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Sample records for splice donor site

  1. Hypophosphatemic rickets caused by a novel splice donor site mutation and activation of two cryptic splice donor sites in the PHEX gene.

    PubMed

    Zou, Minjing; Buluş, Derya; Al-Rijjal, Roua A; Andıran, Nesibe; BinEssa, Huda; Kattan, Walaa E; Meyer, Brian; Shi, Yufei

    2015-01-01

    X-linked hypophosphatemic rickets (XLH) is the most common inherited form of rickets. XLH is caused by inactivating mutations in the PHEX gene and is transmitted as an X-linked dominant disorder. We investigated PHEX mutation in a sporadic Turkish girl with hypophosphatemic rickets. The patient was 2 years of age with a complaint of inability to walk. She had bowing of legs and growth retardation. Laboratory data showed normal calcium, low phosphate with markedly elevated ALP, and low phosphate renal tubular reabsorption. She was treated with Calcitriol 0.5 mg/kg/day and oral phosphate supplement with good response. The entire coding region of PHEX gene was sequenced from patient's peripheral leukocyte DNA and a novel 13 bp deletion at the donor splice site of exon5 was found (c.663+12del). Instead of using the donor splice site of intron 4 to splice out exon 5 and intron 5, the spliceosome utilized two nearby cryptic donor splice sites (5' splice site) to splice out intron 4, resulting in two smaller transcripts. Both of them could not translate into functional proteins due to frameshift. Her parents did not carry the mutation, indicating that this is a de novo PHEX mutation likely resulting from mutagenesis of X chromosome in paternal germ cells. We conclude that c.663+12del is a novel mutation that can activate nearby cryptic 5' splice sites. The selection of cryptic 5' splice sites adds the complexity of cell's splicing mechanisms. The current study extends the database of PHEX mutation and cryptic 5' splice sites.

  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. 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.

  4. 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

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

    PubMed Central

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

    2009-01-01

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

  6. 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

  7. A splice donor site mutation in HOXD13 underlies synpolydactyly with cortical bone thinning.

    PubMed

    Shi, Xiuyan; Ji, Chunyan; Cao, Lihua; Wu, Yuhong; Shang, Yuyang; Wang, Wei; Luo, Yang

    2013-12-15

    Synpolydactyly 1(SPD1) is a dominantly inherited distal limb anomaly that is characterized by incomplete digit separation and increased number of digits. SPD1 is most commonly caused by polyalanine repeat expansions and mutations in the homeodomain of the HOXD13. We report a splice donor site mutation in HOXD13 associated in most cases with cortical bone thinning. In vitro study of transcripts and truncated protein analysis indicated that c.781+1G>A mutation results in truncated HOXD13 protein p.G190fsX4. Luciferase assay indicated that the truncated HOXD13 protein failed to bind to DNA. The mechanism for this phenotype was truncated protein loss of function.

  8. 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.

  9. Importance of RNA secondary structure information for yeast donor and acceptor splice site predictions by neural networks.

    PubMed

    Marashi, Sayed-Amir; Goodarzi, Hani; Sadeghi, Mehdi; Eslahchi, Changiz; Pezeshk, Hamid

    2006-02-01

    Previously, Patterson et al. showed that mRNA structure information aids splice site prediction in human genes [Patterson, D.J., Yasuhara, K., Ruzzo, W.L., 2002. Pre-mRNA secondary structure prediction aids splice site prediction. Pac. Symp. Biocomput. 7, 223-234]. Here, we have attempted to predict splice sites in selected genes of Saccharomyces cerevisiae using the information obtained from the secondary structures of corresponding mRNAs. From Ares database, 154 genes were selected and their structures were predicted by Mfold. We selected a 20-nucleotide window around each site, each containing 4 nucleotides in the exon region. Based on whether the nucleotide is in a stem or not, the conventional four-letter nucleotide alphabet was translated into an eight-letter alphabet. Two different three-layer-based perceptron neural networks were devised to predict the 5' and 3' splice sites. In case of 5' site determination, a network with 3 neurons at the hidden layer was chosen, while in case of 3' site 20 neurons acted more efficiently. Both neural nets were trained applying Levenberg-Marquardt backpropagation method, using half of the available genes as training inputs and the other half for testing and cross-validations. Sequences with GUs and AGs non-sites were used as negative controls. The correlation coefficients in the predictions of 5' and 3' splice sites using eight-letter alphabet were 98.0% and 69.6%, respectively, while these values were 89.3% and 57.1% when four-letter alphabet is applied. Our results suggest that considering the secondary structure of mRNA molecules positively affects both donor and acceptor site predictions by increasing the capacity of neural networks in learning the patterns.

  10. 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.

  11. 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

  12. Donor Splice-Site Mutation in CUL4B is Likely Cause of X-Linked Intellectual Disability

    PubMed Central

    Londin, Eric R.; Adijanto, Jeffrey; Philp, Nancy; Novelli, Antonio; Vitale, Emilia; Perria, Chiara; Serra, Gigliola; Alesi, Viola; Surrey, Saul; Fortina, Paolo

    2015-01-01

    X-linked intellectual disability is the most common form of cognitive disability in males. Syndromic intellectual disability encompasses cognitive deficits with other medical and behavioral manifestations. Recently, a large family with a novel form of syndromic X-linked intellectual disability was characterized. Eight of 24 members of the family are male and had cognitive dysfunction, short stature, aphasia, skeletal abnormalities, and minor anomalies. To identify the causative gene(s), we performed exome sequencing in three affected boys, both parents, and an unaffected sister. We identified a haplotype consisting of eight variants located in cis within the linkage region that segregated with affected members in the family. Of these variants, two were novel. The first was at the splice-donor site of intron 7 (c.974+1G>T) in the cullin-RING ubiquitin ligase (E3) gene, CUL4B. This variant is predicted to result in failure to splice and remove intron 7 from the primary transcript. The second variant mapped to the 3′-UTR region of the KAISO gene (c.1127T>G). Sanger sequencing validated the variants in these relatives as well as in three affected males and five carriers. The KAISO gene variant was predicted to create a binding site for the microRNAs miR-4999 and miR-4774; however, luciferase expression assays failed to validate increased targeting of these miRNAs to the variant 3′-UTR. This SNP may affect 3′-UTR structure leading to decreased mRNA stability. Our results suggest that the intellectual disability phenotype in this family is caused by aberrant splicing and removal of intron 7 from CUL4B gene primary transcript. PMID:24898194

  13. 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.

  14. Monocyte function in a severe combined immunodeficient patient with a donor splice site mutation in the Jak3 gene.

    PubMed

    Villa, A; Sironi, M; Macchi, P; Matteucci, C; Notarangelo, L D; Vezzoni, P; Mantovani, A

    1996-08-01

    Janus kinase-3 (Jak3) is a nonreceptor tyrosine kinase functionally coupled to cytokine receptors which share a "common" gamma chain (gamma c). Mutations in gamma c and Jak3 genes have been identified in X-linked and autosomal severe combined immuno deficiency (SCID), respectively. Jak3 is expressed and activated in myelomonocytic cells. The present study was designed to define the structural alteration responsible for lack of Jak3 in a patient with autosomal SCID and to characterize monocyte function in the absence of this signal transduction element, as well as to establish the whole exon-intron structure. Polymerase chain reaction analysis, performed with primers designed on exon sequences, identified 20 exons spanning approximately 15 kb. These primers, or others designed on the flanking sequences provided in the present report, can be used to amplify the whole gene, allowing the definition of the molecular defects in all cases, including prenatal diagnosis, in which transcript analysis is not possible. On this basis, the deletion transcript found at the homozygous state in patient CM, with both his consanguineous parents being heterozygous for the deletion, was associated with mutation (T to C) of a splice donor site of intron 16 that was also detected in his mother's DNA. Monocytes from Jak3-SCID showed normal cytokine production in response to interleukin-4 (IL-4) (release of IL-1 receptor antagonist) and IL-2 (release of tumor necrosis factor-alpha and IL-8). Lipopolysaccharide-induced cytokine production was also normal and was blocked by IL-4 in Jak3- SCID monocytes. Interferon-gamma induced augmented expression of major histocompatibility class II in Jak3-SCID monocytes. These data indicate that Jak3, expressed and activated in myelomonocytic cells, is dispensable for monocyte differentiation and responsiveness to cytokines that interact with gamma c receptors as well as to other regulatory signals.

  15. 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.

  16. 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

  17. Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA.

    PubMed Central

    Chen, C D; Helfman, D M

    1999-01-01

    The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1-5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3' splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5' splice site of exon 7 in nonmuscle cells. The 5' splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5' splice site of exon 7 does not result from the sequences at the 3' end of intron 6 that block the use of the 3' splice site of exon 7. However, mutating two conserved nucleotides GU at the 5' splice site of exon 6 results in the efficient use of the 5' splice site of exon 7. In addition, a mutation that changes the 5' splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5' splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells. PMID:10024180

  18. 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

    Solera, J; Magallón, 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' 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.

  19. 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

  20. Our favourite alternative splice site.

    PubMed

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

    2006-05-01

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

  1. 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.

  2. 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.

  3. The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

    PubMed Central

    Roller, A B; Hoffman, D C; Zahler, A M

    2000-01-01

    Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors. PMID:10757761

  4. A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation.

    PubMed

    Lal, S; Choi, J H; Shaw, J R; Hannah, L C

    1999-10-01

    DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5' terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5' to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5' to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030-2031) led to the identification of 3' intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing. PMID:10517832

  5. Cryptic splice sites and split genes

    PubMed Central

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

    2011-01-01

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

  6. Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis

    PubMed Central

    Schommartz, Tim; Loroch, Stefan; Alawi, Malik; Grundhoff, Adam; Sickmann, Albert

    2016-01-01

    ABSTRACT Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. IMPORTANCE Herpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when

  7. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site.

    PubMed

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

    2015-07-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins. PMID:25779589

  8. 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.

  9. 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

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

    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+1.6kbA-->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.

  10. 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

  11. 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

  12. Splice Sites Seldom Slide: Intron Evolution in Oomycetes.

    PubMed

    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

  13. 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

  14. 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

  15. Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption.

    PubMed

    Wimmer, K; Roca, X; Beiglböck, H; Callens, T; Etzler, J; Rao, A R; Krainer, A R; Fonatsch, C; Messiaen, L

    2007-06-01

    We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.

  16. Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

    PubMed Central

    Chen, I T; Chasin, L A

    1993-01-01

    A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures. Images PMID:8417332

  17. [Statistical analysis of DNA sequences nearby splicing sites].

    PubMed

    Korzinov, O M; Astakhova, T V; Vlasov, P K; Roĭtberg, M A

    2008-01-01

    Recognition of coding regions within eukaryotic genomes is one of oldest but yet not solved problems of bioinformatics. New high-accuracy methods of splicing sites recognition are needed to solve this problem. A question of current interest is to identify specific features of nucleotide sequences nearby splicing sites and recognize sites in sequence context. We performed a statistical analysis of human genes fragment database and revealed some characteristics of nucleotide sequences in splicing sites neighborhood. Frequencies of all nucleotides and dinucleotides in splicing sites environment were computed and nucleotides and dinucleotides with extremely high\\low occurrences were identified. Statistical information obtained in this work can be used in further development of the methods of splicing sites annotation and exon-intron structure recognition.

  18. 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

  19. 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

  20. 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

    2016-07-21

    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.

  1. Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5' splice site to the internal branch acceptor site.

    PubMed Central

    Köhrer, K; Domdey, H

    1988-01-01

    Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing. Images PMID:3054807

  2. 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

  3. 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.

  4. 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

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

    PubMed Central

    Zafrir, Zohar; Tuller, Tamir

    2015-01-01

    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

  6. Factors influencing alternative splice site utilization in vivo.

    PubMed Central

    Fu, X Y; Manley, J L

    1987-01-01

    To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

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

    PubMed Central

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

    2014-01-01

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

  8. 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.

  9. 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. PMID:27625873

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

    PubMed

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

    2014-09-01

    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. PMID:27625873

  11. Splicing-coupled 3' end formation requires a terminal splice acceptor site, but not intron excision.

    PubMed

    Davidson, Lee; West, Steven

    2013-08-01

    Splicing of human pre-mRNA is reciprocally coupled to 3' end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3' end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3' end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3' end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3' end formation, but that on-going intron removal is less critical. PMID:23716637

  12. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    NASA Astrophysics Data System (ADS)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  13. Sequence-specific flexibility organization of splicing flanking sequence and prediction of splice sites in the human genome.

    PubMed

    Zuo, Yongchun; Zhang, Pengfei; Liu, Li; Li, Tao; Peng, Yong; Li, Guangpeng; Li, Qianzhong

    2014-09-01

    More and more reported results of nucleosome positioning and histone modifications showed that DNA structure play a well-established role in splicing. In this study, a set of DNA geometric flexibility parameters originated from molecular dynamics (MD) simulations were introduced to discuss the structure organization around splice sites at the DNA level. The obtained profiles of specific flexibility/stiffness around splice sites indicated that the DNA physical-geometry deformation could be used as an alternative way to describe the splicing junction region. In combination with structural flexibility as discriminatory parameter, we developed a hybrid computational model for predicting potential splicing sites. And the better prediction performance was achieved when the benchmark dataset evaluated. Our results showed that the mechanical deformability character of a splice junction is closely correlated with both the splice site strength and structural information in its flanking sequences.

  14. Alginate dressing as a donor site haemostat.

    PubMed Central

    Groves, A. R.; Lawrence, J. C.

    1986-01-01

    An alginate fibre dressing has been used to reduce blood loss from skin graft donor sites. Significant haemostasis has been achieved in the immediate post surgery phase and no adverse reactions observed. Images Fig. 1 PMID:3511833

  15. Novel splice site mutation in keratin 1 underlies mild epidermolytic palmoplantar keratoderma in three kindreds.

    PubMed

    Hatsell, S J; Eady, R A; Wennerstrand, L; Dopping-Hepenstal, P; Leigh, I M; Munro, C; Kelsell, D P

    2001-04-01

    We report a novel mutation in the exon 6 splice donor site of keratin 1 (G4134A) that segregates with a palmoplantar keratoderma in three kindreds. The nucleotide substitution leads to the utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent insertion of 18 amino acids into the 2B rod domain. This mutation appears to have a milder effect than previously described mutations in the helix initiation and termination sequence on the function of the rod domain, with regard to filament assembly and stability. Affected individuals displayed only mild focal epidermolysis in the spinous layer of palmoplantar epidermis, in comparison with cases of bullous congenital ichthyosiform erythroderma also due to keratin 1 mutations, which show widespread and severe epidermolysis. This study describes a novel mutation in KRT1 that results in a phenotype distinct from classical bullous congenital ichthyosiform erythroderma.

  16. Analysis by illegitimate transcription of a mutation in the 5{prime} splice site in exon 8 of the PAH gene

    SciTech Connect

    Desviat, L.R.; Perez, B.; Ugarte, M.

    1994-09-01

    Up to now, 12 splice defects have been described within the PAH gene. Using PCR-SSCP and sequence analysis we have found a point mutation involving the last nucleotide in exon 8 (CAG/CAA). The G to A substitution does not alter the amino acid (Q204Q), but it may cause a splice defect, as it is included in the 5{prime} splice donor site, and the G at this position is highly conserved (80%) in all eukaryotic genes. We have analyzed by illegitimate transcription the PAH mRNA in lymphocytes of a patient bearing the mutation in a heterozygous fashion. After RT-PCR we observed once the appearance of an extra larger band, which could be due to the use of a cryptic splice site instead of the mutated one. Furthermore, sequencing of 6 clones of the band of expected size in the patient revealed that all had the normal sequence, in spite of the G to A substitution being found in the genomic DNA. In view of these results, we believe that the larger extra band represents the allele with the mutation which causes a highly unstable mis-spliced RNA. This splice defect could be, therefore, the disease causing mutation in the patient.

  17. 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.

  18. Optimizing aesthetic and functional outcomes at donor sites.

    PubMed

    Jeng, Seng-Feng; Tan, Ngian-Chye

    2012-01-01

    In recent years, there has been increasing interest by reconstructive surgeons in improving the aesthetic and functional outcomes of donor sites. As the success rate of free tissue transfers has exceeded more than 95% in most microsurgical centers, more emphasis can be shifted to the donor site. However, morbidities of donor sites can occur not only in free tissue transfers, but in locoregional flaps as well. In reconstructive procedures, the main principle is to mobilize normal tissue and utilize it to reconstruct an area of defect. The donor site, of course has no pathology, but is a previously healthy area. Therefore, it is of paramount importance to not only minimize postoperative complications at recipient sites, but also pay attention to donor sites. Just as in organ transplantation where efforts are made to ensure the safety and a good outcome for a donor patient, outcomes should be improved and morbidity reduced at donor sites in reconstructive surgery.

