Alexandra E; Holterhus, Paul-Martin; Chao, Mei-Chyn; Kuo, Mei-Chuan; Hwang, Shang-Jyh; Chen, Hung-Chun

    2011-01-01

    17α-Hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination. PMID:21966534

  11. CYP17A1 Intron Mutation Causing Cryptic Splicing in 17α-Hydroxylase Deficiency

    PubMed Central

    Hwang, Daw-Yang; Hung, Chi-Chih; Riepe, Felix G.; Auchus, Richard J.; Kulle, Alexandra E.; Holterhus, Paul-Martin; Chao, Mei-Chyn; Kuo, Mei-Chuan; Hwang, Shang-Jyh; Chen, Hung-Chun

    2011-01-01

    17α-hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination. PMID:21966534

  12. Donor site morbidity with reamer-irrigator-aspirator (RIA) use for autogenous bone graft harvesting in a single centre 204 case series.

    PubMed

    Qvick, Lars M; Ritter, Christopher A; Mutty, Christopher E; Rohrbacher, Bernhard J; Buyea, Cathy M; Anders, Mark J

    2013-10-01

    Donor site morbidity and complication rate using the reamer-irrigator-aspirator (RIA) system for intramedullary, non-structural autogenous bone graft harvesting were investigated in a retrospective chart and radiographic review at a University affiliated Level-1 Trauma Centre. 204 RIA procedures in 184 patients were performed between 1/1/2007 and 12/31/2010. RIA-indication was bone graft harvesting in 201 (98.5%), and intramedullary irrigation and debridement in 3 (1.5%) cases. Donor sites were: femur - antegrade 175, retrograde 4, tibia - antegrade 7, retrograde 18. Sixteen patients had undergone two RIA procedures, two had undergone three procedures, all using different donor sites. In 4 cases, same bone harvesting was done twice. Mean volume of bone graft harvested was 47 ± 22ml (20-85 ml). The complication rate was 1.96% (N=4). Operative revisions included 2 retrograde femoral nails for supracondylar femur fractures 6 and 41 days postoperatively (antegrade femoral RIA), 1 trochanteric entry femoral nail (subtrochanteric fracture) 17 days postoperatively (retrograde femoral RIA) and 1 prophylactic stabilization with a trochanteric entry femoral nail for intraoperative posterior femoral cortex penetration without fracture. In our centre, the RIA technique has a low donor site morbidity and has been successfully implemented for harvesting large volumes of nonstructural autogenous bone graft. PMID:23845569

  13. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    SciTech Connect

    Tan, Chye Ling; Gunaratne, Jayantha; Lai, Deborah; Carthagena, Laetitia; Wang, Qian; Xue, Yue Zhen; Quek, Ling Shih; Doorbar, John; Bachelerie, Francoise; Thierry, Francoise; Bellanger, Sophie

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  14. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members.

    PubMed

    Thomassen, Mads; Blanco, Ana; Montagna, Marco; Hansen, Thomas V O; Pedersen, Inge S; Gutiérrez-Enríquez, Sara; Menéndez, Mireia; Fachal, Laura; Santamariña, Marta; Steffensen, Ane Y; Jønson, Lars; Agata, Simona; Whiley, Phillip; Tognazzo, Silvia; Tornero, Eva; Jensen, Uffe B; Balmaña, Judith; Kruse, Torben A; Goldgar, David E; Lázaro, Conxi; Diez, Orland; Spurdle, Amanda B; Vega, Ana

    2012-04-01

    Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of

  15. The evolution of spliced leader trans-splicing in nematodes.

    PubMed

    Pettitt, Jonathan; Harrison, Neale; Stansfield, Ian; Connolly, Bernadette; Müller, Berndt

    2010-08-01

    Spliced leader trans-splicing occurs in many primitive eukaryotes including nematodes. Most of our knowledge of trans-splicing in nematodes stems from the model organism Caenorhabditis elegans and relatives, and from work with Ascaris. Our investigation of spliced leader trans-splicing in distantly related Dorylaimia nematodes indicates that spliced-leader trans-splicing arose before the nematode phylum and suggests that the spliced leader RNA gene complements in extant nematodes have evolved from a common ancestor with a diverse set of spliced leader RNA genes. PMID:20659016

  16. Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana

    PubMed Central

    Raczynska, Katarzyna Dorota; Simpson, Craig G.; Ciesiolka, Adam; Szewc, Lukasz; Lewandowska, Dominika; McNicol, Jim; Szweykowska-Kulinska, Zofia; Brown, John W. S.; Jarmolowski, Artur

    2010-01-01

    The nuclear cap-binding protein complex (CBC) participates in 5′ splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT–PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5′ and 3′ splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5′ splice site. PMID:19864257

  17. A general role for splicing enhancers in exon definition.

    PubMed Central

    Lam, Bianca J; Hertel, Klemens J

    2002-01-01

    Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection. PMID:12403462

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

    PubMed

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

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence. PMID:24749484

  19. Alternative splicing generates a second isoform of the catalytic A subunit of the vacuolar H(+)-ATPase.

    PubMed Central

    Hernando, N; Bartkiewicz, M; Collin-Osdoby, P; Osdoby, P; Baron, R

    1995-01-01

    We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597085

  20. 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

    2013-01-01

    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 < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01). Conclusion The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. Clinical trial registry information Protocol Record Identification Number: 132/03 Registry URL: http://www.clinicaltrials.gov PMID:23570605

  1. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  2. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

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

  3. A G{sub +3}-to-T donor splice site mutation leads to skipping of exon 50 in von Willebrand factor mRNA

    SciTech Connect

    Mertes, G.; Schwaab, R.; Brackmann, H.H.; Ludwig, M.

    1994-11-01

    Genomic DNA was prepared from leukocytes, and platelet-derived total mRNA was isolated by lysis of thrombocytes in 4 M guanidinium hydrochloride followed by precipitation in 2 M lithium chloride twice. PCR reactions were performed using either RT-PCR primers or genomic PCR-primers. Preferential signle-strand amplification for both PCR products was carried out with nested RT-PCR primers.

  4. Receptor mutations and haplotypes in growth hormone receptor deficiency: a global survey and identification of the Ecuadorean E180splice mutation in an oriental Jewish patient.

    PubMed

    Berg, M A; Peoples, R; Pérez-Jurado, L; Guevara-Aguirre, J; Rosenbloom, A L; Laron, Z; Milner, R D; Francke, U

    1994-04-01

    Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel. PMID:7949594

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

    PubMed

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

    2014-01-01

    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

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

    PubMed

    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

    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

  7. An evaluation of five different dressing materials on split-thickness skin graft donor site and full-thickness cutaneous wounds: an experimental study.

    PubMed

    Uraloğlu, Muhammet; Livaoğlu, Murat; Agdoğan, Özgür; Mungan, Sevdegül; Alhan, Etem; Karaçal, Naci

    2014-02-01

    The objective of this study was to investigate the healing effect of five different products on split-thickness skin graft (STSG) donor sites and full-thickness cutaneous wounds (FTCWs) using an occlusive dressing model. Six groups were included: 1 control and 5 experimental groups, with a total of 24 rats, using an occlusive dressing model. STSG donor sites and FTCWs were established in two separate areas, to the right and left on the animals' backs. Wound sites were dressed with one of the following materials: fine mesh gauze, microporous polysaccharide hemosphere (MPH), clinoptilolite, alginate, hydrogel or biosynthetic wound dressing (Biobran(®) ). These materials were compared in terms of healing rate, healing quality and histopathological findings. Occlusive dressings were applied to each wound on days 0, 3, 5, 7, 10 and 14. Area measurements were taken using images of each dressing. The alginate and clinoptilolite groups gave the best healing rate results for both STSG donor sites (P = 0·003) and FTCWs (P = 0·003). MPH came third in each group. The alginate group produced better results in terms of healing quality criteria, followed by hydrogel, MPH, clinoptilolite and Biobran(®) , in that order. Statistically significant results were obtained in all groups compared to the control group (P < 0·0007). Rapid and good healing quality for both the STSG donor sites and FTCWs were obtained with alginate. Healing with clinoptilolite and MPH was rapid, but poor quality, while slower but good healing quality was obtained with hydrogel. Slower and worse quality healing was obtained with Biobran(®) . PMID:22943661

  8. Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI).

    PubMed

    Schafer, Sebastian; Miao, Kui; Benson, Craig C; Heinig, Matthias; Cook, Stuart A; Hubner, Norbert

    2015-01-01

    Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon-centric manner and compared across conditions. PMID:26439713

  9. 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...

  10. Group II Intron Self-Splicing.

    PubMed

    Pyle, Anna Marie

    2016-07-01

    Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays a major role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns have played a major role in genome evolution, as they are likely progenitors of spliceosomal introns, retroelements, and other machinery that controls genetic variation and stability. The structure and catalytic mechanism of group II introns have recently been elucidated through a combination of genetics, chemical biology, solution biochemistry, and crystallography. These studies reveal a dynamic machine that cycles progressively through multiple conformations as it stimulates the various stages of splicing. A central active site, containing a reactive metal ion cluster, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions about the behavior of RNA machines. PMID:27391926

  11. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  12. Genomic features defining exonic variants that modulate splicing

    PubMed Central

    2010-01-01

    Background Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative. Results We assessed the features of single-nucleotide genomic variants verified to cause exon skipping and compared them to a large set of coding SNPs common in the human population, which are likely to have no effect on splicing. Our findings implicate a number of features important for their ability to discriminate splice-affecting variants, including the naturally occurring density of exonic splicing enhancers and exonic splicing silencers of the exon and intronic environment, extensive changes in the number of predicted exonic splicing enhancers and exonic splicing silencers, proximity to the splice junctions and evolutionary constraint of the region surrounding the variant. By extending this approach to additional datasets, we also identified relevant features of variants that cause increased exon inclusion and ectopic splice site activation. Conclusions We identified a number of features that have statistically significant representation among exonic variants that modulate splicing. These analyses highlight putative mechanisms responsible for splicing outcome and emphasize the role of features important for exon definition. We developed a web-tool, Skippy, to score coding variants for these relevant splice-modulating features. PMID:20158892

  13. Complications in the use of the mandibular body, ramus and symphysis as donor sites in bone graft surgery. A systematic review

    PubMed Central

    Cobo-Vázquez, Carlos; Monteserín-Matesanz, Marta; López-Quiles, Juan

    2016-01-01

    Background To develop a systematic review by assessing and comparing the different complications that occurs in bone graft surgery using the mandibular body, ramus and symphysis as donor sites. Material and Methods In order to respond to the following question, a systematic review was developed: does the use of intraoral mandibular body and ramus as donor sites in bone graft surgery, produce fewer and less severe complications in comparison to the use of the mandibular symphysis in patients that present bone resorption that needs augmentation using autologous grafts? The review was carried out between January 1990 and 2015, during which only clinical essays with a minimum follow-up period of six months were included. Results The initial search yielded a total of 2912 articles, of which 6 were finally selected. In total, 259 graft surgeries were performed; 118 using the mandibular body and ramus as donor sites, and 141, the symphysis. The most frequent complications that arose when using the mandibular symphysis were temporary sensory alterations in the anterior teeth (33.87%), followed by sensory alterations of the skin and mucosa (18.57%). As for the mandibular body and ramus donor sites, the most frequent complications relate to temporary sensory alterations of the mucosa (8.19%) and to minor postoperative bleeding (6.55%). Conclusions The analyzed results show a higher prevalence and severity of complications when using mandibular symphysis bone grafts, producing more discomfort for the patient. Therefore, it would be advisable to perform further clinical essays due to the lack of studies found. Key words:Alveolar ridge augmentation, autogenous bone, mandibular bone grafts, chin, mandibular symphysis, mandibular ramus. PMID:26827063

  14. cis-Acting sequences in addition to donor and acceptor sites are required for template switching during synthesis of plus-strand DNA for duck hepatitis B virus.

    PubMed Central

    Havert, M B; Loeb, D D

    1997-01-01

    A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3' end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis. PMID:9188603

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

    USGS Publications Warehouse

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

    2013-01-01

    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.

  16. Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

    PubMed

    Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

    2016-01-01

    The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1(+∕+)-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca(2+) sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady

  17. Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants

    PubMed Central

    Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

    2016-01-01

    The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and

  18. Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

    PubMed

    Pearson, M N; Rohrmann, G F

    1997-08-18

    A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of the L dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer did not show increased activity when cotransfected with LdMNPV ie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system. LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line. PMID:9300047

  19. A{sup -2} {yields} G transition at the 3{prime} acceptor splice site of IVS17 characterizes the COL2A1 gene mutation in the original Stickler syndrome kindred

    SciTech Connect

    Williams, C.J.; Ganguly, A.; Considine, E.

    1996-06-14

    Hereditary progressive arthro-ophthalmopathy, or {open_quotes}Stickler syndrome,{close_quotes} is an autosomal dominant osteochondrodysplasia characterized by a variety of ocular and skeletal anomalies which frequently lead to retinal detachment and precocious osteoarthritis. A variety of mutations in the COL2A1 gene have been identified in {open_quotes}Stickler{close_quotes} families; in most cases studied thus far, the consequence of mutation is the premature generation of a stop codon. We report here the characterization of a COL2A1 gene mutation in the original kindred described by Stickler et al. Conformational sensitive gel electrophoresis (CSGE) was used to screen for mutations in the entire COL2A1 gene in an affected member from the kindred. A prominent heteroduplex species was noted in the polymerase chain reaction (PCR) product from a region of the gene including exons 17 to 20. Direct sequencing of PCR-amplified genomic DNA resulted in the identification of a base substitution at the A{sup -2} position of the 3{prime} splice acceptor site of IVS17. Sequencing of DNA from affected and unaffected family members confirmed that the mutation segregated with the disease phenotype. Reverse transcriptase-PCR analysis of poly A+ RNA demonstrated that the mutant allele utilized a cryptic splice site in exon 18 of the gene, eliminating 16 bp at the start of exon 18. This frameshift eventually results in a premature termination codon. These findings are the first report of a splice site mutation in classical Stickler syndrome and they provide a satisfying historical context in which to view COL2A1 mutations in this dysplasia. 25 refs., 3 figs., 1 tab.

  20. Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants

    PubMed Central

    Vallée, Maxime P.; Di Sera, Tonya L.; Nix, David A.; Paquette, Andrew M.; Parsons, Michael T.; Bell, Russel; Hoffman, Andrea; Hogervorst, Frans B. L.; Goldgar, David E.; Spurdle, Amanda B.

    2016-01-01

    ABSTRACT Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs—the “integrated evaluation”—that combines a sequence analysis‐based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis‐based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice‐site fitness program MaxEntScan to generate spliceogenicity‐based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity‐based probability of pathogenicity for BRCA gene single‐nucleotide substitutions. We also updated the BRCA gene Ex‐UV LOVD, available at http://hci‐exlovd.hci.utah.edu, with 77 re‐evaluable variants. PMID:26913838

  1. Effect of Intraoperative Platelet-Rich-Plasma Treatment on Post Operative Donor Site Knee Pain in Patellar Tendon Autograft ACL Reconstruction

    PubMed Central

    Walters, Brian L.; Hobart, Sarah; Porter, David; Hogan, Daniel E.; McHugh, Malachy P.; Bedford, Benjamin B.; Nicholas, Stephen J.; Klein, Devon; Harousseau, Kendall

    2016-01-01

    Objectives: Donor site morbidity in the form of anterior knee pain is a frequent complication after bone-patellar tendon-bone (BPTB) autograft ACL reconstruction. The purpose of this Level I study was to examine the effect of the intraoperative administration of platelet-rich plasma on post operative knee pain and patellar defect healing. Methods: Fifty-nine patients (29±12 y/o) undergoing BPTB ACL reconstruction and eligible to enter the study, were randomized to the treatment (PRP; n=31) or non treatment (sham n=28) arms of the study just prior to surgery. In either case, 10 cc of venous blood was drawn prior to the induction of anesthesia and either discarded (sham) or processed (PRP) for preparation of a PRP gel to be later mixed with donor site bone chips and inserted into the patellar defect. At 12 weeks and 6 months after surgery, patients completed IKDC forms and VAS pain scores for ADLs and kneeling (0-10 scale). Healing indices at the donor site were assessed by MRI at 6 months and included the following measurements taken from axial sequences: AP tendon dimensions at the level of the superior tibial cortex, roof of the intercondylar notch and width at the largest patella graft deficit. Mixed model ANOVA was used to assess the effect of PRP on patient symptoms and MRI indices of donor site healing. The primary dependent variable was VAS kneeling pain. It was estimated that with 25 patients per group there would be 80% power to detect a 1.5-point difference in kneeling pain between treatments at P<0.05. A between group difference of 1.5-points in VAS for kneeling pain was deemed to represent a clinically relevant difference. Results: VAS Kneeling Pain at 12 weeks tended to be lower in the PRP versus placebo group (4.5±3.6 vs. 6.2±2.4, P=0.051) but no difference was apparent at 6 months (3.7±3.2 vs. 4.4±2.9, P=0.41). Kneeling pain decreased from 12 weeks to 6 months (P<0.001) with a trend for a greater decrease in the placebo group (Time by Treatment P

  2. Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-ibeta gene.

    PubMed Central

    Yu, G S; Lu, Y C; Gulick, T

    1998-01-01

    Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors. PMID:9693124

  3. Variation in alternative splicing across human tissues

    PubMed Central

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

    2004-01-01

    Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793

  4. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    PubMed

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  5. Splicing mutation in MVK is a cause of porokeratosis of Mibelli.

    PubMed

    Zeng, Kang; Zhang, Qi-Guo; Li, Li; Duan, Yan; Liang, Yan-Hua

    2014-10-01

    Porokeratosis is a chronic skin disorder characterized by the presence of patches with elevated, thick, keratotic borders, with histological cornoid lamella. Classic porokeratosis of Mibelli (PM) frequently appears in childhood with a risk of malignant transformation. Disseminated superficial actinic porokeratosis (DSAP) is the most common subtype of porokeratosis with genetic heterogeneities, and mevalonate kinase gene (MVK) mutations have been identified in minor portion of DSAP families of Chinese origin. To confirm the previous findings about MVK mutations in DSAP patients and test MVK's role(s) in PM development, we performed genomic sequence analysis for 3 DSAP families and 1 PM family of Chinese origin. We identified a splicing mutation of MVK gene, designated as c.1039+1G>A, in the PM family. No MVK mutations were found in three DSAP families. Sequence analysis for complementary DNA templates from PM lesions of all patients revealed a mutation at splice donor site of intron 10, designated as c.1039+1G>A, leading to the splicing defect and termination codon 52 amino acids after exon 10. Although no MVK mutations in DSAP patients were found as reported previously, we identified MVK simultaneously responsible for PM development. PMID:24781643

  6. Exon circularization requires canonical splice signals.

    PubMed

    Starke, Stefan; Jost, Isabelle; Rossbach, Oliver; Schneider, Tim; Schreiner, Silke; Hung, Lee-Hsueh; Bindereif, Albrecht

    2015-01-01

    Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode. PMID:25543144

  7. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    PubMed

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  8. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  9. The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene.

    PubMed Central

    Menssen, A; Höhmann, S; Martin, W; Schnable, P S; Peterson, P A; Saedler, H; Gierl, A

    1990-01-01

    The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene. Images Fig. 3. Fig. 4. Fig. 6. Fig. 8. PMID:2170105

  10. An artificial riboswitch for controlling pre-mRNA splicing.

    PubMed

    Kim, Dong-Suk; Gusti, Veronica; Pillai, Sailesh G; Gaur, Rajesh K

    2005-11-01

    Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant. PMID:16244133

  11. Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age

    PubMed Central

    Ivanov, Nikolay A.; Tao, Ran; Chenoweth, Joshua G.; Brandtjen, Anna; Mighdoll, Michelle I.; Genova, John D.; McKay, Ronald D.; Jia, Yankai; Weinberger, Daniel R.; Kleinman, Joel E.; Hyde, Thomas M.; Jaffe, Andrew E.