  19. A Novel Otoferlin Splice-Site Mutation in Siblings with Auditory Neuropathy Spectrum Disorder

    PubMed Central

    Runge, Christina L.; Erbe, Christy B.; McNally, Mark T.; Van Dusen, Courtney; Friedland, David R.; Kwitek, Anne E.; Kerschner, Joseph E.

    2013-01-01

    We characterize a novel otoferlin (OTOF) mutation discovered in a sibling pair diagnosed with ANSD and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the OTOF splice donor site of exon 28 (IVS28+1 G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (.78 ms) compared to subjects with sensorineural hearing loss (SNHL) (average: adults .71 ms; children .85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered. PMID:24135434

  20. hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes

    PubMed Central

    Buratti, Emanuele; Baralle, Marco; De Conti, Laura; Baralle, Diana; Romano, Maurizio; Ayala, Youhna M.; Baralle, Francisco E.

    2004-01-01

    We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA–protein complexes at several 5′ splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5′ splice site (5′ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5′ss of TSHβ exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts. PMID:15299088

  1. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5'-splice site and the creation of a de novo 3'-splice site.

    PubMed

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5'-splice site and the creation of a newly 3'-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2013-03-01

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

  4. 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.

  5. Management of the difficult split-thickness donor site.

    PubMed

    Wood, R J; Peltier, G L; Twomey, J A

    1989-01-01

    Split-thickness skin graft donor sites are often areas of significant morbidity in the elderly, in immunocompromised patients, and in steroid-dependent patients. We found that managing these donor sites with split-thickness skin and transparent dressings greatly increases the rate of healing and diminishes morbidity.

  6. 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

  7. 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

  8. 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.

  9. Donor-site morbidity after osteochondral autograft transfer procedures.

    PubMed

    LaPrade, Robert F; Botker, Jesse C

    2004-09-01

    We report on 2 patients who had donor-site morbidity after an autogenous osteochondral grafting was performed. Both patients had fibrocartilage hypertrophy at the donor sites that contributed to knee pain and occasional locking; the second patient also had a lack of fibrocartilaginous regrowth with symptomatic residual osteocartilaginous defects. Additional arthroscopic surgery was required in both cases to trim the fibrocartilage. In addition, for the second case, a fresh osteoarticular allograft was used to transfer osteocartilaginous plugs back into the original knee donor sites due to continued knee pain. When performing an osteochondral autograft transfer, the benefits provided at the recipient site must be weighed against the possible donor-site morbidity that may result.

  10. Subcostal Skin Graft Donor Site for Autologous Ear Construction.

    PubMed

    Hassanein, Aladdin H; Greene, Arin K

    2015-06-01

    Autologous ear construction for microtia creates an auricle using a costal cartilage framework. To separate the construct from the mastoid, a skin graft is required to form a retroauricular sulcus. Skin graft donor sites that have been described include the inguinal area (split or full-thickness) or scalp (split-thickness). The purpose of this study is to report a novel skin graft donor site for ear construction. We harvest a full-thickness graft from the subcostal area based on the previous scar from the cartilage harvest. Unlike the inguinal donor site, this method does not place an additional scar on the child. In contrast to the scalp donor site, the technique is simpler and a full-thickness graft minimizes contraction of the retroauricular sulcus. PMID:26080199

  11. 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.

  12. 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

  13. Donor site morbidity after articular cartilage repair procedures: a review.

    PubMed

    Matricali, Giovanni A; Dereymaeker, Greta Ph E; Luyten, Frank P

    2010-10-01

    In order to perform an Osteochondral Autologous Transplantation (OAT) or an Autologous Chondrocyte Implantation (ACI), the integrity of healthy intact articular cartilage at a second location needs to be violated. This creates the possibility for donor site morbidity. Only recently have any publications addressed this issue. The aim of this manuscript is to review the current knowledge on donor site morbidity after an OAT or an ACI. Reports were identified by searching Medline and Pubmed up to March 2010. Donor site morbidity was described mostly considering a clinical outcome, both in a qualitative (parameters in history or physical examination) and/or quantitative way (knee status reported by means of a numerical score). An increasing rate of problems is noted when using quantitative instead of qualitative parameters, and when donor site morbidity is the focus of attention, affecting up to more than half of the patients, in particular for an OAT procedure. The decision to harvest an osteochondral or cartilage biopsy to perform a repair procedure should therefore be taken with caution. This also underscores the need for further research to identify safe donor sites or to develop techniques that eliminate the need for a formal biopsy ccompletely.

  14. 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

  15. Splice Site Strength-Dependent Activity and Genetic Buffering by Poly-G Runs

    PubMed Central

    Xiao, Xinshu; Wang, Zefeng; Jang, Minyoung; Nutiu, Razvan; Wang, Eric T.; Burge, Christopher B.

    2009-01-01

    Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of the G-run class of SREs is ∼4-fold higher when adjacent to intermediate strength 5'ss relative to weak 5'ss, and ∼1.3-fold higher relative to strong 5'ss. This dependence on 5'ss strength was observed in splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNAi against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which crosslinked to G-runs adjacent to many regulated exons. An exon’s responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5'ss strength, and other splicing factors may function similarly. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting the frequency of 5'ss polymorphism and the evolution of new splicing patterns. PMID:19749754

  16. A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin

    SciTech Connect

    Watkins, S.; Madison, J.; Davis, E.; Sakamoto, Yasushi; Putnam, F.W. ); Galliano, M.; Minchiotti, L. )

    1991-07-15

    At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5{prime} donor splice junction of intron 14 and the 3{prime} end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.

  17. 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

  18. 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

  19. Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.

    PubMed Central

    Okagaki, R J; Sullivan, T D; Schiefelbein, J W; Nelson, O E

    1992-01-01

    The defective Suppressor-mutator (dSpm)-induced allele bronze1-mutable 13 (bz1-m13) and many of its derivative alleles are leaky mutants with measurable levels of flavonol O3-glucosyltransferase activity. This activity results from splicing at acceptor site-1, one of two cryptic 3' splice sites within the dSpm insertion in bz1-m13. In this study, splicing in bz1-m13 change-in-state (CS) alleles CS-3 and CS-64 was shown to be altered from bz1-m13; previous work found altered splicing in CS-9. CS-64 is a null allele and lacks the acceptor site-1-spliced transcript because this site is deleted. CS-3 and CS-9 had increased levels of the acceptor site-1 transcript relative to bz1-m13 and increased enzymic activities. A deletion in CS-9 altered splicing by eliminating acceptor site-2. Both acceptor sites were intact in CS-3, but a deletion removed most of a 275-bp GC-rich sequence in dSpm. This suggests that GC-rich sequences affect splicing and is consistent with models postulating a role for AU content in the splicing of plant introns. Splicing does not necessarily occur, however, at the junction of AU-rich intron sequences and GC-rich exon sequences. PMID:1477558

  20. 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

  1. Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.

    PubMed

    Cohen, J B; Snow, J E; Spencer, S D; Levinson, A D

    1994-10-25

    One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.

  2. Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs.

    PubMed

    Vreeswijk, Maaike P G; Kraan, Jaennelle N; van der Klift, Heleen M; Vink, Geraldine R; Cornelisse, Cees J; Wijnen, Juul T; Bakker, Egbert; van Asperen, Christi J; Devilee, Peter

    2009-01-01

    A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis.

  3. 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

  4. 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.

  5. Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts.

    PubMed

    Lualdi, Susanna; Pittis, Maria G; Regis, Stefano; Parini, Rossella; Allegri, Anna E; Furlan, Francesca; Bembi, Bruno; Filocamo, Mirella

    2006-08-01

    Mutations in the gene encoding the enzyme iduronate-2-sulfatase (IDS) were reported as the cause of the X-linked recessive lysosomal disease, mucopolysaccharidosis II (MPS II). Amongst the different mutations, it emerges that nearly 10% are nucleotide substitutions causing splicing mutations. We now report the molecular characterisation of three MPS II patients with multiple aberrant transcripts due to three different point mutations. The c.418+1G>C that occurred in the invariant splice-site motif, produced only aberrantly spliced transcripts. Whilst the mutations affecting variant motifs (c.419G>T) or coding regions (c.245C>T) led to aberrantly spliced transcripts in addition to correctly spliced transcripts with the respective predicted missense mutation, p.G140V or p.A82V. A combination of experimental tests and computational approaches were used to understand the molecular basis underlying the altered transcription patterns. In addition, by using real-time reverse transcriptase polymerase chain reaction, the reduction of mRNA amount in two patients observed was likely due to nonsense-mediated mRNA decay pathway. Overall, our results further emphasised the importance of cloning and sequencing independent transcripts to reveal less abundant, aberrant products, which often could not be detected by direct sequencing. Moreover, the different splicing patterns observed in the three patients as a consequence of point mutations show how sensitive the balance is between constitutive and cryptic splice sites in the IDS gene. The generation of such diverse transcripts, together with their level of expression, could contribute to the profound phenotypic variability reported in MPS II.

  6. Characterisation and quantification of F8 transcripts of ten putative splice site mutations.

    PubMed

    Liang, Qian; Xiang, Mingjie; Lu, Yeling; Ruan, Yufeng; Ding, Qiulan; Wang, Xuefeng; Xi, Xiaodong; Wang, Hongli

    2015-03-01

    Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %, 14.2 %, 5.2 %, 19.2 %, 1.8 % and 2.5 % of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

  7. The consensus 5' splice site motif inhibits mRNA nuclear export.

    PubMed

    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.

  8. Structural and functional analyses of the sixth site of neurexin alternative splicing.

    PubMed

    Serova, O V; Radionov, N V; Shayahmetova, D M; Deyev, I E; Petrenko, A G

    2015-01-01

    In this study, we found the sixth site of alternative splicing (SS6) of neurexin 1a from the rat brain. This site is located between the fifth LNS and the third EGF-like domains. The insertion in the SS6 site corresponds to the 9-residue peptide VALMKADLQ, which is conserved among animals. We demonstrated that the SS6 insertion regulates tissue-specific expression of neurexin 1α.

  9. 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.

  10. A novel splice site mutation of CDHR1 in a consanguineous Israeli Christian Arab family segregating autosomal recessive cone-rod dystrophy

    PubMed Central

    Cohen, Ben; Chervinsky, Elena; Jabaly-Habib, Haneen; Shalev, Stavit A.; Briscoe, Daniel

    2012-01-01

    Purpose To investigate the genetic basis for autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. Methods Patients underwent a detailed ophthalmic examination, including funduscopy, electroretinography (ERG), visual field testing, and optical coherence tomography. Genome-wide homozygosity mapping using a single nucleotide polymorphism array was performed to identify homozygous regions shared between the two affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico analysis was used to predict the effect of the mutation on splicing. Results The family included two affected individuals. Clinical findings included progressive deterioration of visual acuity, photophobia, defective color vision, loss of central visual fields, pigmentary deposits localized mainly in the peripheral retina, a thinned and atrophic macular region, retinal vessel attenuation, absent ERG cone responses, and reduced ERG rod responses. Homozygosity mapping revealed several homozygous intervals shared among the affected individuals. One, a 12Mb interval on chromosome 10, included the CDHR1 gene. Direct sequencing revealed a single base transversion, c.1485+2T>G, located in the conserved donor splice site of Intron 13. This mutation cosegregated with the disease in the family, and was not detected in 208 Israeli Christian Arab control chromosomes. In silico analysis predicted that this mutation eliminates the Intron 13 donor splice site. Conclusions Only three distinct pathogenic mutations of CDHR1 have been reported to date in patients with autosomal recessive retinal degeneration. Here we report a novel splice site mutation of CDHR1, c.1485+2T>G, underlying autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. This report expands the spectrum of pathogenic mutations of the CDHR1 gene. PMID:23233793

  11. Haemophilia B caused by a point mutation in a donor splice junction of the human factor IX gene.

    PubMed

    Rees, D J; Rizza, C R; Brownlee, G G

    Haemophilia B (Christmas disease) is an inherited, recessive, sex-linked, haemorrhagic condition caused by a defect in the intrinsic clotting factor IX. This disease occurs in males at a frequency of approximately 1 in 30,000. Patients differ in the severity of their clinical symptoms, and variation in the clotting activity and in the concentration of factor IX antigen in their plasma has been demonstrated. There is probably heterogeneity in the molecular defects of the factor IX gene causing the disease. Here we study a severely affected, antigen-negative patient, and show that the only significant sequence difference from the normal factor IX gene is a point mutation changing the obligatory GT to a TT within the donor splice junction of exon f. We infer that this change is the cause of the disease in this individual. In addition, we have used oligodeoxynucleotide probes specific for this mutation to demonstrate the feasibility of carrier detection and prenatal diagnosis for relatives of the patient.

  12. Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

    PubMed Central

    Liu, Yuying; Conaway, LaShardai; Rutherford Bethard, Jennifer; Al-Ayoubi, Adnan M.; Thompson Bradley, Amber; Zheng, Hui; Weed, Scott A.; Eblen, Scott T.

    2013-01-01

    Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1. PMID:23519612

  13. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5′-splice site and the creation of a de novo 3′-splice site

    PubMed Central

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5′-splice site and the creation of a newly 3′-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies. PMID:27081538

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Comparison of splice sites reveals that long noncoding RNAs are evolutionarily well conserved.

    PubMed

    Nitsche, Anne; Rose, Dominic; Fasold, Mario; Reiche, Kristin; Stadler, Peter F

    2015-05-01

    Large-scale RNA sequencing has revealed a large number of long mRNA-like transcripts (lncRNAs) that do not code for proteins. The evolutionary history of these lncRNAs has been notoriously hard to study systematically due to their low level of sequence conservation that precludes comprehensive homology-based surveys and makes them nearly impossible to align. An increasing number of special cases, however, has been shown to be at least as old as the vertebrate lineage. Here we use the conservation of splice sites to trace the evolution of lncRNAs. We show that >85% of the human GENCODE lncRNAs were already present at the divergence of placental mammals and many hundreds of these RNAs date back even further. Nevertheless, we observe a fast turnover of intron/exon structures. We conclude that lncRNA genes are evolutionary ancient components of vertebrate genomes that show an unexpected and unprecedented evolutionary plasticity. We offer a public web service (http://splicemap.bioinf.uni-leipzig.de) that allows to retrieve sets of orthologous splice sites and to produce overview maps of evolutionarily conserved splice sites for visualization and further analysis. An electronic supplement containing the ncRNA data sets used in this study is available at http://www.bioinf.uni-leipzig.de/publications/supplements/12-001.

  2. A 3' splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II.

    PubMed

    Jin, Ping; Hao, Jing-Wen; Chen, Ke; Dong, Chang-Sheng; Yang, You-Bo; Mo, Zhao-Hui

    2013-10-10

    The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site. A 3' splice site mutation c.880-8A>G (IVS 6-8A>G) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted.

  3. 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

  4. 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.

  5. SQSTM1 splice site mutation in distal myopathy with rimmed vacuoles

    PubMed Central

    Bucelli, Robert C.; Arhzaouy, Khalid; Pestronk, Alan; Pittman, Sara K.; Rojas, Luisa; Sue, Carolyn M.; Evilä, Anni; Hackman, Peter; Udd, Bjarne; Harms, Matthew B.

    2015-01-01

    Objective: To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy. Methods: We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies. Results: Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization. Conclusions: SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes. PMID:26208961

  6. The posterior interosseous artery flap: clinical results with special emphasis on donor site morbidity.

    PubMed

    Neuwirth, Maximilian; Hubmer, Martin; Koch, Horst

    2013-05-01

    This study evaluated the clinical results, and especially the donor site morbidity of the posterior interosseous artery flap. A retrospective study included 40 patients with defects covered with posterior interosseous flaps. Twenty-one patients were available for a follow-up examination to assess donor site morbidity by evaluating the dimensions and quality of the donor site scar and the forearm contour as well as complaints and subjective satisfaction with the aesthetic result. The flaps and related donor sites healed uneventfully in 29 cases (72.5%); healing was delayed in 11 cases (27.5%), with total flap loss in two cases. Further surgery was required in six cases. The quality of the donor site scar rated with the Vancouver Scar Scale averaged 2.4 points. Eleven patients (55%) reported impaired sensibility around the donor site and four patients (20%) had physical complaints. Subjective and objective donor site evaluation revealed significantly lower donor site morbidity for directly closed as opposed to skin grafted donor sites, although subjectively, there was a high level of satisfaction in both groups. Our data indicated that the posterior interosseous flap is a valuable option for the management of soft-tissue defects on the dorsum of the hand, due to its anatomical reliability and soft and pliable tissue, its low donor site morbidity and high patient acceptance.