    2016-01-01

    Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), “block” (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts—83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation—while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p

  12. SR proteins are required for nematode trans-splicing in vitro.

    PubMed Central

    Sanford, J R; Bruzik, J P

    1999-01-01

    SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts. PMID:10411135

  13. Species-specific signals for the splicing of a short Drosophila intron in vitro.

    PubMed Central

    Guo, M; Lo, P C; Mount, S M

    1993-01-01

    The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species. Images PMID:8423778

  14. Electrical-splicing connector

    NASA Technical Reports Server (NTRS)

    Stringer, E. J.

    1977-01-01

    Connection can be made without removing insulation, and connector case insulates splice. Device can be made in various sizes and saves time, especially when working on prototype boards with several interconnecting test leads.

  15. What Is the Ideal Free Flap for Soft Tissue Reconstruction? A Ten-Year Experience of Microsurgical Reconstruction Using 334 Latissimus Dorsi Flaps From a Universal Donor Site.

    PubMed

    Kim, Jeong Tae; Kim, Sang Wha; Youn, Seungki; Kim, Youn Hwan

    2015-07-01

    Microsurgical free tissue transfer is regarded as the best available method of tissue reconstruction for intractable defects. The ideal soft tissue flap is thought to be the anterolateral thigh flap. On the basis of 334 procedures involving the latissimus dorsi (LD) flap, we discuss the advantages of the LD flap over the current universal option, and we aimed to establish whether the LD could also gain universal status in all reconstructive fields.Three hundred thirty-four reconstructive procedures using the LD flap were performed in 322 patients between September 2002 and July 2012. In accordance with defect characteristics, we performed 334 procedures using flaps, which included the LD muscle flap with skin graft, the myocutaneous flap, the muscle-sparing flap, the perforator flap, the chimeric flap, and the 2-flap technique using the serratus anterior branch.Flap-related complications occurred in 21 patients (6.3%), including total and partial flap failure. In 253 cases, the donor site was closed primarily, and in the remaining cases, we used split-thickness skin grafts. Donor-site complications occurred in 20 cases (6%). In 11 of the 182 cases, no suitable perforators were identified during surgery.The advantages of the LD as a donor site include the possibility of various harvesting positions without position change, versatility of components, availability of muscle to fill extensive defects, and presence of thick fascia to enable full abdominal reconstruction. On the basis of our experience, we concluded that this flap has the potential to be used as widely as, or in preference to, the anterolateral thigh flap in most reconstructive areas. PMID:25785382

  16. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

    PubMed

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease. PMID:19495418

  17. Viral interactions with components of the splicing machinery.

    PubMed

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses. PMID:27571697

  18. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    SciTech Connect

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. ); Schweikert, H.U. ); Zegers, N.D. ); Hodgins, M.B. )

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  19. A randomized comparison study of Aquacel Ag and Glucan II as donor site dressings with regard to healing time, cosmesis, infection rate, and patient's perceived pain: a pilot study.

    PubMed

    Bailey, Suzanne; Carmean, Melissa; Cinat, Marianne; Burton, Kimberly; Lane, Christopher; Malinoski, Darren

    2011-01-01

    This study was undertaken to compare pain, healing time, infection rate, and cosmetic outcome between Aquacel Ag (Convatec) and Glucan II (Brennan Medical) as donor site dressings. The authors performed a prospective, randomized, patient-controlled study. Eligible patients had two donor sites harvested. One site was dressed with Aquacel Ag and the other site with Glucan II. Patients were followed at set time points for 6 months to determine the rate of epithelialization, patient's perceived pain, infection rate, and the cosmetic outcome. A total of 20 patients were enrolled in the study. All patient data were collected through reepithelialization. The average time to wound healing for Aquacel Ag was 12.5 ± 2.07 days compared with Glucan II 12.7 ± 1.99 days. Perceived pain scores for each donor site were recorded. On postoperative day 5, patients reported significantly less pain with the Aquacel Ag site (Aquacel Ag 1.75 vs Glucan II 2.5, P = .02). Three donor sites showed clinical signs of infection (two Glucan II and one Aquacel Ag) prompting culture and dressing removal. There was no statistically significant difference in cosmetic outcomes of the donor sites at any time point. When comparing Aquacel Ag and Glucan II, our study has determined that there is no significant difference with regard to healing time, infection rates, and cosmetic outcomes. Both dressings are comparable with regard to ease of application and postoperative care. PMID:21844815

  20. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  1. Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells.

    PubMed

    Barbour, Suzanne E; Nguyen, Phuong T; Park, Margaret; Emani, Bhargavi; Lei, Xiaoyong; Kambalapalli, Mamatha; Shultz, Jacqueline C; Wijesinghe, Dayanjan; Chalfant, Charles E; Ramanadham, Sasanka

    2015-04-24

    Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2β (iPLA2β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA2β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2β transgenic mice, whereas islets from global iPLA2β(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2β/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2β. PMID:25762722

  2. Laparoscopic donor nephrectomy.

    PubMed

    Deger, S; Giessing, M; Roigas, J; Wille, A H; Lein, M; Schönberger, B; Loening, S A

    2005-01-01

    Laparoscopic live donor nephrectomy (LDN) has removed disincentives of potential donors and may bear the potential to increase kidney donation. Multiple modifications have been made to abbreviate the learning curve while at the same time guarantee the highest possible level of medical quality for donor and recipient. We reviewed the literature for the evolution of the different LDN techniques and their impact on donor, graft and operating surgeon, including the subtleties of different surgical accesses, vessel handling and organ extraction. We performed a literature search (PubMed, DIMDI, medline) to evaluate the development of the LDN techniques from 1995 to 2003. Today more than 200 centres worldwide perform LDN. Hand-assistance has led to a spread of LDN. Studies comparing open and hand-assisted LDN show a reduction of operating and warm ischaemia times for the hand-assisted LDN. Different surgical access sites (trans- or retroperitoneal), different vessel dissection approaches, donor organ delivery techniques, delivery sites and variations of hand-assistance techniques reflect the evolution of LDN. Proper techniques and their combination for the consecutive surgical steps minimize both warm ischaemia time and operating time while offering the donor a safe minimally invasive laparoscopic procedure. LDN has breathed new life into the moribund field of living kidney donation. Within a few years LDN could become the standard approach in living kidney donation. Surgeons working in this field must be trained thoroughly and well acquainted with the subtleties of the different LDN techniques and their respective advantages and disadvantages. PMID:16754618

  3. A point mutation in an intronic branch site results in aberrant splicing of COL5A1 and in Ehlers-Danlos syndrome type II in two British families.

    PubMed

    Burrows, N P; Nicholls, A C; Richards, A J; Luccarini, C; Harrison, J B; Yates, J R; Pope, F M

    1998-08-01

    Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective-tissue disorders characterized by skin fragility, joint laxity, and skeletal deformities. Type V collagen appears to have a causal role in EDS types I and II, which show phenotypic overlap and may sometimes be allelic. Type V collagen can exist as a heterotrimer, [alpha1(V)]2alpha2(V), and it both coassembles with and regulates type I collagen-fibril diameter. Using an intragenic COL5A1 polymorphism, we have demonstrated linkage, at zero recombination, to the same allele in two large British EDS type II families (LOD scores 4.1 and 4.3). Affected members from each family were heterozygous for a point mutation in intron 32 (IVS32:T-25G), causing the 45-bp exon 33 to be lost from the mRNA in approximately 60% of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. Although both families shared the same marker allele, we have been unable to identify a common genealogy. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, in a collagen gene. Very probably, the resulting in-frame exon skip has a dominant-negative effect due to incorporation of the mutant proalpha chain into the triple-helical molecule. These findings further confirm the importance of type V collagen in the causation of EDS type II, and the novel collagen mutation indicates the importance of the lariat branch site in splicing. PMID:9683580

  4. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.  PMID:21637003

  5. Identification of RNA splicing errors resulting in human ornithine transcarbamylase deficiency.

    PubMed Central

    Carstens, R P; Fenton, W A; Rosenberg, L R

    1991-01-01

    Ornithine transcarbamylase (OTC) is an X-linked, liver-specific enzyme that catalyzes the second step of the urea cycle. In humans, inherited deficiency of OTC in hemizygous affected males usually results in severe ammonia intoxication and early death. To characterize mutations responsible for OTC deficiency, we used the PCR to amplify cDNAs prepared from patient livers which demonstrated no OTC enzyme activity and no OTC cross-reacting material on western blots. In three of seven cases, smaller than normal products were observed. Sequencing of these cDNAs revealed that two were missing exon 7 of the OTC gene and that the other was missing the first 12 bp of exon 5. Sequencing of genomic DNA from these three patients revealed that one mutant missing exon 7 had a T-to-C substitution in the 5' splice donor site of intron 7. The other mutant missing exon 7 had an A-to-G change in the third position of intron 7. It is interesting that both of these mutations resulted in skipping the preceding exon rather than in inclusion of some or all of the affected intron. In the third mutant, an A-to-T substitution was found in the 3' splice acceptor site at the end of intron 4. Here, a cryptic splice acceptor site within exon 5 was used. Northern blotting of liver RNA from these patients demonstrated (a) reduced, but significant, amounts of OTC mRNA in one of the patients who had a deleted exon 7 but (b) very little OTC mRNA in the other two patients. We propose that these point mutations, which result in aberrant splicing of the OTC pre-mRNAs, lead to OTC deficiency through either decreased efficiency of mRNA export from the nucleus to the cytosol or synthesis of enzyme subunits that are unstable and rapidly degraded. We speculate that abnormal mRNA splicing may represent a relatively common mechanism in the pathogenesis of this disease. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2035531

  6. Novel splice-site mutation in WDR62 revealed by whole-exome sequencing in a Sudanese family with primary microcephaly.