  7. An Interspecific Plant Hybrid Shows Novel Changes in Parental Splice Forms of Genes for Splicing Factors

    PubMed Central

    Scascitelli, Moira; Cognet, Marie; Adams, Keith L.

    2010-01-01

    Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization. PMID:20100939

  8. 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

  9. 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

  10. 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

  11. Weak negative and positive selection and the drift load at splice sites.

    PubMed

    Denisov, Stepan V; Bazykin, Georgii A; Sutormin, Roman; Favorov, Alexander V; Mironov, Andrey A; Gelfand, Mikhail S; Kondrashov, Alexey S

    2014-05-14

    Splice sites (SSs) are short sequences that are crucial for proper mRNA splicing in eukaryotic cells, and therefore can be expected to be shaped by strong selection. Nevertheless, in mammals and in other intron-rich organisms, many of the SSs often involve nonconsensus (Nc), rather than consensus (Cn), nucleotides, and beyond the two critical nucleotides, the SSs are not perfectly conserved between species. Here, we compare the SS sequences between primates, and between Drosophila fruit flies, to reveal the pattern of selection acting at SSs. Cn-to-Nc substitutions are less frequent, and Nc-to-Cn substitutions are more frequent, than neutrally expected, indicating, respectively, negative and positive selection. This selection is relatively weak (1 < |4Nes| < 4), and has a similar efficiency in primates and in Drosophila. Within some nucleotide positions, the positive selection in favor of Nc-to-Cn substitutions is weaker than the negative selection maintaining already established Cn nucleotides; this difference is due to site-specific negative selection favoring current Nc nucleotides. In general, however, the strength of negative selection protecting the Cn alleles is similar in magnitude to the strength of positive selection favoring replacement of Nc alleles, as expected under the simple nearly neutral turnover. In summary, although a fraction of the Nc nucleotides within SSs is maintained by selection, the abundance of deleterious nucleotides in this class suggests a substantial genome-wide drift load.

  12. Reconstruction of the denuded nasoseptal flap donor site with a free fascia lata graft: technical note.

    PubMed

    Zeinalizadeh, Mehdi; Sadrehosseini, Seyed Mousa; Barkhoudarian, Garni; Carrau, Ricardo L

    2016-10-01

    The nasoseptal flap provides hearty vascularized tissue for the reconstruction of skull base defects subsequent to expanded endonasal approaches; however, it leads to exposure of the cartilage at the septal donor site producing crusting and discomfort while it remucosalizes. We report an alternative technique to reconstruct the denuded nasal septal donor site by means of a free fascia lata graft. Fascia lata grafting of the nasoseptal flap donor site showed evidence of revascularization 4 weeks after initial surgery. Re-epithelialization was complete 4-12 weeks postoperation. Although the nasoseptal flap provides a versatile reconstructive technique, its harvest results in significant donor site morbidity. A free fascia lata graft accelerates the rate of donor site remucosalization; thus, decreasing the nasal complications.

  13. Reconstruction of the denuded nasoseptal flap donor site with a free fascia lata graft: technical note.

    PubMed

    Zeinalizadeh, Mehdi; Sadrehosseini, Seyed Mousa; Barkhoudarian, Garni; Carrau, Ricardo L

    2016-10-01

    The nasoseptal flap provides hearty vascularized tissue for the reconstruction of skull base defects subsequent to expanded endonasal approaches; however, it leads to exposure of the cartilage at the septal donor site producing crusting and discomfort while it remucosalizes. We report an alternative technique to reconstruct the denuded nasal septal donor site by means of a free fascia lata graft. Fascia lata grafting of the nasoseptal flap donor site showed evidence of revascularization 4 weeks after initial surgery. Re-epithelialization was complete 4-12 weeks postoperation. Although the nasoseptal flap provides a versatile reconstructive technique, its harvest results in significant donor site morbidity. A free fascia lata graft accelerates the rate of donor site remucosalization; thus, decreasing the nasal complications. PMID:26951218

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

    PubMed Central

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

    2007-01-01

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

  15. 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.

  16. Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene

    PubMed Central

    Khare, Parul; Mortimer, Sandra I.; Cleto, Cynthia L.; Okamura, Kohji; Suzuki, Yutaka; Kusakabe, Takehiro; Nakai, Kenta; Meedel, Thomas H.; Hastings, Kenneth E. M.

    2011-01-01

    In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5′-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5′-leader trans-splicing (SL trans-splicing) because the original 5′-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5′-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA’s original 5′-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla. PMID:21109525

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Novel NPHS1 splice site mutations in a Chinese child with congenital nephrotic syndrome.

    PubMed

    Fu, R; Gou, M F; Ma, W H; He, J J; Luan, Y; Liu, J

    2015-01-01

    Congenital nephrotic syndrome (CNS) is defined as heavy proteinuria or nephrotic syndrome occurring before 3 months of age. It is characterized by early onset and progresses to end-stage renal disease. Recently, several genes associated with CNS have been identified, including NPHS1 and NPHS2. Mutations in the NPHS1 gene have been identified in patients with CNS in Finland with relatively high frequency. Thus far, only a few case reports about CNS have described an NPHS1 mutation in China. In this study, mutational analyses of NPHS1 and NPHS2 were performed in a Chinese child with CNS. Mutations were analyzed in all exons and exon/intron boundaries of NPHS1 and NPHS2 in the patient and his parents as well as in 50 unrelated controls using polymerase chain reaction and direct sequencing techniques. No mutations were detected in NPHS2. A novel splice site mutation (IVS11+1G>A) within intron 11 and a missense mutation within exon 8 (c.928G>A) in the NPHS1 gene were detected in the child. The child's mother had normal urinalysis and a c.928G>A (D310N) heterozygous mutation, and his father had normal urinalysis and IVS11+1G>A. These were not identified in the 50 unrelated controls. The novel splice site mutation of IVS11+1G>A and a missense mutation at c.928G>A in NPHS1 were found to cause CNS in this Chinese child.

  2. 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

  3. Changes in Surgical Site Infections after Living Donor Liver Transplantation

    PubMed Central

    Yamamoto, Masaki; Takakura, Shunji; Iinuma, Yoshitsugu; Hotta, Go; Matsumura, Yasufumi; Matsushima, Aki; Nagao, Miki; Ogawa, Kohei; Fujimoto, Yasuhiro; Mori, Akira; Ogura, Yasuhiro; Kaido, Toshimi; Uemoto, Shinji; Ichiyama, Satoshi

    2015-01-01

    Surgical site infections (SSIs) are a major threat for liver transplant recipients. We prospectively studied SSIs after living donor liver transplantation (LDLT) at Kyoto University Hospital from April 2001 to March 2002 (1st period) and from January 2011 to June 2012 (2nd period). We investigated the epidemiology of SSIs after LDLT and determined the differences between the two periods. A total of 129 adult recipients (66 during the 1st period and 63 during the 2nd period) and 72 pediatric recipients (39 and 33) were included in this study. The SSI rates for each period were 30.3% (1st period) and 41.3% (2nd period) among the adult recipients and 25.6% and 30.3% among the pediatric recipients. The overall rates of 30-day mortality among adult transplant recipients with SSIs were 10.0% (1st period) and 3.9% (2nd period). No pediatric recipient died from SSIs after LDLT in either period. The incidence of Enterococcus faecium increased from 5.0% to 26.9% in the adults and from 10.0% to 40.0% in the pediatric patients. Extended-spectrum β-lactamase-producing Enterobacteriaceae were emerging important isolates during the 2nd period. For this period, a univariate analysis showed that ABO incompatibility (P = 0.02), total operation duration (P = 0.01), graft-to-recipient body weight ratio (GRWR [P = 0.04]), and Roux-en-Y biliary reconstruction (P<0.01) in the adults and age (P = 0.01) and NHSN risk index (P = 0.02) in the children were associated with SSI development. In a multivariate analysis, lower GRWR (P = 0.02) and Roux-en-Y biliary reconstruction (P<0.01) in the adults and older age (P = 0.01) in the children were independent risk factors for SSIs during the 2nd period. In conclusion, SSIs caused by antibiotic resistant bacteria may become a major concern. Lower GRWR and Roux-en-Y biliary reconstruction among adult LDLT recipients and older age among pediatric LDLT recipients increased the risk of developing SSIs after LDLT. PMID:26322891

  4. A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time.

    PubMed

    Yuan, Yu-Xiang; Wu, Jian; Sun, Ri-Fei; Zhang, Xiao-Wei; Xu, Dong-Hui; Bonnema, Guusje; Wang, Xiao-Wu

    2009-01-01

    FLOWERING LOCUS C (FLC), encoding a MADS-domain transcription factor in Arabidopsis, is a repressor of flowering involved in the vernalization pathway. This provides a good reference for Brassica species. Genomes of Brassica species contain several FLC homologues and several of these colocalize with flowering-time QTL. Here the analysis of sequence variation of BrFLC1 in Brassica rapa and its association with the flowering-time phenotype is reported. The analysis revealed that a G-->A polymorphism at the 5' splice site in intron 6 of BrFLC1 is associated with flowering phenotype. Three BrFLC1 alleles with alternative splicing patterns, including two with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in addition to alleles with normal splicing. It was inferred that aberrant splicing of the pre-mRNA leads to loss-of-function of BrFLC1. A CAPS marker was developed for this locus to distinguish Pi6+1(G) and Pi6+1(A). The polymorphism detected with this marker was significantly associated with flowering time in a collection of 121 B. rapa accessions and in a segregating Chinese cabbage doubled-haploid population. These findings suggest that a naturally occurring splicing mutation in the BrFLC1 gene contributes greatly to flowering-time variation in B. rapa.

  5. How did alternative splicing evolve?

    PubMed

    Ast, Gil

    2004-10-01

    Alternative splicing creates transcriptome diversification, possibly leading to speciation. A large fraction of the protein-coding genes of multicellular organisms are alternatively spliced, although no regulated splicing has been detected in unicellular eukaryotes such as yeasts. A comparative analysis of unicellular and multicellular eukaryotic 5' splice sites has revealed important differences - the plasticity of the 5' splice sites of multicellular eukaryotes means that these sites can be used in both constitutive and alternative splicing, and for the regulation of the inclusion/skipping ratio in alternative splicing. So, alternative splicing might have originated as a result of relaxation of the 5' splice site recognition in organisms that originally could support only constitutive splicing. PMID:15510168

  6. Tissue expansion techniques to minimize morbidity of the anterolateral thigh perforator flap donor site.

    PubMed

    Hallock, Geoffrey G

    2013-11-01

    Selection of any free flap donor site must not only meet the requirements of the recipient site but also minimize untoward sequela at the donor site itself. Although the anterolateral thigh (ALT) perforator flap is an ideal soft tissue donor site, a major drawback can be its nonesthetic appearance if a skin graft was needed. This detriment can be ameliorated by using traditional tissue expansion techniques. In a retrospective review over the past decade, 14 patients had ALT free flap donor site tissue expansion. These were subcategorized as pretransfer, concurrent, or posttransfer tissue expansion. In this group, mean ALT flap width was 12.2 ± 4.2 cm, which precluded direct donor site closure. Rectangular expanders were generally recommended. Multiple expanders are suggested for larger defects. The duration of expansion averaged 291.4 ± 163.9 days. The mean instilled volume ratio exceeded 2.43 ± 0.9 times the maximum vendor recommendation. Small skin graft residua were still left in four patients. Tissue expansion proved to be an important modality to consider for minimizing the stigmata of the skin grafted ALT free flap donor site. However, this process is time consuming and requires an additional surgical procedure. As such, this option must be reserved for the most motivated and compliant patients.

  7. Donor-site giant cell reaction following backfill with synthetic bone material during osteochondral plug transfer.

    PubMed

    Fowler, Donald E; Hart, Joseph M; Hart, Jennifer A; Miller, Mark D

    2009-10-01

    Osteochondral defects are common in younger, active patients. Multiple strategies have been used to treat these lesions, including microfracture and osteochondral plug transfer. We describe a patient experiencing chronic knee pain and a full-thickness cartilage defect on the lateral femoral condyle. After failing conservative management and microfracture surgery, the patient underwent osteochondral autograft plug transfer, with backfilling of the donor sites using synthetic bone graft substitute. Initial recovery was uncomplicated until the patient experienced pain following a twist of the knee. Magnetic resonance imaging for the subsequent knee injury revealed poor healing at the donor sites. The donor sites were debrided, and specimens revealed a foreign body giant cell reaction. Donor-site morbidity is of primary concern during osteochondral plug transfer; however, insufficient data exist to support the use of synthetic bone graft material. Our results indicate that off-label use of synthetic bone graft substitute during a primary procedure requires further investigation.

  8. 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

  9. Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT.

    PubMed

    Spivack, J G; Woods, G M; Fraser, N W

    1991-12-01

    Herpes simplex virus type 1 establishes latent infection in trigeminal ganglia of mice infected via the eye. A family of three colinear viral transcripts (LATs), 2.0, 1.5, and 1.45 kb, is present in latently infected ganglia. To characterize these LATs, lambda gt10 cDNA libraries were constructed with RNAs isolated from the trigeminal ganglia of latently infected mice. A series of recombinant bacteriophage were isolated containing cDNA inserts covering 1.7 kb of the 2.0-kb LAT. Splice junctions of the smaller LATs and the 3' end of the 2.0-kb LAT were identified by sequence analysis of RNA polymerase chain reaction products. No splice acceptor site, which does not support the hypotheses that the 2.0-kb LAT is an intron. However, the data are consistent with the possibility of a short leader sequence or multiple LAT transcription start sites. To generate the smaller 1.5- and 1.45-kb LATs, there is a 559-nucleotide intron spliced from the 2.0-kb LAT in strain F and a 556-nucleotide intron in strain 17+. The nucleotide sequences at the 5' and 3' ends of these introns are characteristic of spliced transcripts from eukaryotic protein-encoding genes, with one significant difference; i.e., the 5' end of the LAT intron is GC instead of the consensus sequence GT. This splice donor sequence is conserved in herpes simplex virus type 1 strains F, 17+, and KOS. Processing of the 2.0-kb LAT to form the spliced LATs preserves two open reading frames (ORFs) at the 3' end of the LATs; no new ORFs are created. Splicing of the LATs positions a 276-nucleotide leader sequence close to these ORFs and removes an intron that inhibits their translation in vitro. The novel 5' structure of the intron within the 2.0-kb LAT may be part of a control mechanism for transcription processing that results in splicing of the LATs only in sensory neurons during latent infection and reactivation but not during the viral replication cycle.

  10. Donor site morbidity of the fasciocutaneous radial forearm flap: what does the patient really bother?

    PubMed

    de Witt, Christien A; de Bree, Remco; Verdonck-de Leeuw, Irma M; Quak, Jasper J; Leemans, C René

    2007-08-01

    The objective of this study was the evaluation of donor site morbidity in head and neck cancer patients after reconstruction using a free vascularized radial forearm flap with emphasis on subjective complaints. Fifty patients who underwent at least 6 months before a reconstruction using a free vascularized radial forearm flap were asked to fill out two questionnaires regarding cosmetics and sensibility and forearm disabilities. Furthermore, a function test including movement extensions (flexion-extension, ulnar-radial deviation and pronation-supination), strength (pinch and grip) and temperature (digiti I and V) of the donor and non-donor site were measured and compared. Thirty-five percent of the patients reported no complaints regarding cosmetics and sensibility and 75% mentioned no forearm disabilities. There was no difference in movement extensions, temperature and grip strength between donor and non-donor sites. The difference in pinch strength appeared to be significant (p < 0.001). The total score of the questionnaire on forearm disabilities correlated significantly with extension, pronation and grip strength of the donor arm. Donor site morbidity of the radial forearm flap measured by objective functional tests was limited but subjective self-ratings revealed complaints regarding cosmestics and sensibility and to a lesser extent regarding forearm disability. The present data may be used for solid patient counselling.

  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. 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.

  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. A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene.

    PubMed

    Rodriguez, I R; Gonzalez, P; Zigler, J S; Borrás, T

    1992-10-13

    A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3' end of the coding region. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the zeta-crystallin gene disclosed a dinucleotide deletion of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.

  17. Genome-wide survey of allele-specific splicing in humans

    PubMed Central

    Nembaware, Victoria; Lupindo, Bukiwe; Schouest, Katherine; Spillane, Charles; Scheffler, Konrad; Seoighe, Cathal

    2008-01-01

    Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST) and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array data, including several

  18. 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

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

    PubMed Central

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

    2013-01-01

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

  1. Iliac crest reconstruction to reduce donor-site morbidity: technical note.

    PubMed

    Dusseldorp, Joseph Richard; Mobbs, Ralph J

    2009-09-01

    The design of the study includes case series, technical note and review of the literature. Autogenous bone graft (autograft) harvest from the iliac crest remains the gold standard substrate for spinal fusion. Persistent donor-site pain is the most common cause of morbidity after autograft harvest, occurring in one-third of patients. Numerous techniques for reducing donor-site morbidity have been reported in the literature, including infusion of analgesics and post-harvest reconstruction of the iliac crest with ceramics, allograft or bone morphogenic protein. A case series of patients undergoing spinal fusion surgery is reported. All patients were treated with iliac crest reconstruction using Calcium Phosphate Cement and follow-up data of persistent donor-site pain was collected. Twelve patients underwent autograft harvest with iliac spine reconstruction using Calcium Phosphate Cement. 42% (5) had persistent donor-site pain (after 3 months). All pain scores were less than or equal to 2 out of 10 (mean 1.25). In conclusion, iliac spine reconstruction using Calcium Phosphate Cement following autogenous bone graft harvest is a relatively simple procedure which failed to decrease the incidence of donor-site morbidity in our patient population.

  2. Local full-thickness skin graft of the donor arm--a novel technique for the reduction of donor site morbidity in radial forearm free flap.

    PubMed

    Riecke, B; Assaf, A T; Heiland, M; Al-Dam, A; Gröbe, A; Blessmann, M; Wikner, J

    2015-08-01

    A novel technique to reduce donor site morbidity after radial forearm free flap (RFFF) harvest, using a local full-thickness skin graft (FTSG), is described. Thirty consecutive patients undergoing RFFF for head and neck reconstruction were enrolled in a prospective study. Donor site defect closure was performed with spindle-shaped FTSGs excised from the wavelike skin incision made for the vascular pedicle. Both the removal site of the FTSG on the volar forearm and the covered RFFF donor site healed uneventfully in 29 cases, with no impairment of function related to the skin graft. No skin graft failure and no exposure, tenting, or adherence of the flexor tendons occurred. All patients expressed satisfaction with postoperative pain, the functional outcome, and cosmetic appearance. Primary donor site defect closure could be achieved in all cases with the use of a local FTSG. This graft can be gained at the access incision for the vascular pedicle, avoids expansion of the incision for a local flap technique, and does not prolong wound healing, and thus reduces both donor site and graft site morbidity of the RFFF. This technique leads to an inconspicuous aesthetic result with no apparent relevant functional deficits and avoids the need for a second donor site.

  3. Comparison of donor-site morbidity and satisfaction between anterolateral thigh and parascapular free flaps in the same patient.

    PubMed

    Fischer, Sebastian; Klinkenberg, Marek; Behr, Bjoern; Hirsch, Tobias; Kremer, Thomas; Hernekamp, Frederick; Kolbenschlag, Jonas; Lehnhardt, Marcus; Kneser, Ulrich; Daigeler, Adrien

    2013-10-01

    The purpose of this study was to compare donor-site morbidity after anterolateral thigh (ALT) and parascapular (PS) free flap harvest in the same patient. A total of 13 patients were included in this study. Because of initial flap loss, each patient received ALT as well as PS free flap transplantation. A total of 10 patients were available for follow-up. The average follow-up time was 44.5 months. Besides physical examination, range of motion and scar dimensions were assessed to objectify donor-site deficiencies. The subjective donor-site morbidity was assessed by the patients using a self-report questionnaire. In addition, patients were requested to state their donor-site preference. ALT donor site revealed more sensitivity deficiencies compared with the PS harvest site (8 vs. 4). The latter provoked less functional impairments (1 vs. 2). Scar dimensions were larger at PS harvest site (25.8 × 4.3 cm vs. 23.3 × 3.6 cm). Patients' satisfaction was in favor of the PS donor site (1.9 vs. 2.7). Among the 10 patients, 7 patients preferred the PS and 3 patients preferred the ALT donor site. Comparison of donor-site morbidities in the same patient reveals a valuable tool to diminish individual bias. Despite the low number of cases, we were able to demonstrate the superiority of PS compared with ALT donor sites.The Level of Evidence of the study is III.

  4. The intronic splicing code: multiple factors involved in ATM pseudoexon definition.

    PubMed

    Dhir, Ashish; Buratti, Emanuele; van Santen, Maria A; Lührmann, Reinhard; Baralle, Francisco E

    2010-02-17

    Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5' splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.

  5. A novel splice-site mutation in ALS2 establishes the diagnosis of juvenile amyotrophic lateral sclerosis in a family with early onset anarthria and generalized dystonias.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. The chin as a donor site in early secondary osteoplasty: a retrospective clinical and radiological evaluation.

    PubMed

    Hoppenreijs, T J; Nijdam, E S; Freihofer, H P

    1992-04-01

    26 unilateral cleft palate patients received an autogenous bicortical chin bone graft for early reconstruction of the alveolar process. In the evaluation of the donor site, 4% of the anterior teeth showed a negative pulpal sensibility, less than 1% a peri-apical granuloma and 12% pulp canal obliteration. The tooth buds of the canines showed developmental disturbances in 6%. Exposure of unerupted canines should be avoided, and a 5 mm safety margin is advised. Based on its architecture, topographic accessibility, minimal post-operative morbidity and absence of visible scars, the chin can be considered to be a very useful donor site in bone grafting procedures.

  8. Perforator arteries of the medial upper arm: anatomical basis of a new flap donor site.

    PubMed

    Perignon, D; Havet, E; Sinna, R

    2013-01-01

    The development of perforator flaps' concept based on knowledge on vascular anatomy of the skin represents a major improvement in reconstructive surgery. Succeeding description about vascular territories and anatomical basics of the main donor sites, the study of hidden donor sites, such as medial upper arm, constitutes a new step and an additional refinement. 20 upper limbs of 10 fresh adult cadavers were studied with colored latex injections. The origin and distribution of the perforator arteries of the superior ulnar collateral artery and the brachial artery were investigated. We have noted constant perforator arteries and described the limits of vascular territories of the medial upper arm.

  9. 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

  10. 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

  11. In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein.

    PubMed

    Rain, J C; Legrain, P

    1997-04-01

    Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.

  12. 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

  13. 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

  14. Desmin splice variants causing cardiac and skeletal myopathy.

    PubMed

    Park, K Y; Dalakas, M C; Goebel, H H; Ferrans, V J; Semino-Mora, C; Litvak, S; Takeda, K; Goldfarb, L G

    2000-11-01

    Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A-->G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G-->A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A-->G and IVS2-1G-->A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.

  15. Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression.

    PubMed

    Ajiro, Masahiko; Jia, Rong; Zhang, Lifang; Liu, Xuefeng; Zheng, Zhi-Ming

    2012-01-01

    HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5' splice sites (5' ss) and three 3' splice sites (3' ss) normally used in HPV16(+) cervical cancer and its derived cell lines. The choice of two novel alternative 5' ss (nt 221 5' ss and nt 191 5' ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5' ss and nt 409 3' ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3' ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3' ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of (91)QYNK(94) to (91)PSFW(94) displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

  16. Splicing of intron 3 of human BACE requires the flanking introns 2 and 4.

    PubMed

    Annies, Maik; Stefani, Muriel; Hueber, Andreas; Fischer, Frauke; Paganetti, Paolo

    2009-10-16

    Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer's disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.

  17. 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

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

    PubMed

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

    2011-09-01

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

  19. 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

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

    PubMed Central

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

    2013-01-01

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

  1. 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.

  2. 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

  3. A novel splicing site mutation of the GPR143 gene in a Chinese X-linked ocular albinism pedigree.

    PubMed

    Cai, C Y; Zhu, H; Shi, W; Su, L; Shi, O; Cai, C Q; Ling, C; Li, W D

    2013-01-01

    Ocular albinism is an X-linked inherited disease characterized by hypopigmentation of the iris and nystagmus. To identify a new disease-causing mutation of ocular albinism, we collected a Han Chinese pedigree with 7 male congenital nystagmus patients over 3 generations. Slit-lamp photography and optical coherence tomography were performed for the proband. Genomic DNA was extracted from a whole blood sample from the proband using the high-salt method. Polymerase chain reaction (PCR) sequencing was carried out for GPR143 and FRMD7 genes. The three-dimensional structures of the wild-type and mutant GPR143 proteins were determined using SWISS-MODEL. The transmission of the disease in the pedigree clearly followed an X-linked pattern. The proband had significant iris and fundus hypopigmentation. Optical coherence tomography showed severe foveal hypoplasias in both eyes of the proband. A novel splicing site (G/C) mutation was found on the boundary of the 6th intron and the 7th exon of the GPR143 gene, resulting in a 9-amino-acid deletion (codons 257-265) in the 6th transmembrane domain of the GPR143 protein. In conclusion, a novel splicing site mutation of the GPR143 gene was found in a Han Chinese congenital ocular albinism pedigree. PMID:24301936

  4. 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.

  5. 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

  6. 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

  7. Structure function and splice site analysis of the synaptogenic activity of the neurexin-1 beta LNS domain.

    PubMed

    Graf, Ethan R; Kang, Yunhee; Hauner, Anna M; Craig, Ann Marie

    2006-04-19

    Recent findings suggest that the neurexin-neuroligin link promotes both GABAergic and glutamatergic synaptogenesis, but the mechanism by which neurexins influence the clustering of appropriate neuroligins and postsynaptic differentiation remains unclear. Previous studies suggested that the presence or absence of alternatively spliced residues at splice site 4 (S4) in the neurexin LNS domain may regulate neurexin function. We demonstrate that addition of the S4 insert selectively reduces the ability of neurexin-1beta to cluster neuroligin-1/3/4 and glutamatergic postsynaptic proteins, although clustering of neuroligin-2 and GABAergic postsynaptic proteins remain strong. Furthermore, addition of the S4 insert decreases the binding affinity of neurexin-1beta to neuroligins-1 and -4 but has little effect on binding to neuroligins-2 and -3. Additional structure-function studies reveal the neurexin binding interface mediating synaptogenic activity to be composed primarily of residues in the beta2beta3, beta6beta7, and beta10beta11 loops on one rim of the LNS domain beta sandwich. Mutation of two predicted Ca(2+)-binding residues disrupts postsynaptic protein clustering and binding to neuroligins, consistent with previous findings that neurexin-neuroligin binding is Ca2+ dependent. Glutamatergic postsynaptic clustering was more readily disrupted by the mutagenesis than GABAergic postsynaptic protein clustering. Perhaps neurexins-neuroligins, or neurexin-1beta at least, is most important for GABA synapse formation or controlling the balance of GABA and glutamate synapses. These results suggest that differential neurexin-neuroligin binding affinities and splice variations may play an instructive role in postsynaptic differentiation.

  8. 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.

  9. 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

  10. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

    PubMed

    Darman, Rachel B; Seiler, Michael; Agrawal, Anant A; Lim, Kian H; Peng, Shouyong; Aird, Daniel; Bailey, Suzanna L; Bhavsar, Erica B; Chan, Betty; Colla, Simona; Corson, Laura; Feala, Jacob; Fekkes, Peter; Ichikawa, Kana; Keaney, Gregg F; Lee, Linda; Kumar, Pavan; Kunii, Kaiko; MacKenzie, Crystal; Matijevic, Mark; Mizui, Yoshiharu; Myint, Khin; Park, Eun Sun; Puyang, Xiaoling; Selvaraj, Anand; Thomas, Michael P; Tsai, Jennifer; Wang, John Y; Warmuth, Markus; Yang, Hui; Zhu, Ping; Garcia-Manero, Guillermo; Furman, Richard R; Yu, Lihua; Smith, Peter G; Buonamici, Silvia

    2015-11-01

    Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer. PMID:26565915

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Splicing site mutations in dentin sialophosphoprotein causing dentinogenesis imperfecta type II.

    PubMed

    Holappa, Heidi; Nieminen, Pekka; Tolva, Liisa; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu

    2006-10-01

    Dentinogenesis imperfecta (DGI) type II (OMIM # 125490) is an inherited disorder affecting dentin. Defective dentin formation results in discolored teeth that are prone to attrition and fracture. To date, several mutations have been described in the dentin sialophosphoprotein (DSPP) gene, causing DGI types II and III and dentin dysplasia type II. DSPP encodes two proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here, we describe a mutational analysis of DSPP in seven Finnish families with DGI type II. We report two mutations and five single nucleotide polymorphisms. In one family we found a mutation that has been described earlier in families with different ethnicity, while in six families we found a novel g.1194C>A (IVS2-3) transversion. Bioinformatic analysis of known DSPP mutations suggests that DGI type II is usually caused by aberration of normal splicing.

  16. 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

  17. Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome

    PubMed Central

    Furukawa, Hiroshi; Murata, Shigeo; Yabe, Toshio; Shimbara, Naoki; Keicho, Naoto; Kashiwase, Kouichi; Watanabe, Kaoru; Ishikawa, Yoshihide; Akaza, Tatsuya; Tadokoro, Kenji; Tohma, Shigeto; Inoue, Tetsufumi; Tokunaga, Katsushi; Yamamoto, Kazuhiko; Tanaka, Keiji; Juji, Takeo

    1999-01-01

    Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient. J. Clin. Invest. 103:649–652 (1999) PMID:10074494

  18. 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.

  19. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

    PubMed Central

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-01-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations. PMID:21407258

  20. Two stages splicing system

    NASA Astrophysics Data System (ADS)

    Mudaber, Mohammad Hassan; Yusof, Yuhani

    2015-05-01

    The study of the biological process of deoxyribonucleic acid (DNA) splicing system in a translucent approach was investigated in 2012 by Yusof under the framework of formal language theory. In this work, the concepts of splicing system in two stages as well as splicing languages are mathematically and biologically discussed. Additionally, some theorems based on recognition site factor of initial strings at the existence of two initial strings and two rules are provided via Yusof-Goode (Y-G) approach. Besides, an example is also given in showing the biological meaning of the introduced concept.

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

    NASA Astrophysics Data System (ADS)

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

    2002-05-01

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

  2. Human palatal and tuberosity mucosa as donor sites for ridge augmentation.

    PubMed

    Dellavia, Claudia; Ricci, Giano; Pettinari, Letizia; Allievi, Cristina; Grizzi, Fabio; Gagliano, Nicoletta

    2014-01-01

    Since different clinical outcomes of periodontal bilaminar surgeries using the palate or the maxillary tuberosity as connective tissue (CT) donor sites have been observed, tissues grafted with CT from the palate or the tuberosity 1 year after surgical procedures for ridge augmentation were compared with nongrafted tissues by using morphologic and molecular methods. Collagen content and matrix metalloproteinases 1 and 2 expression were similar in tissues and cultured fibroblasts from the palate and tuberosity, although with interindividual differences. In contrast, differences in collagen cross-linking and maturation in the tuberosity fibroblasts were observed, suggesting a possible role in determining hyperplastic responses in some patients. PMID:24600654

  3. Clinical, in silico, and experimental evidence for pathogenicity of two novel splice site mutations in the SH3TC2 gene.

    PubMed

    Laššuthová, Petra; Gregor, Martin; Sarnová, Lenka; Machalová, Eliška; Sedláček, Radek; Seeman, Pavel

    2012-09-01

    Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676-8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676-8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676-8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676-8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.

  4. 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.

  5. A novel splicing mutation alters DSPP transcription and leads to dentinogenesis imperfecta type II.

    PubMed

    Zhang, Jun; Wang, Jiucun; Ma, Yanyun; Du, Wenqi; Zhao, Siyang; Zhang, Zuowei; Zhang, Xiaojiao; Liu, Yue; Xiao, Huasheng; Wang, Hongyan; Jin, Li; Liu, Jie

    2011-01-01

    Dentinogenesis imperfecta (DGI) type II is an autosomal dominant disease characterized by a serious disorders in teeth. Mutations of dentin sialophosphoprotein (DSPP) gene were revealed to be the causation of DGI type II (DGI-II). In this study, we identified a novel mutation (NG_011595.1:g.8662T>C, c.135+2T>C) lying in the splice donor site of intron 3 of DSPP gene in a Chinese Han DGI-II pedigree. It was found in all affected subjects but not in unaffected ones or other unrelated healthy controls. The function of the mutant DSPP gene, which was predicted online and subsequently confirmed by in vitro splicing analysis, was the loss of splicing of intron 3, leading to the extended length of DSPP mRNA. For the first time, the functional non-splicing of intron was revealed in a novel DSPP mutation and was considered as the causation of DGI-II. It was also indicated that splicing was of key importance to the function of DSPP and this splice donor site might be a sensitive mutation hot spot. Our findings combined with other reports would facilitate the genetic diagnosis of DGI-II, shed light on its gene therapy and help to finally conquer human diseases.