    PubMed

    Bastaki, Fatma; Mohamed, Madiha; Nair, Pratibha; Saif, Fatima; Tawfiq, Nafisa; Aithala, Gururaj; El-Halik, Majdi; Al-Ali, Mahmoud; Hamzeh, Abdul Rezzak

    2016-05-01

    The WDR62 gene encodes a scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. It plays a critical role in laying out various cellular layers in the cerebral cortex during embryogenesis, and hence the dramatic clinical features resulting from WDR62 mutations. These mutations are associated with autosomal recessive primary microcephaly 2, with or without cortical malformations (MCPH2). Using whole exome sequencing we uncovered a novel WDR62 variant; c.390G > A, from two Sudanese siblings whose parents are first cousins. The patients suffered MCPH2 with incomplete lissencephaly and developmental delay. The mutation affects the last nucleotide of exon4, and probably leads to aberrant splicing, which may result in a truncated protein lacking all functional domains. PMID:26577670

  7. Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders.

    PubMed

    Pettitt, Jonathan; Müller, Berndt; Stansfield, Ian; Connolly, Bernadette

    2008-04-01

    The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda. PMID:18256244

  8. Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells

    PubMed Central

    Turton, Keren B.; Annis, Douglas S.; Rui, Lixin; Esnault, Stephane; Mosher, Deane F.

    2015-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3β, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-β, and ΔS-β) rather than the three reported in publically available databases from which ΔS-β is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-β. ΔS-α and ΔS-β together comprised 15.6±4.0 % (mean±SD, n=21) of the total. The percentage of STAT3β variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina’s “BodyMap” RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-β, and ΔS-β variants of STAT3. PMID:25984943

  9. Mechanisms and Regulation of Alternative Pre-mRNA Splicing

    PubMed Central

    Lee, Yeon

    2015-01-01

    Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

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

    SciTech Connect

    Markus, M. Andrea; Heinrich, Bettina; Raitskin, Oleg; Adams, David J.; Mangs, Helena; Goy, Christine; Ladomery, Michael; Sperling, Ruth; Stamm, Stefan; Morris, Brian J. . E-mail: brianm@medsci.usyd.edu.au

    2006-10-15

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

  11. Splice Form Dependence of b-Neurexin/Neuroligin Binding Interactions

    SciTech Connect

    Koehnke, J.; Katsamba, P; Ahlsen, G; Bahna, F; Vendome, J; Honig, B; Shapiro, L; Jin, X

    2010-01-01

    Alternatively spliced {beta}-neurexins ({beta}-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a {beta}-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced {beta}-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all {beta}-NRX/NL pairs. The presence of the NL1 B splice insertion lowers {beta}-NRX binding affinity by 2-fold, while {beta}-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated {beta}-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new {beta} strand that replaces the {beta}10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in {beta}-NRX/NL-mediated synaptic recognition.

  12. Multifunctional in situ photopolymerized semi-interpenetrating network system is an effective donor site dressing: a cross comparison study in a swine model.

    PubMed

    Kleinbeck, Kyle R; Faucher, Lee; Kao, Weiyuan John

    2009-01-01

    Effective dressings for donor sites or other partial thickness wounds must promote removal of nonviable or necrotic tissue, eradication and prevention of microbial infiltrate, exudate absorbance, and regrowth of healthy epidermis and dermis. There are many commonly used products that facilitate these processes. Established properties of an in situ photopolymerizable semi-interpenetrating network (sIPN) suggest that it is also a viable treatment option. The widely varying material properties suggest that these dressing treatments may elicit different healing responses via different cellular mechanisms. In this study, we sought to resolve the differences in healing between Acticoat, sIPN, nonadherent dressing with Tisseel, and Xeroform dressing treatments in a porcine partial thickness wound model. Donor site wounds were produced on pigs at two cut depths and dressed with Acticoat, sIPN, nonadherent dressing with Tisseel, and Xeroform with alternatively placed autografts to provide a control area between each test site. Pigs were euthanized at 4, 7, 14, and 42 days for macroscopic examination and biopsy collection. Biopsies were analyzed histologically by two blinded observers for cellular densities and regional thicknesses within the tissue. sIPN- and Xeroform-treated wounds were healed by 7 days, and Acticoat- and nonadherent dressing with Tisseel-treated wounds were healed by 14 days. Inflammatory responses were between comparable treatment type across all time periods. Dermal granulation features increased with time but were not significantly different. All dressing treatments elicited wound healing without outstanding toxicity or pathology indicating that sIPN is a comparable and viable treatment for partial thickness wounds. PMID:19131760

  13. Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.

    PubMed

    Ramalho, Anabela S; Clarke, Luka A; Sousa, Marisa; Felicio, Verónica; Barreto, Celeste; Lopes, Carlos; Amaral, Margarida D

    2016-01-01

    The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis. PMID:25735457

  14. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    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.

  15. A novel synonymous mutation in the MPZ gene causing an aberrant splicing pattern and Charcot-Marie-Tooth disease type 1b.

    PubMed

    Corrado, L; Magri, S; Bagarotti, A; Carecchio, M; Piscosquito, G; Pareyson, D; Varrasi, C; Vecchio, D; Zonta, A; Cantello, R; Taroni, F; D'Alfonso, S

    2016-08-01

    Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy with a heterogeneous genetic background. Here, we describe two CMT1B families with a mild sensory-motor neuropathy and a novel synonymous variant (c.309G > T, p.G103G) in exon 3 of the MPZ gene. Next generation sequencing analysis on a 94 CMT gene panel showed no mutations in other disease genes. In vitro splicing assay and mRNA expression analysis indicated that the c.309T variant enhances a cryptic donor splice site at position c.304 resulting in the markedly increased expression of the r.304_448del alternative transcript in patients' cells. This transcript is predicted to encode a truncated P0 protein (p.V102Cfs11*) lacking the transmembrane domain, thus suggesting a possible haploinsufficiency mechanism for this mutation. This is the third reported synonymous MPZ variant associated with CMT1 and affecting splicing. These data confirm the functional impact of synonymous variants on MPZ splicing and their possible role as disease-causing mutations rather than silent polymorphisms. PMID:27344971

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

    PubMed Central

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

    2014-01-01

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

  17. Functional studies on the ATM intronic splicing processing element

    PubMed Central

    Lewandowska, Marzena A.; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E.; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing. PMID:16030351

  18. Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila

    PubMed Central

    Duff, Michael O.; Olson, Sara; Wei, Xintao; Garrett, Sandra C.; Osman, Ahmad; Bolisetty, Mohan; Plocik, Alex; Celniker, Susan; Graveley, Brenton R.

    2015-01-01

    Recursive splicing is a process in which large introns are removed in multiple steps by resplicing at ratchet points - 5′ splice sites recreated after splicing1. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene1 and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing2,3. Here, we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues, and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identified four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together these results indicate that recursive splicing is commonly used in Drosophila, occurs in human and provides insight into the mechanisms by which some large introns are removed. PMID:25970244

  19. Technical refinements of composite thoracodorsal system free flaps for 1-stage lower extremity reconstruction resulting in reduced donor-site morbidity.

    PubMed

    Bannasch, Holger; Strohm, Peter C; Al Awadi, Khalid; Stark, G Björn; Momeni, Arash

    2008-04-01

    A multitude of local flaps has been suggested for lower extremity reconstruction. However, the gold standard for defect coverage remains free tissue transfer. In this regard, the scapular vascular axis is a well-established source of expendable skin, fascia, muscle, and bone for use in free flap reconstruction of defects requiring bone and soft tissue in complex 3-dimensional relationships. Composite bone and soft-tissue flaps derived from the subscapular vascular axis include the osteocutaneous scapular flap, the "latissimus/bone flap," and the thoracodorsal artery perforator-scapular osteocutaneous flap.Patient outcome following reconstruction of lower extremity defects with composite free flaps from the thoracodorsal system were analyzed. Here, we demonstrate the execution of technical refinements on free composite flap transfers based on the thoracodorsal vascular axis, thus resulting in a stepwise reduction of donor-site morbidity. PMID:18362565

  20. Copper-Sulfur Complexes Supported by N-Donor Ligands: Towards Models of the Cu(Z) Site in Nitrous Oxide Reductase.

    PubMed

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

    2008-03-01

    The distinctive structure of the [(his)(7)Cu(4)(μ-S)](n+) cluster in the "Cu(Z)" 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

  1. In vitro splicing of fibronectin pre-mRNAs.

    PubMed Central

    Norton, P A; Hynes, R O

    1990-01-01

    We have investigated the alternative splicing of the EIIIB exon of the rat fibronectin gene. Mini-gene constructs containing this exon and portions of adjacent introns and exons, when transfected into HeLa cells, are transcribed and spliced, but omit the EIIIB exon. In vitro, HeLa nuclear extracts similarly splice out (skip) the EIIIB exon from similarly structured transcripts. Therefore, the HeLa splicing apparatus recognizes as atypical the EIIIB exon and its flanking intron sequences, both in vivo and in vitro. We also report that alterations in the ionic conditions of the in vitro splicing reaction can promote the initiation of EIIIB exon inclusion, as reflected by the formation of intermediate and product RNAs related to the removal of the intron upstream of EIIIB. Processing of this intron correlates with the formation of complexes resembling intermediates in spliceosome assembly. The branch sites involved in this alternative processing pathway are rather distant from the EIIIB 3' splice site, and lie within a region which is well conserved in the fibronectin genes of other species. Thus, the intron upstream of EIIIB shows singular structure and behavior which probably have a bearing on the regulated alternative splicing of this exon. Images PMID:2377454

  2. Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations

    PubMed Central

    Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P

    2012-01-01

    UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5′ and 3′ of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype. PMID:22189268

  3. The transcription factor c-Myb affects pre-mRNA splicing

    SciTech Connect

    Orvain, Christophe; Matre, Vilborg; Gabrielsen, Odd S.

    2008-07-25

    c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF{sup 65} and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.

  4. Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.

    PubMed

    Hawke, Lindsey; Bowman, Mackenzie L; Poon, Man-Chiu; Scully, Mary-Frances; Rivard, Georges-Etienne; James, Paula D

    2016-07-28

    Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells. PMID:27317792

  5. Effect of Schneiderian membrane perforation on sinus lift graft outcome using two different donor sites: a retrospective study of 105 maxillary sinus elevation procedures

    PubMed Central

    Sakkas, Andreas; Konstantinidis, Ioannis; Winter, Karsten; Schramm, Alexander; Wilde, Frank

    2016-01-01

    Background: Sinuslift is meanwhile an established method of bone augmentation in the posterior maxilla. Aim of the study was to evaluate the significance of intraoperative Schneiderian membrane perforations during maxillary sinus floor elevation surgery using autogenous bone harvested from two different donor sites using a Safescraper device on the success rate, graft survival and implant integration. Methods: The investigators conducted a retrospective cohort study at the Department of Oral and Maxillofacial Surgery of Military Hospital Ulm composed of patients with severe maxillary atrophy who underwent sinus augmentation from January 2011 until December 2011. Ninety-nine consecutive patients (89 men, 10 women) with a mean age of 43.1 years underwent sinus graft procedures in a 2-stage procedure using the lateral wall approach, as described by Tatum (1986). Data on patient age, smoking status, donor site and surgical complications were recorded and the relationship between Schneiderian membrane perforation and complication rate was evaluated. Dental implants were inserted 4 months after grafting. Results: A total of 105 sinus lift procedures were performed in 99 patients. Sixty-one patients (61.6%) underwent sinus elevation with autogenous bone from the buccal sinus wall, while 38 patients (38.4%) bone harvesting from the iliac crest. Intraoperative perforation of the Schneiderian membrane was observed in 11 of the 105 sinuses (10.4%). These perforations resulted in 4 (36.3%) of the cases in major postoperative complications accompanied by swelling and wound infection. Membrane perforations were slightly associated with the appearance of postoperative complications (p=0.0762). In 2.4% of all cases, regarding 2 patients the final rehabilitation with dental implants was not possible because of extensive bone resorption. Conclusion: Intraoperative complications performing sinus augmentation may lead to postoperative complications. With careful clinical and

  6. Long-term effect on foot and ankle donor site following vascularized fibular graft resection in children.

    PubMed

    Sulaiman, Abdul Razak; Wan, Zulmi; Awang, Shukrimi; Che Ahmad, Aminudin; Halim, Ahmad Sukari; Ahmad Mohd Zain, Rajaei

    2015-09-01

    This study was carried out to evaluate the long-term effect on the donor side of the foot and ankle following vascularized fibular graft resection in children. Eight patients underwent resection of the fibula for the purpose of a vascularized fibular graft by a surgical team who practiced leaving at least 6 cm residual distal fibula. The age of these children at the time of surgery was between 3 and 12 years. They were reviewed between 3 and 12 years after surgery. Two patients who underwent resection of the middle shaft of the fibula at 3 and 5 years of age developed abnormal growth of the distal tibia, leading to ankle valgus. They were treated with growth modulation of the distal tibial physis and supramalleolar osteotomy with tibiofibular synostosis. Another patient who underwent the entire proximal fibula resection at the age of 6 years had developed hindfoot valgus because of weakness of the tibialis posterior muscle. He required talonavicular fusion and flexor hallucis to tibialis posterior muscle transfer. Patients operated at the age of older than 8 years neither had ankle nor hindfoot deformity. We concluded that resection of the middle shaft of the fibula for the purpose of a vascularized fibula graft, leaving a 6 cm distal fibular stump in children younger than 6 years old, may give rise to abnormal growth of the distal tibial physis, leading to valgus ankle. The entire proximal fibular resection for the similar purpose in a 6-year-old child may give rise to weakness of tibialis posterior and hindfoot valgus. PMID:26049965

  7. A novel SMARCAL1 missense mutation that affects splicing in a severely affected Schimke immunoosseous dysplasia patient.

    PubMed

    Barraza-García, Jimena; Rivera-Pedroza, Carlos I; Belinchón, Alberta; Fernández-Camblor, Carlota; Valenciano-Fuente, Blanca; Lapunzina, Pablo; Heath, Karen E

    2016-08-01

    Schimke immunoosseous dysplasia (SIOD) is an autosomal recessive disease characterized by skeletal dysplasia, focal segmental glomerulosclerosis, renal failure and immunodeficiency. In this work, we report the molecular studies undertaken in a severely affected SIOD patient that died at six years old due to nephropathy. The patient was screened for mutations using a targeted skeletal dysplasias panel. A homozygous novel missense mutation was identified, c.1615C > G (p.[Leu539Val]) that was predicted as mildly pathogenic by in silico pathogenicity prediction tools. However, splicing prediction software suggested that this variant may create a new splicing donor site in exon 9, which was subsequently confirmed using a minigene assay in HEK293 cells. Thus, the splicing alteration, c.1615C > G; r.1615c > g, 1615_1644del; (p.[Leu539_Ile548del]), results in the loss of 10 amino acids of the HARP-ATPase catalytic domain and the RPA-binding domain. Several studies have demonstrated a weak genotype-phenotype correlation among such patients. Thus, the molecular characterization has helped us to understand why a predicted weakly pathogenic missense mutation results in severe SIOD and should be considered in similar scenarios. PMID:27282802

  8. SPA: A Probabilistic Algorithm for Spliced Alignment

    PubMed Central

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

    2006-01-01

    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

  9. Unexpected CEP290 mRNA Splicing in a Humanized Knock-In Mouse Model for Leber Congenital Amaurosis

    PubMed Central

    Garanto, Alejandro; van Beersum, Sylvia E. C.; Peters, Theo A.; Roepman, Ronald; Cremers, Frans P. M.; Collin, Rob W. J.

    2013-01-01

    Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations. PMID:24223178

  10. 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

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

    2015-02-01

    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

  11. Using patient-specific induced pluripotent stem cells to interrogate the pathogenicity of a novel retinal pigment epithelium-specific 65 kDa cryptic splice site mutation and confirm eligibility for enrollment into a clinical gene augmentation trial.

    PubMed

    Tucker, Budd A; Cranston, Cathryn M; Anfinson, Kristin A; Shrestha, Suruchi; Streb, Luan M; Leon, Alejandro; Mullins, Robert F; Stone, Edwin M

    2015-12-01

    Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However, the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations, and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an A>G change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE, control iPSC-RPE cells, and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study, we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations, which can enable positive clinical outcomes. PMID:26364624

  12. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  13. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    PubMed Central

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  14. RNA structure in splicing: An evolutionary perspective.

    PubMed

    Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G

    2016-09-01

    Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements. PMID:27454491

  15. A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

    PubMed Central

    Hastings, M L; Wilson, C M; Munroe, S H

    2001-01-01

    The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA. PMID:11421362

  16. 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)

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

    2011-12-01

    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.

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

    PubMed

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

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

  18. Purifying Selection on Exonic Splice Enhancers in Intronless Genes

    PubMed Central

    Savisaar, Rosina; Hurst, Laurence D.

    2016-01-01

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

  19. BUILDING ROBUST TRANSCRIPTOMES WITH MASTER SPLICING FACTORS

    PubMed Central

    Jangi, Mohini; Sharp, Phillip A.

    2014-01-01

    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

  20. Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy

    PubMed Central

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

    2013-01-01

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

  1. Genome-wide survey of Alternative Splicing in Sorghum Bicolor.

    PubMed

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

    2014-07-01

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

  2. Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis

    PubMed Central

    Stadler, Michael B; Shomron, Noam; Yeo, Gene W; Schneider, Aniket; Xiao, Xinshu; Burge, Christopher B

    2006-01-01

    Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences. PMID:17121466

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

    PubMed Central

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

    2011-01-01

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

  4. The landscape of alternative splicing in cervical squamous cell carcinoma

    PubMed Central

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

    2015-01-01

    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

  5. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  6. Unusual Phenotypic Features in a Patient with a Novel Splice Mutation in the GHRHR Gene

    PubMed Central

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (−5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  7. Unusual phenotypic features in a patient with a novel splice mutation in the GHRHR gene.

    PubMed

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (-5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  8. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  9. Sip1, a novel RS domain-containing protein essential for pre-mRNA splicing.

    PubMed

    Zhang, W J; Wu, J Y

    1998-02-01

    Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5' and 3' splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors. PMID:9447963

  10. Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

    PubMed Central

    McNeer, Nicole A.; Schleifman, Erica B.; Cuthbert, Amy; Brehm, Michael; Jackson, Andrew; Cheng, Christopher; Anandalingam, Kavitha; Kumar, Priti; Shultz, Leonard D.; Greiner, Dale L.

    2013-01-01

    In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2rγnull mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo. PMID:23076379

  11. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells

    PubMed Central

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  12. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

    PubMed

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  13. Entropic contributions to the splicing process

    NASA Astrophysics Data System (ADS)

    Osella, Matteo; Caselle, Michele

    2009-12-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model.