  6. Mitogen-Activated Protein Kinase Phosphorylation of Splicing Factor 45 (SPF45) Regulates SPF45 Alternative Splicing Site Utilization, Proliferation, and Cell Adhesion

    PubMed Central

    Al-Ayoubi, Adnan M.; Zheng, Hui; Liu, Yuying; Bai, Tao

    2012-01-01

    The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38α also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H2O2, UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation. PMID:22615491

  7. 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

  8. Role of helical constraints of the EBS1-IBS1 duplex of a group II intron on demarcation of the 5' splice site.

    PubMed

    Popovic, Milena; Greenbaum, Nancy L

    2014-01-01

    Recognition of the 5' splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3' end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5' splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns. PMID:24243113

  9. 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

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

    PubMed

    Acar, Mustafa; Yazıcı, Demet; San, Turhan; Muluk, Nuray Bayar; Cingi, Cemal

    2015-04-01

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

  11. Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

    PubMed

    Gilfoyle, D J; Rodriguez-Lopez, J N; Smith, A T

    1996-03-01

    The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why

  12. 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

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

    PubMed Central

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

    2016-01-01

    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

  14. Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

    SciTech Connect

    Cizdziel, P.E.; De Mars, M.; Murphy, E.C. Jr.

    1988-04-01

    The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33/sup 0/C or lower than at 37 to 41/sup 0/C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41/sup 0/C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. The authors exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39/sup 0/C. However, after a short (about 30-min) lag following a shift to 33/sup 0/C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA.

  15. Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

    PubMed

    Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

    2005-06-01

    In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed

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

    PubMed Central

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

    2015-01-01

    This study describes a new mechanism controlling the production of alternatively-spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively-spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5’ splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5’ splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the α3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative

  18. Two siblings with homozygous pathogenic splice-site variant in mitochondrial asparaginyl-tRNA synthetase (NARS2).

    PubMed

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

    2015-02-01

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

  19. Two siblings with homozygous pathogenic splice-site variant in mitochondrial asparaginyl-tRNA synthetase (NARS2).

    PubMed

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

    2015-02-01

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

  20. 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

  1. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Henseler, M.; Klein, A.; Reber, M.; Vanier, M. T.; Landrieu, P.; Sandhoff, K.

    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 "saposins," 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-->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. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

  4. 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

  5. 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

  6. RNA splicing in a new rhabdovirus from Culex mosquitoes.

    PubMed

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-07-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

  7. 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.

  8. Evolution of splicing regulatory networks in Drosophila

    PubMed Central

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

    2014-01-01

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

  9. 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.

  10. Late quadriceps tendon rupture at the donor site following cruciate ligament reconstruction using central quadriceps tendon graft.

    PubMed

    Pandey, Vivek; Madi, Sandesh; Joseph, Amy; Acharya, Kiran

    2015-10-16

    Central quadriceps tendon (CQT) graft has been successfully used as a viable autograft option in cruciate ligament reconstruction of the knee. The prime emphasis in the majority of the literature is given to surgical details of quadriceps graft harvesting and outcome of cruciate ligament reconstruction. There is less discussion about donor site morbidity in CQT graft, and it is less frequent as compared to that in bone patellar tendon bone graft. We report an extremely unusual case of late quadriceps tendon rupture at the donor site following anterior cruciate ligament reconstruction using CQT graft.

  11. Large exon size does not limit splicing in vivo.

    PubMed

    Chen, I T; Chasin, L A

    1994-03-01

    Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

  12. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    PubMed Central

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  13. 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/.

  14. 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

  15. Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome

    PubMed Central

    Shah, Mohd Hussain; Bhat, Vishwanath; Shetty, Jyoti S.

    2014-01-01

    Purpose Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, microspherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Methods Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)–PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. Results The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT–PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. Conclusions The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six. PMID:24940034

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

    PubMed

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

    2014-09-01

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

  17. 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

  18. Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

    PubMed Central

    Garbarz, M; Tse, W T; Gallagher, P G; Picat, C; Lecomte, M C; Galibert, F; Dhermy, D; Forget, B G

    1991-01-01

    The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain. Images PMID:2056132

  19. Unusual Splice-Site Mutations in the RSK2 Gene and Suggestion of Genetic Heterogeneity in Coffin-Lowry Syndrome

    PubMed Central

    Zeniou, Maria; Pannetier, Solange; Fryns, Jean-Pierre; Hanauer, André

    2002-01-01

    Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation that is characterized, in male patients, by psychomotor and growth retardation and various skeletal anomalies. Typical facial changes and specific clinical and radiological hand aspects exhibited by patients are essential clues for the diagnosis. CLS is caused by mutations in a gene that is located in Xp22.2 and that encodes RSK2, a growth-factor–regulated protein kinase. RSK2 mutations are extremely heterogeneous and lead to premature termination of translation and/or loss of phosphotransferase activity. Surprisingly, among a series of 250 patients screened by single-strand conformation polymorphism (SSCP) analysis, in whom a clinical diagnosis of CLS was made, no mutations were detected in 66% (165) of the patients. To determine what proportion of these latter patients have a RSK2 mutation that has not been detected and what proportion have different disorders that are phenotypically similar to CLS, we have, in the present article, investigated, by western blot analysis and in vitro kinase assay, cell lines from 26 patients in whom no mutation was previously identified by SSCP analysis. This approach allowed us to identify seven novel RSK2 mutations: two changes in the coding sequence of RSK2, one intragenic deletion, and four unusual intronic nucleotide substitutions that do not affect the consensus GT or AG splice sites. We have also determined the nucleotide sequence of the promoter region of the RSK2 gene, and we have screened it for mutations. No disease-causing nucleotide change was identified, suggesting that mutations affecting the promoter region are unlikely to account for a large number of patients with CLS. Finally, our results provide evidence that some patients have a disease that is phenotypically very similar to CLS, which is not caused by RSK2 defects. This suggests that there are defects in either additional genes or combinations of genes that may result in a CLS

  20. 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

  1. 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

  2. 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.

  3. Alternative autogenous bone graft donor sites in brachymetatarsia reconstruction: a review of the literature with clinical presentations.

    PubMed

    Kashuk, K B; Hanft, J R; Schabler, J A; Kopelman, J

    1991-01-01

    The authors present a literature review on the surgical treatment of brachymetatarsia. They discuss the identification of donor sites for autogenous bone graft harvesting from the foot. Three case reports of brachymetatarsia, one of iatrogenic, and two of congenital origin are presented. PMID:1874999

  4. The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

    PubMed

    Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

    2015-05-01

    When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

  5. 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

  6. RNA splicing and genes

    SciTech Connect

    Sharp, P.A.

    1988-11-25

    The splicing of long transcripts RNA (copied from DNA in the cell nucleus) into smaller specific mRNA is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing 5' and 3' splice sites. This complex contains the evolutionary highly conserved small nuclear RNAs (snRNAs) Us, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRANs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

  7. 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.

  8. 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

  9. Discovery and Mass Spectrometric Analysis of Novel Splice-junction Peptides Using RNA-Seq*

    PubMed Central

    Sheynkman, Gloria M.; Shortreed, Michael R.; Frey, Brian L.; Smith, Lloyd M.

    2013-01-01

    Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Eighty million paired-end Illumina reads and ∼500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and

  10. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    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.

  11. 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

  12. Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3' splice site of intron 1)

    PubMed

    Aldred, A R; Prapunpoj, P; Schreiber, G

    1997-06-01

    Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406], showed that the N-terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N-termini of transthyretin evolved by successive shifts of the 3' splice site of intron 1 in the 3' direction, resulting in successive shortening of the 5' end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N-terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.

  13. Compound heterozygote for lipoprotein lipase deficiency: Ser----Thr244 and transition in 3' splice site of intron 2 (AG----AA) in the lipoprotein lipase gene.

    PubMed Central

    Hata, A; Emi, M; Luc, G; Basdevant, A; Gambert, P; Iverius, P H; Lalouel, J M

    1990-01-01

    Cloning and sequencing of translated exons and intron-exon boundaries of the lipoprotein lipase gene in a patient of French descent who has the chylomicronemia syndrome revealed that he was a compound heterozygote for two nucleotide substitutions. One (TCC----ACC) leads to an amino acid substitution (Ser----Thr244), while the other alters the 3' splice site of intron 2 (AG----AA). The functional significance of the Thr244 amino acid substitution was established by in vitro expression in cultured mammalian cells. Images Figure 1 Figure 2 PMID:2121025

  14. Species-dependent splice recognition of a cryptic exon resulting from a recurrent intronic CEP290 mutation that causes congenital blindness.

    PubMed

    Garanto, Alejandro; Duijkers, Lonneke; Collin, Rob W J

    2015-01-01

    A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo.

  15. 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

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

    PubMed

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

    2015-02-01

    Nipple reconstruction is of importance in achieving the best possible aesthetic outcome after breast reconstruction. Nipple sharing is a common technique; this study focused on the potential morbidity at the donor nipple. Between 2008 and 2012, 26 patients underwent nipple sharing at our institution. The donor nipple was examined before and after the procedure (mean follow-up of 21 months). Sensitivity, projection, diameter, and patient satisfaction were evaluated. The sensitivity in the donor nipple decreased, albeit insignificantly, from 1.2 g/mm2 (0.8-1.6) to 1.8 g/mm2 (0.8-4.8) (p=0.054, n=26). The projection due to graft removal decreased from 8.0 mm (6.8-10.0) to 4.5 mm (4.0-5.0) (p=0.001). Of the patients, 88% were "very satisfied" or "somewhat satisfied" with the sensitivity and 89% with the symmetry between the donor and reconstructed nipple. At least 60% of the patients were "very satisfied" with all aesthetic outcome parameters (projection, appearance, naturalness, color, and shape). All patients would agree to undergo this procedure again, if necessary. Nipple sharing was associated with minimal morbidity at the donor nipple. The postoperative projection was adequate. Regardless of whether simultaneous mastopexy was performed, the loss of sensitivity was minimal and presumably imperceptible to the patient. By using no sutures after graft removal and letting the donor nipple heal spontaneously, scarring was minimized and the natural appearance and good sensitivity of the donor nipple were preserved.

  17. 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.

  18. Homozygosity for the common GAA gene splice site mutation c.-32-13T>G in Pompe disease is associated with the classical adult phenotypical spectrum.

    PubMed

    Musumeci, Olimpia; Thieme, Andrea; Claeys, Kristl G; Wenninger, Stephan; Kley, Rudolf A; Kuhn, Marius; Lukacs, Zoltan; Deschauer, Marcus; Gaeta, Michele; Toscano, Antonio; Gläser, Dieter; Schoser, Benedikt

    2015-09-01

    Homozygosity for the common Caucasian splice site mutation c.-32-13T>G in intron 1 of the GAA gene is rather rare in Pompe patients. We report on the clinical, biochemical, morphological, muscle imaging, and genetic findings of six adult Pompe patients from five unrelated families with the c.-32-13T>G GAA gene mutation in homozygous state. All patients had decreased GAA activity and elevated creatine kinase levels. Five patients, aged between 43 and 61 years (median 53 years), initially presented with myalgia, hyperCKaemia, and/or exercise induced fatigue at an age of onset (12-55 years). All but one had proximal lower limb weakness combined with axial weakness and moderate respiratory insufficiency; the sixth patient presented with hyperCKaemia only. Muscle biopsies showed PAS-positive vacuolar myopathy with lysosomal changes and reduced GAA activity. Muscle MRI of lower limb muscles revealed a moderate adipose substitution of the gluteal muscles, biceps femoris and slight fatty infiltration of all thigh muscles. One MRI of the respiratory muscles revealed a diaphragmatic atrophy with unilateral diaphragm elevation. So, the common Caucasian, so called mild, splice site mutation c.-32-13T>G in intron 1 of the GAA gene in a homozygote status reflects the full adult Pompe disease phenotype severity spectrum. PMID:26231297

  19. 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

  20. Functional analysis of a C. elegans trans-splice acceptor.

    PubMed Central

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

    1993-01-01

    The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site. Images PMID:8451190

  1. Computation of direct and inverse mutations with the SEGM web server (Stochastic Evolution of Genetic Motifs): an application to splice sites of human genome introns.

    PubMed

    Benard, Emmanuel; Michel, Christian J

    2009-08-01

    We present here the SEGM web server (Stochastic Evolution of Genetic Motifs) in order to study the evolution of genetic motifs both in the direct evolutionary sense (past-present) and in the inverse evolutionary sense (present-past). The genetic motifs studied can be nucleotides, dinucleotides and trinucleotides. As an example of an application of SEGM and to understand its functionalities, we give an analysis of inverse mutations of splice sites of human genome introns. SEGM is freely accessible at http://lsiit-bioinfo.u-strasbg.fr:8080/webMathematica/SEGM/SEGM.html directly or by the web site http://dpt-info.u-strasbg.fr/~michel/. To our knowledge, this SEGM web server is to date the only computational biology software in this evolutionary approach.

  2. Splicing of the Large Intron Present in the Nonstructural Gene of Minute Virus of Mice Is Governed by TIA-1/TIAR Binding Downstream of the Nonconsensus Donor▿

    PubMed Central

    Choi, Eun-Young; Pintel, David

    2009-01-01

    The essential proteins NS1 and NS2 of minute virus of mice are encoded by mRNAs R1 and R2, respectively. R2 is derived from R1 by excision of a large intron and thus splicing governs the relative ratios of NS1 and NS2. Excision of the large intron utilizes a nonconsensus 5′ donor site. We identified a U-rich and A-rich intronic sequence immediately downstream of the nonconsensus 5′ donor site that functions as an intronic splicing enhancer (ISE) required for efficient large-intron excision. The ISE binds the cellular RNA-processing proteins TIA-1 and TIAR, which enhance usage of the nonconsensus donor. PMID:19339348

  3. 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

  4. 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.

  5. The effect of conventional surgery and piezoelectric surgery bone harvesting techniques on the donor site morbidity of the mandibular ramus and symphysis.

    PubMed

    Altiparmak, N; Soydan, S S; Uckan, S

    2015-09-01

    The aim of this study was to evaluate the morbidity following bone harvesting at two different intraoral donor sites, mandibular symphysis and ramus, and to determine the effects of piezoelectric and conventional surgical graft harvesting techniques on donor site morbidity. Intraoral block bone grafts were harvested from the symphysis (n=44) and ramus (n=31). The two donor site groups were divided into two subgroups according to the surgical graft harvesting method used (conventional or piezoelectric surgery). Intraoperative and postoperative pain was assessed using a visual analogue scale (VAS). Donor site morbidity and the harvesting techniques were compared statistically. Of 290 teeth evaluated in the symphysis group, four needed root canal treatment after surgery. The incidence of transient paresthesia in the mucosa was significantly higher in the symphysis group than in the ramus group (P=0.004). In the symphysis group, the incidence of temporary skin and mucosa paresthesia was lower in the piezoelectric surgery subgroup than in the conventional surgery subgroup (P=0.006 and P=0.001, respectively). No permanent anaesthesia of any region of the skin was reported in either donor site group. VAS scores did not differ between the ramus and symphysis harvesting groups, or between the piezoelectric and conventional surgery subgroups. When the symphysis was chosen as the donor site, minor sensory disturbances of the mucosa and teeth were recorded. The use of piezoelectric surgery during intraoral harvesting of bone blocks, especially from the symphysis, can reduce these complications.

  6. Hemicellulose dressing versus rayon dressing in the re-epithelialization of split-thickness skin graft donor sites: a multicenter study.

    PubMed

    Ferreira, Lydia M; Blanes, Leila; Gragnani, Alfredo; Veiga, Daniela F; Veiga, Frederico P; Nery, Gilka B; Rocha, Gustavo Henrique H R; Gomes, Heitor C; Rocha, Mario G; Okamoto, Regina

    2009-08-01

    The aim of this study was to compare the effectiveness of a hemicellulose dressing with that of rayon dressing in the healing of split-thickness skin graft donor sites. Twenty-eight patients were selected from five different hospitals and randomized into two groups: hemicellulose dressing group and rayon dressing group. All patients underwent split-thickness skin grafting for various reasons, and the skin graft donor site wounds were covered with hemicellulose dressing (n=14) or rayon dressing (n=14). The donor site was assessed on postoperative days 1, 7, 14, 21, and 28 for hyperemia, pruritus, pain, exudate level, and adherence of the wound dressing. At the 60-day follow-up visit, the donor site was assessed again for pruritus and pain. Touch-pressure, thermal, and pain sensibility tests were performed preoperatively and on postoperative day 60 together with the assessment of color and texture of the re-epithelialized area. In all patients, re-epithelialization was completed between 14 and 21 days after surgery. There were no significant differences between the two groups with regard to pain, hyperemia, pruritus, exudate, and final appearance (color and texture) of the skin graft donor site. The rayon dressing provided significantly better adherence than the hemicellulose dressing, and both dressings showed similar results with regard to the parameters evaluated when used in the treatment of split-thickness skin graft donor sites.