  14. Extensive aggregation of α-synuclein and tau in juvenile-onset neuroaxonal dystrophy: an autopsied individual with a novel mutation in the PLA2G6 gene-splicing site

    PubMed Central

    2013-01-01

    Background Infantile neuroaxonal dystrophy (INAD) is a rare autosomal-recessive neurodegenerative disorder. Patients with INAD usually show neurological symptoms with infant onset and die in childhood. Recently, it was reported that mutations in the PLA2G6 gene cause INAD, but neuropathological analysis of genetically confirmed individuals with neuroaxonal dystrophy has been limited. Results Here, we report a Japanese individual with neuroaxonal dystrophy associated with compound heterozygous mutations in the PLA2G6 gene. A novel splice-site mutation resulting in skipping and missense mutations (p.R538C) in exon 9 was identified in the patient. This patient initially presented with cerebellar ataxia at the age of 3 years, which was followed by symptoms of mental retardation, extrapyramidal signs, and epileptic seizure. The patient survived until 20 years of age. Neuropathological findings were characterized by numerous axonal spheroids, brain iron deposition, cerebellar neuronal loss, phosphorylated alpha-synuclein-positive Lewy bodies (LBs), and phosphorylated-tau-positive neurofibrillary tangles. In particular, LB pathology exhibited a unique distribution with extremely severe cortical involvement. Conclusions Our results support a genetic clinical view that compound heterozygous mutations with potential residual protein function are associated with a relatively mild phenotype. Moreover, the severe LB pathology suggests that dysfunction of the PLA2G6 gene primarily contributes to LB formation. PMID:24252552

  15. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    SciTech Connect

    Morcos, Paul A. . E-mail: pmorcos@gene-tools.com

    2007-06-29

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a {beta}-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

  16. Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma

    PubMed Central

    Fu, Yao; Zhao, Xingli; Li, Zhaohui; Wei, Jun; Tian, Yu

    2016-01-01

    The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma. PMID:27446352

  17. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  18. Therapeutic targeting of splicing in cancer.

    PubMed

    Lee, Stanley Chun-Wei; Abdel-Wahab, Omar

    2016-09-01

    Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer. PMID:27603132

  19. Regulation of alternative pre-mRNA splicing during erythroid differentiation.

    PubMed

    Hou, V C; Conboy, J G

    2001-03-01

    Although the mature enucleated erythrocyte is no longer active in nuclear processes such as pre-mRNA splicing, the function of many of its major structural proteins is dependent on alternative splicing choices made during the earlier stages of erythropoiesis. These splicing decisions fundamentally regulate many aspects of protein structure and function by governing the inclusion or exclusion of exons that encode protein interaction domains, regulatory signals, or translation initiation or termination sites. Alternative splicing events may be partially or entirely erythroid-specific, ie, distinct from the splicing patterns imposed on the same transcripts in nonerythroid cells. Moreover, differentiation stage-specific splicing "switches" may alter the structure and function of erythroid proteins in physiologically important ways as the cell is morphologically and functionally remodeled during normal differentiation. Derangements in the splicing of individual mutated pre-mRNAs can produce synthesis of truncated or unstable proteins that are responsible for numerous erythrocyte disorders. This review will summarize the salient features of regulated alternative splicing in general, review existing information concerning the widespread extent of alternative splicing among erythroid genes, and describe recent studies that are beginning to uncover the mechanisms that regulate an erythroid splicing switch in the protein 4.1R gene. PMID:11224680

  20. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

    NASA Astrophysics Data System (ADS)

    Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  1. Accurate Splicing of HDAC6 Pre-mRNA Requires SON

    PubMed Central

    Battini, Vishnu Priya; Bubulya, Athanasios; Bubulya, Paula A.

    2015-01-01

    Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Our lab previously reported that the SR-like protein SON maintains organization of pre-mRNA splicing factors in nuclear speckles as well as splicing of many human transcripts including mRNAs coding for the chromatin-modifying enzymes HDAC6, ADA and SETD8. However, the mechanism by which SON maintains accurate splicing is unknown. To build tools for understanding SON-dependent pre-mRNA splicing, we constructed a minigene reporter plasmid driving expression of the genomic sequence spanning exons 26 through 29 of HDAC6. Following SON depletion, we observed altered splicing of HDAC6 reporter transcripts that showed exclusion of exons 27 and 28, reflecting the splicing patterns of endogenous HDAC6 mRNA. Importantly, loss of HDAC6 biological function was also observed, as indicated by truncated HDAC6 protein and corresponding absence of aggresome assembly activities of HDAC6 binding-of-ubiquitin zinc finger (BUZ) domain. We therefore propose that SON-mediated splicing regulation of HDAC6 is essential for supporting protein degradation pathways that prevent human disease. PMID:25782155

  2. Trans-splicing of pre-mRNA is predicted to occur in a wide range of organisms including vertebrates.

    PubMed Central

    Dandekar, T; Sibbald, P R

    1990-01-01

    Several known trans-splicing RNA structures were used to define a canonical trans-splicing structure which was then used to perform a computer search of the EMBL nucleotide database. In addition to most known trans-splicing structures, many putative new trans-splicing sites were detected. These were found in a broad range of organisms including the vertebrates. Control experiments indicate that the search predicts known false positives at a rate of only 20%. Trans-splicing may therefore be a very wide-spread phenomenon. PMID:2395638

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

    PubMed Central

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

    2014-01-01

    The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ. PMID:25025302

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

    PubMed

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

    2015-01-01

    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

  5. nagnag: Identification and quantification of NAGNAG alternative splicing using RNA-Seq data.

    PubMed

    Yan, Xiaoyan; Sablok, Gaurav; Feng, Gang; Ma, Jiaxin; Zhao, Hongwei; Sun, Xiaoyong

    2015-07-01

    Regulation of proteome diversity by alternative splicing has been widely demonstrated in plants and animals. NAGNAG splicing, which was recently defined as a tissue specific event, results in the production of two distinct isoforms that are distinguished by three nucleotides (NAG) as a consequence of the intron proximal or distal to the splice site. Since the NAGNAG mechanism is not well characterized, tools for the identification and quantification of NAGNAG splicing events remain under-developed. Here we report nagnag, an R-based NAGNAG splicing detection tool, which accurately identifies and quantifies NAGNAG splicing events using RNA-Seq. Overall, nagnag produces user-friendly visualization reports and highlights differences between the DNA/RNA/protein across the identified isoforms of the reported gene. The package is available on https://sourceforge.net/projects/nagnag/files/; or http://genome.sdau.edu.cn/research/software/nagnag.html. PMID:26028313

  6. Characterization of the Regulation of CD46 RNA Alternative Splicing.

    PubMed

    Tang, Sze Jing; Luo, Shufang; Ho, Jia Xin Jessie; Ly, Phuong Thao; Goh, Eling; Roca, Xavier

    2016-07-01

    Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5' splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease. PMID:27226545

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

    PubMed

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

    2015-03-17

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

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

    PubMed Central

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

    2015-01-01

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

  9. Evolutionarily conserved autoregulation of alternative pre-mRNA splicing by ribosomal protein L10a

    PubMed Central

    Takei, Satomi; Togo-Ohno, Marina; Suzuki, Yutaka; Kuroyanagi, Hidehito

    2016-01-01

    Alternative splicing of pre-mRNAs can regulate expression of protein-coding genes by generating unproductive mRNAs rapidly degraded by nonsense-mediated mRNA decay (NMD). Many of the genes directly regulated by alternative splicing coupled with NMD (AS-NMD) are related to RNA metabolism, but the repertoire of genes regulated by AS-NMD in vivo is to be determined. Here, we analyzed transcriptome data of wild-type and NMD-defective mutant strains of the nematode worm Caenorhabditis elegans and demonstrate that eight of the 82 cytoplasmic ribosomal protein (rp) genes generate unproductively spliced mRNAs. Knockdown of any of the eight rp genes exerted a dynamic and compensatory effect on alternative splicing of its own transcript and inverse effects on that of the other rp genes. A large subunit protein L10a, termed RPL-1 in nematodes, directly and specifically binds to an evolutionarily conserved 39-nt stretch termed L10ARE between the two alternative 5′ splice sites in its own pre-mRNA to switch the splice site choice. Furthermore, L10ARE-mediated splicing autoregulation of the L10a-coding gene is conserved in vertebrates. These results indicate that L10a is an evolutionarily conserved splicing regulator and that homeostasis of a subset of the rp genes are regulated at the level of pre-mRNA splicing in vivo. PMID:26961311

  10. Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation, and tumor characteristics.

    PubMed

    Arnold, Sven; Buchanan, Daniel D; Barker, Melissa; Jaskowski, Lesley; Walsh, Michael D; Birney, Genevieve; Woods, Michael O; Hopper, John L; Jenkins, Mark A; Brown, Melissa A; Tavtigian, Sean V; Goldgar, David E; Young, Joanne P; Spurdle, Amanda B

    2009-05-01

    Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice-site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data. PMID:19267393

  11. Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation and tumor characteristics

    PubMed Central

    Arnold, Sven; Buchanan, Daniel D.; Barker, Melissa; Jaskowski, Lesley; Walsh, Michael D.; Birney, Genevieve; Woods, Michael O.; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tavtigian, Sean V.; Goldgar, David E.; Young, Joanne P.; Spurdle, Amanda B.

    2009-01-01

    Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for ten variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16/19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach which includes in vitro, tumor pathology, clinical, and evolutionary conservation data. PMID:19267393

  12. Genomic functions of U2AF in constitutive and regulated splicing

    PubMed Central

    Wu, Tongbin; Fu, Xiang-Dong

    2015-01-01

    The U2AF heterodimer is generally accepted to play a vital role in defining functional 3′ splice sites in pre-mRNA splicing. Given prevalent mutations in U2AF, particularly in the U2AF1 gene (which encodes for the U2AF35 subunit) in blood disorders and other human cancers, there are renewed interests in these classic splicing factors to further understand their regulatory functions in RNA metabolism in both physiological and disease settings. We recently reported that U2AF has a maximal capacity to directly bind ˜88% of functional 3′ splice sites in the human genome and that numerous U2AF binding events also occur in various exonic and intronic locations, thus providing additional mechanisms for the regulation of alternative splicing besides their traditional role in titrating weak splice sites in the cell. These findings, coupled with the existence of multiple related proteins to both U2AF65 and U2AF35, beg a series of questions on the universal role of U2AF in functional 3′ splice site definition, their binding specificities in vivo, potential mechanisms to bypass their requirement for certain intron removal events, contribution of splicing-independent functions of U2AF to important cellular functions, and the mechanism for U2AF mutations to invoke specific diseases in humans. PMID:25901584

  13. NOUGHT MAY ENDURE BUT MUTABILITY*: SPLICEOSOME DYNAMICS AND THE REGULATION OF SPLICING

    PubMed Central

    Smith, Duncan J.; Query, Charles C.; Konarska, Maria M.

    2008-01-01

    SUMMARY The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction. PMID:18570869

  14. Minimum Factorization Agreement of Spliced ESTs

    NASA Astrophysics Data System (ADS)

    Bonizzoni, Paola; Della Vedova, Gianluca; Dondi, Riccardo; Pirola, Yuri; Rizzi, Raffaella

    Producing spliced EST sequences is a fundamental task in the computational problem of reconstructing splice and transcript variants, a crucial step in the alternative splicing investigation. Now, given an EST sequence, there can be several spliced EST sequences associated to it, since the original EST sequences may have different alignments against wide genomic regions.

  15. LDLR gene synonymous mutation c.1813C>T results in mRNA splicing variation in a kindred with familial hypercholesterolaemia.

    PubMed

    Ho, Clement K M; Musa, Fathel Rahman; Bell, Christine; Walker, Simon W

    2015-11-01

    Familial hypercholesterolaemia, one of the most common inherited diseases in the general population, is associated with mutations in at least three different genes including the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and protein convertase subtilisin/kexin type 9 (PCSK9) genes. In this report, we describe an unclassified DNA variant (c.1813C>T; p.Leu605Leu) within exon 12 of the LDLR gene in a kindred in which familial hypercholesterolaemia is associated with c.1813C>T heterozygosity. In silico analysis suggested that c.1813C>T might affect splicing of the LDLR gene by creating a cryptic donor splice site, which was confirmed by RT-PCR coupled with cDNA sequencing, to result in the loss of 34 base pairs in the coding sequence. The latter truncated mRNA is predicted to generate a frameshift leading to a premature stop at codon 652 and early termination of the low density lipoprotein receptor polypeptide chain, and thus provides a molecular basis for the hypercholesterolaemic phenotype. This case report highlights the emerging utility of RNA studies for the molecular diagnosis of familial hypercholesterolaemia in patients with potential mRNA splicing variants. PMID:25624525

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

    USGS Publications Warehouse

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

    2012-01-01

    The gene encoding IgH δ 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 δ-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 δ 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 δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ 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.

  17. Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

    PubMed Central

    Leprince, D; Duterque-Coquillaud, M; Li, R P; Henry, C; Flourens, A; Debuire, B; Stehelin, D

    1988-01-01

    Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence. Images PMID:2841475

  18. Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death.

    PubMed

    Zaragoza, Michael V; Fung, Lianna; Jensen, Ember; Oh, Frances; Cung, Katherine; McCarthy, Linda A; Tran, Christine K; Hoang, Van; Hakim, Simin A; Grosberg, Anna

    2016-01-01

    The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency. PMID:27182706

  19. Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death

    PubMed Central

    Zaragoza, Michael V.; Fung, Lianna; Jensen, Ember; Oh, Frances; Cung, Katherine; McCarthy, Linda A.; Tran, Christine K.; Hoang, Van; Hakim, Simin A.; Grosberg, Anna

    2016-01-01

    The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency. PMID:27182706

  20. Sec16 alternative splicing dynamically controls COPII transport efficiency.

    PubMed

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  1. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  2. Splicing Efficiently Couples Optical Fibers

    NASA Technical Reports Server (NTRS)

    Lutes, G. F.

    1985-01-01

    Method of splicing single-mode optical fibers results in very low transmission losses through joined fiber ends. Coupling losses between joined optical-fiber ends only 0.1 dB. Method needs no special operator training.

  3. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.

    PubMed

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-07-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  4. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  5. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

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

    PubMed

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

    2014-05-01

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

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

    PubMed Central

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

    2015-01-01

    The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5′ splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3′ splice site. The 5′ and 3′ splice site complexes are thought to be joined together by protein–protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF65. Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5′ and 3′ splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival. PMID:25605964

  8. Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

    PubMed Central

    2012-01-01

    Introduction The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. PMID:22632462

  9. Hyperfine and Nuclear Quadrupole Tensors of Nitrogen Donors in the QA Site of Bacterial Reaction Centers: Correlation of the Histidine Nδ Tensors with Hydrogen Bond Strength

    PubMed Central

    2015-01-01

    X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the QA site semiquinone (SQA) with nitrogens from the local protein environment in natural abundance 14N and in 15N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 Nδ and Ala-M260 peptide nitrogen (Np) were estimated through simultaneous simulation of the Q-band 15N Davies ENDOR, X- and Q-band 14,15N HYSCORE, and X-band 14N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(14N) = 2.3 MHz, T = 0.3 MHz for His-M219 Nδ and a(14N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 Nδ is established as the stronger of the two H-bond donors, Ala-M260 Np is found to have the larger value of a(14N). The nuclear quadrupole coupling constants were estimated as e2Qq/4h = 0.38 MHz, η = 0.97 and e2Qq/4h = 0.74 MHz, η = 0.59 for His-M219 Nδ and Ala-M260 Np, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes–Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter η to be a sensitive probe of the histidine Nδ–semiquinone hydrogen bond strength. This is supported by a strong correlation observed between η and the isotropic coupling constant a(14N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(14N) and η will provide an important structural characterization tool in future studies of semiquinone-binding proteins. PMID:25026433

  10. SpliceVista, a Tool for Splice Variant Identification and Visualization in Shotgun Proteomics Data*

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-06-01

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

  12. The mammalian homolog of suppressor-of-white-apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS.

    PubMed

    Sarkissian, M; Winne, A; Lafyatis, R

    1996-12-01

    We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(wa) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5' splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5' splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire IIICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors. PMID:8940107

  13. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing. PMID:15972844

  14. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing “minigene” in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  15. Convergent origins and rapid evolution of spliced leader trans-splicing in Metazoa: Insights from the Ctenophora and Hydrozoa

    PubMed Central

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

    2010-01-01

    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

  16. Alternative Splice Transcripts for MHC Class I-like MICA Encode Novel NKG2D Ligands with Agonist or Antagonist Functions.

    PubMed

    Gavlovsky, Pierre-Jean; Tonnerre, Pierre; Gérard, Nathalie; Nedellec, Steven; Daman, Andrew W; McFarland, Benjamin J; Charreau, Béatrice

    2016-08-01

    MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins are ligands of the activating NKG2D receptor involved in cancer and immune surveillance of infection. Structurally, MICA/B proteins contain an α3 domain, whereas UL16-binding proteins do not. We identified novel alternative splice transcripts for MICA encoding five novel MICA isoforms: MICA-A, -B1, -B2, -C, and -D. Alternative splicing associates with MICA*015 and *017 and results from a point deletion (G) in the 5' splice donor site of MICA intron 4 leading to exon 3 and exon 4 skipping and/or deletions. These changes delete the α3 domain in all isoforms, and the α2 domain in the majority of isoforms (A, B1, C, and D). Endothelial and hematopoietic cells contained endogenous alternative splice transcripts and isoforms. MICA-B1, -B2, and -D bound NKG2D by surface plasmon resonance and were expressed at the cell surface. Functionally, MICA-B2 contains two extracellular domains (α1 and α2) and is a novel potent agonist ligand for NKG2D. We found that MICA-D is a new truncated form of MICA with weak affinity for NKG2D despite lacking α2 and α3 domains. MICA-D may functionally impair NKG2D activation by competing with full-length MICA or MICA-B2 for NKG2D engagement. Our study established NKG2D binding for recombinant MICA-B1 but found no function for this isoform. New truncated MICA isoforms exhibit a range of functions that may drive unexpected immune mechanisms and provide new tools for immunotherapy. PMID:27342847

  17. Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

    PubMed Central

    Ohno, K; Tsujino, A; Shen, X; Milone, M; Engel, A

    2005-01-01

    Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing. PMID:16061559

  18. Identifying splicing regulatory elements with de Bruijn graphs.

    PubMed

    Badr, Eman; Heath, Lenwood S

    2014-12-01

    Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation. PMID:25393830

  19. Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers. PMID:10022858

  20. Alternatively Spliced Androgen Receptor Variants

    PubMed Central

    Dehm, Scott M.; Tindall, Donald J.

    2011-01-01

    Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. De-regulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS, and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. More recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen-depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa. PMID:21778211

  1. A Bidirectional SF2/ASF- and SRp40-Dependent Splicing Enhancer Regulates Human Immunodeficiency Virus Type 1 rev, env, vpu, and nef Gene Expression

    PubMed Central

    Caputi, Massimo; Freund, Marcel; Kammler, Susanne; Asang, Corinna; Schaal, Heiner

    2004-01-01

    The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3′ splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3′ splice site usage and binding of the U1 snRNP to the downstream 5′ splice site no. 4. U1 snRNP binding to the 5′ splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5′ splice site no. 4, even when the 5′ splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes. PMID:15163745

  2. Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments.

    PubMed

    Englert, Markus; Latz, Andreas; Becker, Dirk; Gimple, Olaf; Beier, Hildburg; Akama, Kazuhito

    2007-11-01

    Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting. PMID:17698277

  3. Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

    PubMed

    Venables, Julian P; Vignal, Emmanuel; Baghdiguian, Stephen; Fort, Philippe; Tazi, Jamal

    2012-01-01

    Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue

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

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

    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.