  7. 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.

  8. 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

  9. 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

  10. A novel splice site mutation of FGD1 gene in an Aarskog-Scott syndrome patient with a large anterior fontanel.

    PubMed

    Parıltay, Erhan; Hazan, Filiz; Ataman, Esra; Demir, Korcan; Etlik, Özdal; Özbek, Erhan; Özkan, Behzat

    2016-09-01

    Aarskog-Scott syndrome (ASS) is a rare X-linked recessive genetic disorder caused by FGD1 mutations. FGD1 regulates the actin cytoskeleton and regulates cell growth and differentiation by activating the c-Jun N-terminal kinase signaling cascade. ASS is characterized by craniofacial dysmorphism, short stature, interdigital webbing and shawl scrotum. However, there is a wide phenotypic heterogeneity because of the additional clinical features. ASS and some syndromes including the autosomal dominant inherited form of Robinow syndrome, Noonan syndrome, pseudohypoparathyroidism, Silver-Russel and SHORT syndrome have some overlapping phenotypic features. Herein, we report a patient with ASS and a large anterior fontanel who was initially diagnosed as Robinow syndrome. He was found to have a novel c.1340+2 T>A splice site mutation on the FGD1 gene. PMID:27544718

  11. A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

    PubMed

    Zhang, Junyu; Liu, Hongbin; Liu, Zhiyuan; Liao, Yong; Guo, Luo; Wang, Honglian; He, Lin; Zhang, Xiaodong; Xing, Qinghe

    2013-01-01

    Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1.

  12. 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.

  13. Elastomeric enriched biodegradable polyurethane sponges for critical bone defects: a successful case study reducing donor site morbidity.

    PubMed

    Lavrador, Catarina; Mascarenhas, Ramiro; Coelho, Paulo; Brites, Cláudia; Pereira, Alfredo; Gogolewski, Sylwester

    2016-03-01

    Bone substitutes have been a critical issue as the natural source can seldom provide enough bone to support full healing. No bone substitute complies with all necessary functions and characteristics that an autograft does. Polyurethane sponges have been used as a surgical alternative to cancellous bone grafts for critical bone defect donor sites. Critical bone defects were created on the tibial tuberosity and iliac crest using an ovine model. In group I (control-untreated), no bone regeneration was observed in any animal. In group II (defects left empty but covered with a microporous polymeric membrane), the new bone bridged the top ends in all animals. In groups III and IV, bone defects were implanted with polyurethane scaffolds modified with biologically active compounds, and bone regeneration was more efficient than in group II. In groups III and IV there were higher values of bone regeneration specific parameters used for evaluation (P < 0.05) although the comparison between these groups was not possible. The results obtained in this study suggest that biodegradable polyurethane substitutes modified with biologically active substances may offer an alternative to bone graft, reducing donor site morbidity associated with autogenous cancellous bone harvesting.

  14. 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

  15. Understanding splicing regulation through RNA splicing maps.

    PubMed

    Witten, Joshua T; Ule, Jernej

    2011-03-01

    Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

  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. 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

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

    PubMed

    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.

  19. 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

  20. 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

  1. 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.

  2. Comparison of the radial forearm flap and the thinned anterolateral thigh cutaneous flap for reconstruction of tongue defects: an evaluation of donor-site morbidity.

    PubMed

    Huang, Chih-Hung; Chen, Hung-Chi; Huang, Yau-Lin; Mardini, Samir; Feng, Guan-Ming

    2004-12-01

    The radial forearm flap is commonly used for reconstruction of tongue defects following tumor extirpation. This flap is easy to harvest and offers thin tissue with large-caliber vessels. However, its use leaves behind a conspicuous aesthetic deformity in the forearm and requires the sacrifice of a major artery of that limb, the radial artery. The anterolateral thigh cutaneous flap has found clinical applications in the reconstruction of soft-tissue defects requiring thin tissue. More recently, in a thinned form, the anterolateral thigh flap has been used for reconstructing defects of the tongue with functional results equivalent to that of the radial forearm flap. For the reconstruction of tongue defects, these two flaps could provide similar soft-tissue coverage, but they seem to result in different donor-site appearances. The donor site is closed primarily, leaving only a linear scar that is inconspicuous with normal clothing, and no functional deficit is left behind in the thigh. Thus, for the supply of flaps for tongue defects, a comparison between the radial forearm flap and the anterolateral thigh flap donor sites is provided in this study. Between December of 2000 and August of 2002, 41 patients who underwent reconstruction of defects of the tongue using either a radial forearm flap or an anterolateral thigh flap were evaluated. The focus was on the evaluation of the functional and aesthetic outcome of the donor site after harvesting these flaps for the purpose of reconstructing either total or partial tongue defects. Finally, a comparison was performed between the donor sites of the two flaps. The disadvantages of the radial forearm flap include the conspicuous unattractive scar in the forearm region, pain, numbness, and the sacrifice of a major artery of the limb. In some patients, the donor-site scar of the forearm acted as a social stigma, preventing these patients from leading a normal life. In contrast, the anterolateral thigh cutaneous flap, after

  3. 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

  4. 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.

  5. Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

    PubMed Central

    Zhang, L; Liu, W; Grabowski, P J

    1999-01-01

    In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are

  6. Tongue reconstruction with minimal donor site morbidity using a deep inferior epigastric perforator (DIEP) free flap in a 6-year-old girl.

    PubMed

    Yano, Tomoyuki; Okazaki, Mutsumi; Kawaguchi, Runa; Suesada, Nobuko; Tanaka, Kentaro; Kishimoto, Seiji

    2013-09-01

    Tongue reconstruction was performed using a deep inferior epigastric perforator (DIEP) free flap in a 6-year-old girl with undifferentiated sarcoma of the tongue. After hemi-glossectomy with upper neck dissection, a 3-lobed DIEP free flap was used for the reconstruction. Donor site was closed primarily with suturing umbilicus in proper position. No flap loss, leakage, or infection occurred. Postoperatively, the patient was able to consume a normal diet without difficulty or aspiration and displayed good speech function. No donor site morbidity, e.g., herniation or bulging, was observed, and the patient was able to perform their normal daily activities. DIEP flaps provide a pliable skin paddle, an adequate amount of adipose tissue, and reduced donor site morbidity, even in children. We did not have any difficulty harvesting the DIEP flap or with the microvascular anastomosis. We consider DIEP free flaps to be the ideal option for pediatric tongue reconstruction.

  7. Genome-Wide Landscape of Alternative Splicing Events in Brachypodium distachyon

    PubMed Central

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

    2013-01-01

    Recently, Brachypodium distachyon has emerged as a model plant for studying monocot grasses and cereal crops. Using assembled expressed transcript sequences and subsequent mapping to the corresponding genome, we identified 1219 alternative splicing (AS) events spanning across 2021 putatively assembled transcripts generated from 941 genes. Approximately, 6.3% of expressed genes are alternatively spliced in B. distachyon. We observed that a majority of the identified AS events were related to retained introns (55.5%), followed by alternative acceptor sites (16.7%). We also observed a low percentage of exon skipping (5.0%) and alternative donor site events (8.8%). The ‘complex event’ that consists of a combination of two or more basic splicing events accounted for ∼14.0%. Comparative AS transcript analysis revealed 163 and 39 homologous pairs between B. distachyon and Oryza sativa and between B. distachyon and Arabidopsis thaliana, respectively. In all, we found 16 AS transcripts to be conserved in all 3 species. AS events and related putative assembled transcripts annotation can be systematically browsed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/plant/). PMID:23297300

  8. 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

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

    PubMed Central

    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

  10. 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.

  11. SpliceJumper: a classification-based approach for calling splicing junctions from RNA-seq data

    PubMed Central

    2015-01-01

    Background Next-generation RNA sequencing technologies have been widely applied in transcriptome profiling. This facilitates further studies of gene structure and expression on the genome wide scale. It is an important step to align reads to the reference genome and call out splicing junctions for the following analysis, such as the analysis of alternative splicing and isoform construction. However, because of the existence of introns, when RNA-seq reads are aligned to the reference genome, reads can not be fully mapped at splicing sites. Thus, it is challenging to align reads and call out splicing junctions accurately. Results In this paper, we present a classification based approach for calling splicing junctions from RNA-seq data, which is implemented in the program SpliceJumper. SpliceJumper uses a machine learning approach which combines multiple features extracted from RNA-seq data. We compare SpliceJumper with two existing RNA-seq analysis approaches, TopHat2 and MapSplice2, on both simulated and real data. Our results show that SpliceJumper outperforms TopHat2 and MapSplice2 in accuracy. The program SpliceJumper can be downloaded at https://github.com/Reedwarbler/SpliceJumper. PMID:26678515

  12. Suppressors of the cdc-25.1(gf)-associated intestinal hyperplasia reveal important maternal roles for prp-8 and a subset of splicing factors in C. elegans

    PubMed Central

    Hebeisen, Michaël; Drysdale, John; Roy, Richard

    2008-01-01

    The maternal contribution of gene products enables embryos to initiate their developmental program in the absence of zygotic gene expression. In Caenorhabditis elegans, maternal CDC-25.1 levels are tightly regulated to promote early cell divisions, while stabilization of this phosphatase by gain-of-function mutations gives rise to intestinal-specific hyperplasia. To identify regulators of CDC-25.1 levels and/or function, we performed a modifier screen of the cdc-25.1(gf)-dependent hyperplasia. One of the isolated suppressor mutants possesses a donor splice site mutation in prp-8, a key splicing factor of the U5-specific snRNP. prp-8(rr40) produces aberrant prp-8 splice variants that generate C-terminal truncations at the expense of wild-type prp-8. Levels of maternal transcripts are reduced, including cdc-25.1, while zygotic transcripts appear unperturbed, suggesting a germ-line-specific role for this splicing factor in regulating the splicing, and consequently, the steady-state levels of maternal transcripts. Using a novel feeding RNAi strategy we found that only a subset of splicing factors suppress cdc-25.1(gf), suggesting that they too may play specific roles in germ-line spliceosome function. In humans, mutations in the corresponding hPrp8 C-terminal domain result in retinitis pigmentosa, a retinal-specific disorder. Intriguingly, despite affecting the general splicing apparatus, both human and C. elegans show tissue-specific defects resulting from mutations in this key splicing component. Our findings suggest that in addition to its important regulatory function in the C. elegans germ line, prp-8(rr40) may provide further insight into the etiology of this splicing-associated human disorder. PMID:18945809

  13. Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin

    PubMed Central

    Wilming, Laurens G.; Liu, Bin; Probst, Frank J.; Harrow, Jennifer; Grafham, Darren; Hentges, Kathryn E.; Woodward, Lanette P.; Maxwell, Andrea; Mitchell, Karen; Risley, Michael D.; Johnson, Randy; Hirschi, Karen; Lupski, James R.; Funato, Yosuke; Miki, Hiroaki; Marin-Garcia, Pablo; Matthews, Lucy; Coffey, Alison J.; Parker, Anne; Hubbard, Tim J.; Rogers, Jane; Bradley, Allan; Adams, David J.; Justice, Monica J.

    2009-01-01

    An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing. PMID:20011118

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

    PubMed

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

    2015-03-01

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

  15. A novel splice-site mutation c.42-2A>T (IVS1-2A>T) of SERPINC1 in a Korean family with inherited antithrombin deficiency.

    PubMed

    Jang, Moon Ju; Lee, Jeong-Guil; Chong, So Young; Huh, Ji Young; Jang, Mi-Ae; Kim, Hee-Jin; Oh, Doyeun

    2011-12-01

    Inherited antithrombin (AT) deficiency (OMIM 107300) is an autosomal dominant disorder and causes a 20-fold increase in the risk of venous thromboembolism. Herein, we describe a case of a novel splice-site mutation in the SERPINC1 gene in a Korean patient with inherited AT deficiency. The patient was a 35-year-old woman who presented with deep vein thrombosis (DVT) and pulmonary embolism and was without a recent history of any precipitating factors. The obtaining of her family history revealed that her mother had an ischemic stroke and a pulmonary embolism and her two sisters both had an episode of DVT during pregnancy. DNA sequencing of SERPINC1 revealed the novel variant IVS1-2A>T (c.42-2A>T), a substitution in intron 1, in the proband and her daughter. The mutation IVS1-2A>T eliminates the acceptor splice-site of intron 1. The present case is the first novel splice-site mutation of SERPINC1 in a Korean family with inherited AT deficiency.

  16. Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.

    PubMed Central

    Whiting, P; McKernan, R M; Iversen, L L

    1990-01-01

    Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes. Images PMID:1702226

  17. 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

  18. 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.

  19. 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

  20. Distinct splicing signatures affect converged pathways in myelodysplastic syndrome patients carrying mutations in different splicing regulators.

    PubMed

    Qiu, Jinsong; Zhou, Bing; Thol, Felicitas; Zhou, Yu; Chen, Liang; Shao, Changwei; DeBoever, Christopher; Hou, Jiayi; Li, Hairi; Chaturvedi, Anuhar; Ganser, Arnold; Bejar, Rafael; Zhang, Dong-Er; Fu, Xiang-Dong; Heuser, Michael

    2016-10-01

    Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS. PMID:27492256

  1. 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.

  2. Maps, codes, and sequence elements: can we predict the protein output from an alternatively spliced locus?

    PubMed

    Sharma, Shalini; Black, Douglas L

    2006-11-22

    Alternative splicing choices are governed by splicing regulatory protein interactions with splicing silencer and enhancer elements present in the pre-mRNA. However, the prediction of these choices from genomic sequence is difficult, in part because the regulators can act as either enhancers or silencers. A recent study describes how for a particular neuronal splicing regulatory protein, Nova, the location of its binding sites is highly predictive of the protein's effect on an exon's splicing.

  3. Deregulation of splicing factors and breast cancer development.

    PubMed

    Silipo, Marco; Gautrey, Hannah; Tyson-Capper, Alison

    2015-10-01

    It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single-nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.

  4. The effect of a single dose of bupivacaine on donor site pain after anterior iliac crest bone harvesting.

    PubMed

    Barkhuysen, R; Meijer, G J; Soehardi, A; Merkx, M A W; Borstlap, W A; Bergé, S J; Bronkhorst, E M; Hoppenreijs, T J M

    2010-03-01

    Transplants from the anterior iliac crest are used for most reconstructive procedures in cranio-maxillofacial surgery. The advantages are easy accessibility, the ability to work in two teams and the amount of corticocancellous bone available; disadvantages are postoperative pain and gait disturbances. To reduce donor-site pain, the effect of a single dose of bupivacaine (10 cc of 2.5mg/cc with 1:80.000 epinephrine) was studied. 200 consecutive patients, who underwent anterior iliac crest bone harvesting for reconstructive procedures, were randomly divided into those receiving bupivacaine and those not. They completed a standardized questionnaire. Patients scored the intensity of the pain and difficulties walking at different times with a visual analogue scale. They recorded analgesics used. 98 questionnaires were eligible for analysis. No differences between the bupivacaine and the control group were detected for postoperative pain and gait disturbance. There is no support for administration of a single dose of bupivacaine to reduce pain in the first postoperative days. The surface area of the removed bone had a significant influence on pain and walking; pain is related to the local osseous damage or periosteal stripping rather than to the length of incision or the operation time.

  5. The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

    PubMed

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

    The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

  6. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    PubMed

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  7. A Novel Aberrant Splice Site Mutation in RAB23 Leads to an Eight Nucleotide Deletion in the mRNA and Is Responsible for Carpenter Syndrome in a Consanguineous Emirati Family

    PubMed Central

    Ben-Salem, S.; Begum, M.A.; Ali, B.R.; Al-Gazali, L.