  5. Alternative splicing of RNAs transcribed from the human c- myb gene

    SciTech Connect

    Shen-Ong, G.L.C.; Skurla, R.M. Jr.; Owens, J.D.; Mushinski, J.F. )

    1990-06-01

    An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-{ital myb} homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express {ital myb}. The authors show that this alternative splicing event is conserved in human c-{ital myb} transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-{ital myb} mRNAs expressed in normal and tumor cells. Although the {ital myb} protein isoform encoded by murine E6A-containing mRNA is larger than the major c-{ital myb} protein, the predicted products of both forms of human alternatively spliced {ital myb} transcripts are 3{prime}-truncated {ital myb} proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral {ital myb} proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-{ital myb} transcripts. The contribution that alternative splicing events in c-{ital myb} expression may make on c-{ital myb} function remains to be elucidated.

  6. TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

    PubMed Central

    Montes, Marta; Cloutier, Alexandre; Sánchez-Hernández, Noemí; Michelle, Laetitia; Lemieux, Bruno; Blanchette, Marco; Hernández-Munain, Cristina; Chabot, Benoit

    2012-01-01

    Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-xS 5′ splice site. PMID:22158966

  7. Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon.

    PubMed Central

    Del Gatto, F; Plet, A; Gesnel, M C; Fort, C; Breathnach, R

    1997-01-01

    The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5' splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5' splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements. PMID:9271388

  8. 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)

    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

    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 τ1=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 ( τ2=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.

  9. Added-value from a multi-criteria selection of donor catchments in the prediction of continuous streamflow series at ungauged pollution control-sites

    NASA Astrophysics Data System (ADS)

    Drogue, Gilles; Ben Khediri, Wiem; Conan, Céline

    2016-05-01

    We explore the potential of a multi-criteria selection of donor catchments in the prediction of continuous streamflow series by the spatial proximity method. Three criteria have been used: (1) spatial proximity; (2) physical similarity; (3) stream gauging network topology. An extensive assessment of our spatial proximity method variant is made on a 149 catchment-data set located in the Rhine-Meuse catchment. The competitiveness of the method is evaluated against spatial interpolation of catchment model parameters with ordinary kriging. We found that the spatial proximity approach is more efficient than ordinary kriging. When distance to upstream/downstream stream gauge stations is considered as a second order criterion in the selection of donor catchments, an unprecedented level of efficiency is reached for nested catchments. Nevertheless, the spatial proximity method does not take advantage from physical similarity between donor catchments and receiver catchments because catchments that are the most hydrologically similar to each catchment poorly match with the catchments that are the most physically similar to each catchment.

  10. Functional Characterization of the spf/ash Splicing Variation in OTC Deficiency of Mice and Man

    PubMed Central

    Viecelli, Hiu Man; Rüfenacht, Veronique; Pérez, Belén; Ugarte, Magdalena; Häberle, Johannes; Thöny, Beat; Desviat, Lourdes Ruiz

    2015-01-01

    The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5’ splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the “spf/ash” mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations. PMID:25853564

  11. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes.

    PubMed

    Machinaga, Akihito; Ishihara, Syuhei; Shirai, Akiko; Takase-Yoden, Sayaka

    2016-01-01

    Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5' long terminal repeat (LTR), 5' leader sequence, gag, pol, env, and 3' LTR. Transcription from proviral DNA begins from the R region of the 5' LTR and ends at the polyadenylation signal located at the R region of the other end of the 3' LTR. There is a 5' splice site in the 5' leader sequence and a 3' splice site at the 3' end of the pol region. Both full-length unspliced mRNAs and a singly spliced mRNA (env-mRNA) are produced in MLV-infected cells. The MLV Env protein plays important roles both in viral adsorption to host cells and in neuropathogenic disease in MLV-infected mice and rats. Understanding the regulatory mechanisms controlling Env expression is important for determining the functions of the Env protein. We have previously shown that splicing increases env-mRNA stability and translation efficiency. Generally, mRNA polysome formation correlates with translation efficiency. Therefore, here we investigated the effects of env-mRNA splicing on polysome formation to identify mechanisms for Env up-regulation due to splicing. We performed polysome profile analyses using Env-expression plasmids producing spliced or unspliced env-mRNA and showed that the former formed polysomes more efficiently than the latter. Thus, splicing of env-mRNA facilitated polysome formation, suggesting that this contributes to up-regulation of Env expression. We replaced the env region of the expression plasmids with a luciferase (luc) gene, and found that in this case both unspliced and spliced luc-mRNA formed polysomes to a similar extent. Thus, we conclude that whether mRNA polysome formation is affected by splicing depends on the structure of gene in question. PMID:26909075

  12. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes

    PubMed Central

    Machinaga, Akihito; Ishihara, Syuhei; Shirai, Akiko; Takase-Yoden, Sayaka

    2016-01-01

    Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5′ long terminal repeat (LTR), 5′ leader sequence, gag, pol, env, and 3′ LTR. Transcription from proviral DNA begins from the R region of the 5′ LTR and ends at the polyadenylation signal located at the R region of the other end of the 3′ LTR. There is a 5′ splice site in the 5′ leader sequence and a 3′ splice site at the 3′ end of the pol region. Both full-length unspliced mRNAs and a singly spliced mRNA (env-mRNA) are produced in MLV-infected cells. The MLV Env protein plays important roles both in viral adsorption to host cells and in neuropathogenic disease in MLV-infected mice and rats. Understanding the regulatory mechanisms controlling Env expression is important for determining the functions of the Env protein. We have previously shown that splicing increases env-mRNA stability and translation efficiency. Generally, mRNA polysome formation correlates with translation efficiency. Therefore, here we investigated the effects of env-mRNA splicing on polysome formation to identify mechanisms for Env up-regulation due to splicing. We performed polysome profile analyses using Env-expression plasmids producing spliced or unspliced env-mRNA and showed that the former formed polysomes more efficiently than the latter. Thus, splicing of env-mRNA facilitated polysome formation, suggesting that this contributes to up-regulation of Env expression. We replaced the env region of the expression plasmids with a luciferase (luc) gene, and found that in this case both unspliced and spliced luc-mRNA formed polysomes to a similar extent. Thus, we conclude that whether mRNA polysome formation is affected by splicing depends on the structure of gene in question. PMID:26909075

  13. Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences.

    PubMed

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

    2013-07-01

    Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal Ca(V) 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 Ca(V) channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of Ca(V) channel structures and functions. The precise composition of Ca(V) 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 Ca(V) splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of Ca(V) 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

  14. Alternative splicing: Functional diversity among voltage-gated calcium channels and behavioral consequences☆

    PubMed Central

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

    2012-01-01

    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

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

    PubMed

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

    2014-10-13

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

  16. Splice form-dependence of β-neurexin/neuroligin binding interactions

    PubMed Central

    Koehnke, Jesko; Katsamba, Phinikoula S.; Ahlsen, Goran; Bahna, Fabiana; Vendome, Jeremie; Honig, Barry; Shapiro, Lawrence; Jin, Xiangshu

    2010-01-01

    Alternatively spliced β-neurexins (β-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a β-NRX/NL synaptic recognition code. We have utilized surface plasmon resonance to measure binding affinities between all sixty combinations of alternatively spliced ectodomains of β-NRXs 1–3 and NLs 1–3. Binding was observed for all β-NRX/NL pairs. The presence of the NL1 B splice insertion lowers β-NRX binding affinity by ~2-fold, while β-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated β-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new β-strand that replaces the β10-strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in β-NRX/NL mediated synaptic recognition. PMID:20624592

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

    PubMed Central

    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

    2014-01-01

    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

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

    PubMed

    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

    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

  19. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

    PubMed Central

    Caminsky, Natasha; Mucaki, Eliseos J.; Rogan, Peter K.

    2014-01-01

    The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368

  20. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis.

    PubMed

    Caminsky, Natasha; Mucaki, Eliseos J; Rogan, Peter K

    2014-01-01

    The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368

  1. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  2. Donor Tag Game

    MedlinePlus

    ... Cross chapter closest to you. Can't Donate Blood? A financial donation can also help save lives. Donate Now Find ... Donation Student Donors Donation Process Eligibility Blood FAQs Blood Donor Community Learn About Blood Blood Facts and Statistics ...

  3. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  4. Defective splicing of mRNA from one COL1A1 allele of type I collagen in nondeforming (type I) osteogenesis imperfecta.

    PubMed Central

    Stover, M L; Primorac, D; Liu, S C; McKinstry, M B; Rowe, D W

    1993-01-01

    Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COL1A1 gene. We studied fibroblasts established from a child with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1 (I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population was confined to the nuclear compartment of the cell, and contained the entire sequence of intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A transition was also identified in the genomic DNA. The retained intron contained an in-frame stop codon and introduced an out-of-frame insertion within the collagen mRNA producing stop codons downstream of the insertion. These changes probably account for the failure of the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript, this mutation did not result in production of a defective collagen pro alpha 1 (I) chain. Instead, the mild nature of the disease in this case reflects failure to process the defective mRNA and thus the absence of a protein product from the mutant allele. Images PMID:8408653

  5. SON Controls Cell Cycle Progression by Coordinated Regulation of RNA Splicing

    PubMed Central

    Ahn, Eun-Young; DeKelver, Russell C.; Lo, Miao-Chia; Nguyen, Tuyet Ann; Matsuura, Shinobu; Boyapati, Anita; Pandit, Shatakshi; Fu, Xiang-Dong; Zhang, Dong-Er

    2011-01-01

    SUMMARY It has been suspected that cell cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing co-factor contributing to efficient splicing of cell cycle regulators. Down-regulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient co-transcriptional RNA processing. These results reveal a mechanism for controlling cell cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases. PMID:21504830

  6. Becoming a Donor

    MedlinePlus

    ... by Organ and Gender. > U.S. Waiting List Candidate Data HOW TO BECOME A DONOR The most important thing to do is to sign up as an organ and tissue donor in your state's donor registry. To cover all bases, it's also helpful to: Designate your decision on ...

  7. Evolution of alternative splicing after gene duplication.

    PubMed

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

    2006-02-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evo