    2013-01-01

    Carpenter syndrome is caused by mutations in the RAB23 gene that encodes a small GTPase of the Rab subfamily of proteins. Rab proteins are known to be involved in the regulation of cellular trafficking and signal transduction. Currently, only few mutations in RAB23 have been reported in patients with Carpenter syndrome. In this paper, we report the clinical features, molecular and functional analysis of 2 children from an Emirati consanguineous family with this syndrome. The affected children exhibit the typical features including craniosynostosis, typical facial appearance, polysyndactyly, and obesity. Molecular analysis of the RAB23 gene revealed a homozygous mutation affecting the first nucleotide of the acceptor splice site of exon 5 (c.482-1G>A). This mutation affects the authentic mRNA splicing and activates a cryptic acceptor site within exon 5. Thus, the erroneous splicing results in an eight nucleotide deletion, followed by a frameshift and premature termination codon at position 161 (p.V161fsX3). Due to the loss of the C-terminally prenylatable cysteine residue, the truncated protein will probably fail to associate with the target cellular membranes due to the absence of the necessary lipid modification. The p.V161fsX3 extends the spectrum of RAB23 mutations and points to the crucial role of prenylation in the pathogenesis of Carpenter syndrome within this family. PMID:23599695

  8. Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

    PubMed Central

    Dikanov, Sergei A.; Holland, J. Todd; Endeward, Burkhard; Kolling, Derrick R. J.; Samoilova, Rimma I.; Prisner, Thomas F.; Antony R., Crofts

    2011-01-01

    The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (~9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nε of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Nδ in other quinone-processing sites. The experiments with 15N showed that the Nε of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nε of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ± 0.13Å. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover. PMID:17616531

  9. Linking Splicing to Pol II Transcription Stabilizes Pre-mRNAs and Influences Splicing Patterns

    PubMed Central

    Hicks, Martin J; Yang, Chin-Rang; Kotlajich, Matthew V

    2006-01-01

    RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs. PMID:16640457

  10. 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.

  11. Tissue-specific splicing mutation in acute intermittent porphyria

    SciTech Connect

    Grandchamp, B.; Picat, C. ); Mignotte, V.; Romeo, P.H.; Goossens, M. ); Wilson, J.H.P.; Sandkuyl, L. ); Te Velde, K. ); Nordmann, Y. )

    1989-01-01

    An inherited deficiency of porphobilinogen deaminase in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G {yields} A) in the canonical 5{prime} splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using olignonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.

  12. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

    PubMed

    Wang, HaoYang; Hou, YanNing; Cui, YingXia; Huang, YuFeng; Shi, YiChao; Xia, XinYi; Lu, HongYong; Wang, YunHua; Li, XiaoJun

    2009-03-01

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

  13. SR protein kinases promote splicing of nonconsensus introns.

    PubMed

    Lipp, Jesse J; Marvin, Michael C; Shokat, Kevan M; Guthrie, Christine

    2015-08-01

    Phosphorylation of the spliceosome is essential for RNA splicing, yet how and to what extent kinase signaling affects splicing have not been defined on a genome-wide basis. Using a chemical genetic approach, we show in Schizosaccharomyces pombe that the SR protein kinase Dsk1 is required for efficient splicing of introns with suboptimal splice sites. Systematic substrate mapping in fission yeast and human cells revealed that SRPKs target evolutionarily conserved spliceosomal proteins, including the branchpoint-binding protein Bpb1 (SF1 in humans), by using an RXXSP consensus motif for substrate recognition. Phosphorylation of SF1 increases SF1 binding to introns with nonconsensus splice sites in vitro, and mutation of such sites to consensus relieves the requirement for Dsk1 and phosphorylated Bpb1 in vivo. Modulation of splicing efficiency through kinase signaling pathways may allow tuning of gene expression in response to environmental and developmental cues. PMID:26167880

  14. 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

  15. 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...

  16. Cryptic exon activation by disruption of exon splice enhancer: novel mechanism causing 3-methylcrotonyl-CoA carboxylase deficiency.

    PubMed

    Stucki, Martin; Suormala, Terttu; Fowler, Brian; Valle, David; Baumgartner, Matthias R

    2009-10-16

    3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha (MCCA) and smaller beta (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G-->A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G-->A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G-->A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G-->A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon.

  17. 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.

  18. 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

  19. 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.

  20. Discover hidden splicing variations by mapping personal transcriptomes to personal genomes.

    PubMed

    Stein, Shayna; Lu, Zhi-Xiang; Bahrami-Samani, Emad; Park, Juw Won; Xing, Yi

    2015-12-15

    RNA-seq has become a popular technology for studying genetic variation of pre-mRNA alternative splicing. Commonly used RNA-seq aligners rely on the consensus splice site dinucleotide motifs to map reads across splice junctions. Consequently, genomic variants that create novel splice site dinucleotides may produce splice junction RNA-seq reads that cannot be mapped to the reference genome. We developed and evaluated an approach to identify 'hidden' splicing variations in personal transcriptomes, by mapping personal RNA-seq data to personal genomes. Computational analysis and experimental validation indicate that this approach identifies personal specific splice junctions at a low false positive rate. Applying this approach to an RNA-seq data set of 75 individuals, we identified 506 personal specific splice junctions, among which 437 were novel splice junctions not documented in current human transcript annotations. 94 splice junctions had splice site SNPs associated with GWAS signals of human traits and diseases. These involve genes whose splicing variations have been implicated in diseases (such as OAS1), as well as novel associations between alternative splicing and diseases (such as ICA1). Collectively, our work demonstrates that the personal genome approach to RNA-seq read alignment enables the discovery of a large but previously unknown catalog of splicing variations in human populations.

  1. Discover hidden splicing variations by mapping personal transcriptomes to personal genomes

    PubMed Central

    Stein, Shayna; Lu, Zhi-xiang; Bahrami-Samani, Emad; Park, Juw Won; Xing, Yi

    2015-01-01

    RNA-seq has become a popular technology for studying genetic variation of pre-mRNA alternative splicing. Commonly used RNA-seq aligners rely on the consensus splice site dinucleotide motifs to map reads across splice junctions. Consequently, genomic variants that create novel splice site dinucleotides may produce splice junction RNA-seq reads that cannot be mapped to the reference genome. We developed and evaluated an approach to identify ‘hidden’ splicing variations in personal transcriptomes, by mapping personal RNA-seq data to personal genomes. Computational analysis and experimental validation indicate that this approach identifies personal specific splice junctions at a low false positive rate. Applying this approach to an RNA-seq data set of 75 individuals, we identified 506 personal specific splice junctions, among which 437 were novel splice junctions not documented in current human transcript annotations. 94 splice junctions had splice site SNPs associated with GWAS signals of human traits and diseases. These involve genes whose splicing variations have been implicated in diseases (such as OAS1), as well as novel associations between alternative splicing and diseases (such as ICA1). Collectively, our work demonstrates that the personal genome approach to RNA-seq read alignment enables the discovery of a large but previously unknown catalog of splicing variations in human populations. PMID:26578562

  2. Tissue-specific alternative splicing of Shaker potassium channel transcripts results from distinct modes of regulating 3' splice choice.

    PubMed

    Iverson, L E; Mottes, J R; Yeager, S A; Germeraad, S E

    1997-05-01

    Alternative splicing of precursor RNA enables a single gene to encode multiple protein isoforms with different functional characteristics and tissue distributions. Differential splicing of Drosophila Shaker (Sh) gene transcripts regulates the tissue-specific expression of kinetically distinct potassium ion channels throughout development. Regulation of Sh alternative splicing is being examined in germline transformants using lacZ as a reporter gene. P-element constructs were generated in which one or both of the two mutually exclusive Sh 3' acceptor sites were positioned in the same translational reading frame as the lacZ coding sequences. The constructs were introduced into the germline and the transgenic animals examined for tissue-specific beta-galactosidase expression patterns. Some tissues exhibit "promiscuous" splicing; these tissues are competent to splice to either 3' acceptor even when both are present on the same pre-mRNA. In other tissues splice choice results from competition between the two 3' sites; these tissues can splice to either site when it is the only available 3' acceptor, but when given a choice will splice to only one of the two 3' acceptors. In some tissues, splicing occurs exclusively at only one of the 3' acceptor sites; these tissues are not competent to splice to one of the sites even if it is the only 3' acceptor present on the pre-mRNA. These results suggests that multiple, distinct regulatory modes are operating to control tissue-specific alternative splicing of Sh 3' domains and are discussed in terms of potential underlying mechanisms for regulating the tissue-specific expression of alternatively spliced genes.

  3. Translational control of intron splicing in eukaryotes.

    PubMed

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

    2008-01-17

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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Introduction to cotranscriptional RNA splicing.

    PubMed

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

    2014-01-01

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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

  16. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  17. Analysis of aberrantly spliced transcripts of a novel de novo GNAS mutant in a male with albright hereditary osteodystrophy and PHP1A.

    PubMed

    Ham, H-J; Baek, K-H; Lee, J-Y; Kim, S Y; Mo, E Y; Kim, E S; Han, J H; Moon, S-D

    2015-07-01

    Pseudohypoparathyroidism (PHP) is a genetic disorder due to target-organ unresponsiveness to parathyroid hormone (PTH). PHP type 1A (PHP1A) is an autosomal dominant disease characterized by Albright hereditary osteodystrophy (AHO) and PTH resistance caused by defects at the GNAS locus. We analyzed the GNAS gene in a male with typical AHO and elevated PTH levels. We identified a novel de novo heterozygous mutation at the splice donor site in intron-7 (IVS7+1G>A, c.585+1G>A) of the GNAS gene. No GNAS mutations were detected in his parents. Our patient was diagnosed with PHP1A due to a heterozygous de novo mutation in the GNAS gene. Reverse transcriptase (RT) PCR analysis and sequencing revealed that this de novo splice mutation generated alternative splicing errors leading to the formation of 2 mutant transcripts: one with exon-7 deleted, the other with whole intron-7 included. To investigate whether these aberrantly spliced transcripts were stable, we assessed the differential expression of GNAS mRNAs in the proband's blood by real-time quantitative RT-PCR. In the proband, the relative expression levels of wild-type, exon-7-deleted, and intron-7-included GNAS mRNAs were 0.21, 6.12E-07, and 1.08E-04, respectively, relative to wild-type GNAS mRNA from a healthy control (set at 1.0). This suggests that this novel de novo splicing mutation generates rapidly decaying mutant transcripts, which might affect stimulatory G-protein activity and give rise to this sporadic case. In conclusion, this is an interesting report of aberrantly spliced mRNAs from a de novo splice mutation of the GNAS gene causing PHP1A in a male. PMID:25502941

  18. Multifunctional Photopolymerized Semi-Interpenetrating Network (sIPN) System Containing Bupivacaine And Silver Sulfadiazine Is An Effective Donor Site Treatment In A Swine Model

    PubMed Central

    Faucher, Lee D.; Kleinbeck, Kyle R.; Kao, Weiyuan John

    2010-01-01

    Introduction Previously, we have shown in a cross-comparison study that multifunctional photopolymerized semi-interpenetrating network (sIPN) system is an effective donor site treatment in a swine model. The advantages of sIPN include spray-on application, in-situ photopolymerization, and ability to cover large contoured areas. sIPN has also been shown to be an effective delivery vehicle for keratinocyte growth factor, dexamethasone, bupivacaine, and silver sulfadiazine in vitro. Our aim for this study was to show that these products delivered to the wound bed with sIPN would not change the wound healing characteristics compared to the control site through qualitative clinical evaluation and to compare the rate and quality of donor site healing through histologic evaluation. Methods Eight Yucatan swine of 40lbs each were randomly divided into four groups of two pigs prior to surgery. Each animal had 5.6 % total body surface area of skin harvested from two different dorsal regions, with one at 22/1000th inch and the other at 30/1000th inch setting on the dermatome. Each test site on each animal was then sequentially dressed with 50 cm2 of Xeroform gauze, sIPN, sIPN loaded with 0.5% bupivacaine, or sIPN loaded with 1% silver sulfadiazine. sIPN with or without soluble drugs were applied as liquid then photopolymerized in situ to form an elastic covering. Each of the test areas was separated by 50 cm2 of autograft which was used to divide the test areas. Wound assessment and euthanasia occurred at days 7, 9, 14, and 21 days. A full thickness biopsy was taken from each of the study areas for histological analysis. Results By 14 days, all areas show complete epidermal coverage histologically. The 30/1000th inch site revealed a thicker, more irregular dermis compared to the 22/1000th site. Evaluation of the day 21 sites revealed equal thinning and flattening of the new epidermis. No site showed full restoration of the rete ridges. No signs of infection were seen in

  19. Long-term functional outcome and donor-site morbidity associated with autogenous iliac crest bone grafts utilizing a modified anterior approach.

    PubMed

    Singh, Jaspal Ricky; Nwosu, Uzoma; Egol, Kenneth A

    2009-01-01

    Prior studies and techniques for harvesting iliac crest bone have shown significant postoperative pain, disability, and poor cosmesis. This retrospective study was conducted to examine bone graft donor-site morbidity by evaluating functional outcomes in patients who have undergone a modified anterior harvesting approach. The medical charts and hospital records of 43 patients were retrospectively reviewed over a 6-year period. Demographic information, operative notes, laboratory results, and the American Society of Anesthesiologists (ASA) classification were recorded. All patients were evaluated retrospectively at a mean 41 months after bone-graft harvesting. Patients available for follow-up were asked to quantify their pain level at the donor-site on a visual analog pain scale (0-10). They also completed SMFA forms, as well as a survey pertaining to sensory deficits, gait disturbances, and cosmetic appearance. Forty-four patients met the inclusion criteria consisting of 25 males and 18 females, mean age 47 years (range, 22 to 80 years). A total of 32 (73%) patients were available for long-term follow-up at a mean of 41.3 months (range, 8 to 83 months). Eight (25%) of these patients reported minimal postoperative pain at time of follow-up. Three of 32 (9%) patients reported minor ambulation difficulty as a result of donor-site pain. Other minor complications included hypertrophic scar formation (7%) and hematoma/seroma (3%). There were no major complications reported, such as deformity at the crest site (0%) or infection (0%). SMFA scores demonstrated a mean dysfunction score of 48.5 (range, 41.8 to 71.1) and a bother index of mean 47.9 (range, 42.6 to 73.9). Utilizing the anterior approach in iliac crest bone harvesting provides an abundant supply of both cortical and cancellous bone, an aesthetically favorable scar, and decreased postoperative donor-site pain. There were very few complications seen in our cohort as compared to previous studies with very good long

  20. Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency.

    PubMed

    Simpson, C G; Clark, G; Davidson, D; Smith, P; Brown, J W

    1996-03-01

    Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.

  1. 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

  2. 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

  3. 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

  4. The splicing landscape is globally reprogrammed during male meiosis.

    PubMed

    Schmid, Ralf; Grellscheid, Sushma Nagaraja; Ehrmann, Ingrid; Dalgliesh, Caroline; Danilenko, Marina; Paronetto, Maria Paola; Pedrotti, Simona; Grellscheid, David; Dixon, Richard J; Sette, Claudio; Eperon, Ian C; Elliott, David J

    2013-12-01

    Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2β, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2β and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5' splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle. PMID:24038356

  5. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

    PubMed Central

    2010-01-01

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

  6. 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.

  7. 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

  8. Chromatin and alternative splicing.

    PubMed

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  9. Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells*

    PubMed Central

    Barbour, Suzanne E.; Nguyen, Phuong T.; Park, Margaret; Emani, Bhargavi; Lei, Xiaoyong; Kambalapalli, Mamatha; Shultz, Jacqueline C.; Wijesinghe, Dayanjan; Chalfant, Charles E.; Ramanadham, Sasanka

    2015-01-01

    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

  10. Splicing enhancement in the yeast rp51b intron.

    PubMed Central

    Libri, D; Lescure, A; Rosbash, M

    2000-01-01

    Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions. It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement. PMID:10744020

  11. Donor site morbidity after reconstruction of alveolar bone defects with mandibular symphyseal bone grafts in cleft patients--111 consecutive patients.

    PubMed

    Andersen, K; Nørholt, S E; Knudsen, J; Küseler, A; Jensen, J

    2014-04-01

    The aim of this study was to assess the objective and subjective morbidity after reconstruction of alveolar bone defects with mandibular symphyseal bone grafts in patients with cleft lip and palate. One hundred and eleven patients born between 1995 and 1999, who had undergone chin bone harvesting for alveolar cleft reconstruction in the period from 2000 through 2011, were included. A survey of medical records was conducted. Subjective morbidity after reconstruction was assessed using a questionnaire. Medical records revealed few postoperative incidents; 5.6% reported persistent sensory disturbances in the donor area. Postoperative pain averaged 3.6 ± 2.1 (scale 0-10). The overall satisfaction with the surgical result was 8.7 ± 1.7 (scale 0-10). This study revealed that chin bone harvesting for reconstruction of alveolar defects in patients with cleft lip and palate is a safe and predictable procedure, highly appreciated by the patients, and characterized by only minor postoperative incidents. Patients must be informed of the risk of sensory disturbances in the donor area.

  12. Donor-site morbidity after fibula free flap transfer: a comparison of subjective evaluation using a visual analogue scale and point evaluation system.

    PubMed

    Vittayakittipong, P

    2013-08-01

    The aims of this study were: (1) to compare the visual analogue scale (VAS) with the point evaluation system (PES) in the subjective evaluation of donor-site morbidity after fibula free flap transfer; (2) to compare the functional outcomes of fibula free flap surgery between patients with a normal body mass index (BMI) and patients with a high BMI, and between skin paddle and non-skin paddle harvesting; and (3) to determine the correlation between functional outcomes and related factors. This study included 15 patients who underwent a vascularized fibula free flap transfer for oral and maxillofacial reconstruction. Demographic data, preoperative, intraoperative, and postoperative data were collected. Subjective self-evaluation of functional outcomes was done using a VAS followed by a PES. Comparison of the VAS and PES scores was assessed with Pearson's correlation coefficient. The statistical significance was set at P<0.05. The VAS score was significantly correlated with the PES score (r=0.63, P=0.01). The tourniquet times for the skin paddle group were longer than for the non-skin paddle group (P=0.02), while the satisfaction score of the non-skin paddle group was higher than that of the skin paddle group (P=0.03). The VAS is a potential option for the subjective evaluation of donor-site morbidity after fibula free flap transfer.

  13. 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

  14. Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

    PubMed

    Ág, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsébet

    2015-12-01

    Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

  15. Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

    PubMed

    Ág, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsébet

    2015-12-01

    Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing. PMID:26514742

  16. 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. 

  17. Some relations between two stages DNA splicing languages

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  18. RNA splicing factors as oncoproteins and tumor suppressors

    PubMed Central

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

    2016-01-01

    Preface 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 critical for the development of cancer therapies targeted to these mutations. PMID:27282250

  19. A rare splicing mutation in the PROS1 gene of a Korean patient with type I hereditary protein S deficiency.

    PubMed

    Choi, Jonghyeon; Kim, Hee-Jin; Chang, Myung Hee; Choi, Jong-Rak; Yoo, Jong-Ha

    2011-01-01

    Hereditary protein S (PS) deficiency (Gene ID: 5627; MIM # 176880) is a notable risk factor for recurrent venous thrombosis, inherited as an autosomal-dominant trait, either homozygous or heterozygous. It may be caused by point mutations in the gene (PROS1) encoding PS, which contains 15 exons on the chromosome 3q11.2. Only a few point mutations associated with the PROS1 gene in patients with hereditary PS deficiency have been reported. A 60-year-old woman was admitted for deep vein thrombosis (DVT) of the right lower extremity. Upon coagulation examination, both the free PS antigen level and the total PS antigen level were decreased, so the DNA-PCR products of all 15 exons, including the exon-intron boundaries of the PROS1, gene were directly sequenced. A substitution from guanine to adenine at position +5 of the donor splice site of intron 10 (c.1155+5G>A) was identified. Further familial study was performed, and the patient's older sister was revealed to have the same mutation; she was already taking warfarin due to diagnosed pulmonary thromboembolism. Here we report a G to A transition at position +5 of intron 10 from the splice donor site as a rare case of a patient with type I hereditary PS deficiency in Korea.

  20. SON controls cell-cycle progression by coordinated regulation of RNA splicing.

    PubMed

    Ahn, Eun-Young; DeKelver, Russell C; Lo, Miao-Chia; Nguyen, Tuyet Ann; Matsuura, Shinobu; Boyapati, Anita; Pandit, Shatakshi; Fu, Xiang-Dong; Zhang, Dong-Er

    2011-04-22

    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 cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation 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 cotranscriptional 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.

  1. 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.

  2. Surprising diversity and distribution of spliced leader RNAs in flatworms.

    PubMed

    Davis, R E

    1997-07-01

    Trans-splicing generates the mature 5' ends of certain mRNAs through the addition of a small spliced leader (SL) exon to pre-mRNAs. To search for novel flatworm spliced leaders, degenerate oligonucleotides and 5' RACE [corrected was used to isolate and characterize the 5' terminal sequences of enolase mRNAs in diverse flatworms. Several new spliced leaders and their SL RNA genes were identified, characterized, and compared. All parasitic trematodes examined trans-splice enolase. A primitive polyclad turbellarian, Stylochus zebra, also contains a trans-spliced enolase mRNA. The S. zebra SL is the longest SL yet identified, 51 nucleotides. Comparison of flatworm SLs indicates that they vary significantly in sequence and length. This suggests that neither spliced leader exon sequence nor size is likely to be essential for trans-splicing in flatworms. Flatworm SL RNAs have unusual Sm binding sites with characteristics distinct from other known flatworm snRNA Sm binding sites. Predicted flatworm SL RNA secondary structures show variation exhibiting 2-4 stem loops. Although limited in sequence similarity, phylogenetically conserved regions within the diverse flatworm SL RNAs suggest that they are likely to be derived from a common ancestor and provide information on potentially important SL RNA elements. The identification of a SL in a primitive flatworm suggests that trans-splicing may have been an ancestral feature in the phylum. Representative species of other early and more recent clades within the phylum, however, do not trans-splice enolase, nor do they or representatives of several other flatworm groups, have an SL RNA with a phylogenetically conserved region identified in the current study.

  3. 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

  4. 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.

  5. Structure-function analysis of the trypanosomatid spliced leader RNA.

    PubMed Central

    Goncharov, I; Xu, Y X; Zimmer, Y; Sherman, K; Michaeli, S

    1998-01-01

    In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA. PMID:9547281

  6. 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.

  7. Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells.

    PubMed

    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.

  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. 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.

  10. SpliceDisease database: linking RNA splicing and disease.

    PubMed

    Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua

    2012-01-01

    RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation, we give direct links of the entry to the respective position/region in the genome browser. The users can freely browse, search and download the data in SpliceDisease at http://cmbi.bjmu.edu.cn/sdisease.

  11. 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

  12. 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

  13. The use of a T-shaped contoured unilocking titanium radial plate for prophylactic internal fixation of the radial osteocutaneous donor site.

    PubMed

    Avery, C M E; Parmar, S; Martin, Tim

    2010-12-01

    The radial osteocutaneous flap retains a limited role in reconstructive maxillofacial surgery The application of prophylactic internal fixation, using straight 3.5 mm plates, has become established to substantially reduce the incidence of fracture at the radial donor site. New lower profile T-shaped 2.4 mm plates and anatomically contoured 3.5 mm plates are now available, both with unilocking screw fixation systems. These plates are easy to apply and allow the removal of up to 50% of the circumference of the radial bone, including the maximum amount of good quality bone from the distal radius. Although there have been no reports of complications as a result of a stress shielding effect with larger plates these refinements in plate design should lessen any remaining concerns.

  14. Past and future of providing matched, unrelated donors for marrow transplantation.

    PubMed

    Roeckel, I E; Baker, J

    1997-01-01

    Prior to 1979, bone marrow transplants were only performed with histocompatible sibling donors. Once it was established that histocompatible, unrelated donors could donate marrow for transplantation, the recruitment of such donors needed to be standardized. Blood donor centers had already identified the histocompatibility locus antigen (HLA) typing for donors who could be recruited to donate bone marrow. Recruiting a large number of donors required systematic evaluation and testing according to defined standards which were published in 1988 by the National Marrow Donor Program (NMDP). Peripheral stem cell collection (PBS) has been added as a transplant source. It promises additional therapeutic modalities, such as gene splicing to address other than cancer therapy.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Inappropriate splicing of a chimeric gene containing a large internal exon results in exon skipping in transgenic mice.

    PubMed

    Davisson, R L; Nuutinen, N; Coleman, S T; Sigmund, C D

    1996-10-15

    We generated transgenic mice containing a chimeric construct consisting of the alpha-cardiac myosin heavy chain (alpha cMHC) promoter and the human renin (hRen) gene in order to target hRen synthesis specifically to the heart. The construct consisted of three segments: (i) an alpha cMHC DNA segment including 4.5 kb of 5' flanking DNA and an additional 1.1 kb of genomic DNA encompassing exons I-III (non-coding) and the first two introns; (ii) a partial hRen cDNA consisting of exons I-VI; and (iii) a hRen genomic segment containing exons VII through IX, their intervening introns, and 400 bp of 3' flanking DNA. This results in the formation of a 909 bp internal fusion exon consisting of alpha cMHC, polylinker, and hRen sequences. Despite the presence of splice acceptor and donor sites bracketing this exon, transcription of this transgene resulted in a major alternatively spliced mRNA lacking the exon and therefore a majority of the hRen coding sequence. Cloning and sequencing of RT-PCR products from several heart samples from two independent transgenic lines confirmed accurate and faithful splicing of alpha cMHC exon II to hRen exon VII thus bypassing the internal fusion exon. All other exons (alpha cMHC exons I and II and hRen exons VII, VIII and IX) were appropriately spliced. These results are consistent with the hypothesis on exon definition which states that internal exons have a size limitation. Moreover, the results demonstrate that transgenes present in the genome at independent insertion sites and in either a single copy or multiple copies can be subject to exon skipping. The implications for transgene design will be discussed.

  4. 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.

  5. 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

  6. 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.

  7. Splicing-related features of introns serve to propel evolution.

    PubMed

    Luo, Yuping; Li, Chun; Gong, Xi; Wang, Yanlu; Zhang, Kunshan; Cui, Yaru; Sun, Yi Eve; Li, Siguang

    2013-01-01

    The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.

  8. 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

  9. Aberrant and alternative splicing in skeletal system disease.

    PubMed

    Fan, Xin; Tang, Liling

    2013-10-01

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

  10. Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

    PubMed

    Coleman, Timothy P; Tran, Quincy; Roesser, James R

    2003-01-27

    The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

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

    PubMed

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

    2014-11-01

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

  12. 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

  13. 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.

  14. During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

    PubMed Central

    Bailleul, B

    1996-01-01

    Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping. PMID:8604331

  15. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  16. Nested introns in an intron: evidence of multi-step splicing in a large intron of the human dystrophin pre-mRNA.

    PubMed

    Suzuki, Hitoshi; Kameyama, Toshiki; Ohe, Kenji; Tsukahara, Toshifumi; Mayeda, Akila

    2013-03-18

    The mechanisms by which huge human introns are spliced out precisely are poorly understood. We analyzed large intron 7 (110199 nucleotides) generated from the human dystrophin (DMD) pre-mRNA by RT-PCR. We identified branching between the authentic 5' splice site and the branch point; however, the sequences far from the branch site were not detectable. This RT-PCR product was resistant to exoribonuclease (RNase R) digestion, suggesting that the detected lariat intron has a closed loop structure but contains gaps in its sequence. Transient and concomitant generation of at least two branched fragments from nested introns within large intron 7 suggests internal nested splicing events before the ultimate splicing at the authentic 5' and 3' splice sites. Nested splicing events, which bring the authentic 5' and 3' splice sites into close proximity, could be one of the splicing mechanisms for the extremely large introns.

  17. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    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.

  18. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  19. 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

    2016-07-14

    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.

  20. 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

  1. 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

  2. Evaluation of the anterior mandibular donor site one year after secondary reconstruction of an alveolar cleft: 3-dimensional analysis using cone-beam computed tomography.

    PubMed

    van Bilsen, M W T; Schreurs, R; Meulstee, J W; Kuijpers, M A R; Meijer, G J; Borstlap, W A; Bergé, S J; Maal, T J J

    2015-10-01

    The aim of this study was to analyse changes in the volume of the chin after harvest of a bone graft for secondary reconstruction of an alveolar cleft. Cone-beam computed tomographic (CT) scans of 27 patients taken preoperatively, and immediately and one year postoperatively, were analysed, and 3-dimensional hard-tissue reconstructions made. The hard-tissue segmentation of the scan taken one year postoperatively was subtracted from the segmentation of the preoperative scan to calculate the alteration in the volume of bone at the donor site (chin). A centrally-orientated persistent concavity at the buccal side of the chin was found (mean (range) 160 (0-500) mm(3)). At the lingual side of the chin, a central concavity remained (mean (range) volume 20 (0-80) mm(3)). Remarkably, at the periphery of this concavity there was overgrowth of new bone (mean (range) volume 350 (0-1600) mm(3)). Re-attachment of the muscles of the tongue resulted in a significantly larger central lingual defect one year postoperatively (p=0.01). We also measured minor alterations in volume of the chin at one year. Whether these alterations influence facial appearance and long term bony quality is to be the subject of further research.

  3. Technique for Minimizing Donor-site Morbidity after Pedicled TRAM-Flap Breast Reconstruction: Outcomes by a Single Surgeon’s Experience

    PubMed Central

    Rietjens, Mario; De Lorenzi, Francesca; Andrea, Manconi; Petit, Jean-Yves; Hamza, Alaa; Martella, Stefano; Barbieri, Benedetta; Gottardi, Alessandra; Giuseppe, Lomeo

    2015-01-01

    Background: Breast reconstruction with pedicled transverse rectus abdominis myocutaneous (TRAM) flap can result in significant abdominal wall donor-site morbidity. We present our technique of transversely dividing the anterior fascia and rectus abdominis combined with reinforcement above the arcuate line for closure of the anterior abdominal wall defect to prevent contour deformities performed by a single senior surgeon and compare these results with those of our prior series. Methods: We described our new technique of closure of the abdominal wall defect and retrospectively performed the comparison between the results of pedicled TRAM flaps using the new closure technique and those of 420 pedicled TRAM flaps from our 2003 publication in terms of abdominal bulging and hernia. Results: Sixty-seven pedicled TRAM flaps in 65 patients were compared with 420 pedicled TRAM flaps of the 2003 series. The new technique was associated with 5 partial TRAM flap necroses (8%). There was no total flap loss with the new technique. The median follow-up period was 13 months (range, 4–36 months). There were no instances of abdominal hernia and bulge during follow-up in the new series. Compared with the previous 2003 series, the new technique was superior in terms of occurrence of abdominal wall hernia or bulging. Conclusions: We are still performing pedicled TRAM flap for autologous breast reconstruction. Using the technique of transversely dividing the anterior fascia and rectus abdominis combined with reinforcement above the arcuate line can reduce the occurrence of abdominal bulging and hernia. PMID:26495189

  4. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators

    PubMed Central

    Llorian, Miriam; Gooding, Clare; Bellora, Nicolas; Hallegger, Martina; Buckroyd, Adrian; Wang, Xiao; Rajgor, Dipen; Kayikci, Melis; Feltham, Jack; Ule, Jernej; Eyras, Eduardo; Smith, Christopher W.J.

    2016-01-01

    Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery. PMID:27317697

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Alternative splicing of TAF6: downstream transcriptome impacts and upstream RNA splice control elements.

    PubMed

    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.

  12. 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

  13. 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

  14. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

    PubMed Central

    Cáceres, J F; Krainer, A R

    1993-01-01

    Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain. Images PMID:8223480

  15. 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

  16. 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

  17. Splicing in the human brain.

    PubMed

    Zaghlool, Ammar; Ameur, Adam; Cavelier, Lucia; Feuk, Lars

    2014-01-01

    It has become increasingly clear over the past decade that RNA has important functions in human cells beyond its role as an intermediate translator of DNA to protein. It is now known that RNA plays highly specific roles in pathways involved in regulatory, structural, and catalytic functions. The complexity of RNA production and regulation has become evident with the advent of high-throughput methods to study the transcriptome. Deep sequencing has revealed an enormous diversity of RNA types and transcript isoforms in human cells. The transcriptome of the human brain is particularly interesting as it contains more expressed genes than other tissues and also displays an extreme diversity of transcript isoforms, indicating that highly complex regulatory pathways are present in the brain. Several of these regulatory proteins are now identified, including RNA-binding proteins that are neuron specific. RNA-binding proteins also play important roles in regulating the splicing process and the temporal and spatial isoform production. While significant progress has been made in understanding the human transcriptome, many questions still remain regarding the basic mechanisms of splicing and subcellular localization of RNA. A long-standing question is to what extent the splicing of pre-mRNA is cotranscriptional and posttranscriptional, respectively. Recent data, including studies of the human brain, indicate that splicing is primarily cotranscriptional in human cells. This chapter describes the current understanding of splicing and splicing regulation in the human brain and discusses the recent global sequence-based analyses of transcription and splicing. PMID:25172473

  18. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position.

    PubMed

    Shen, Manli; Mattox, William

    2012-01-01

    SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.

  19. One single nucleotide difference alters the differential expression of spliced RNAs between HBV genotypes A and D.

    PubMed

    Huang, Chien-Chiao; Kuo, Tzer-Min; Yeh, Chau-Ting; Hu, Cheng-Po; Chen, Ya-Ling; Tsai, Yue-Lin; Chen, Mong-Liang; Chou, Yu-Chi; Chang, Chungming

    2013-06-01

    Hepatitis B virus (HBV) is generally classified into eight genotypes (A to H) based on genomic sequence divergence. The sequence variation among the different HBV genotypes suggests that the spliced RNAs should be different from genotype to genotype. However, the cis-acting element involved in the modulation of the distinct expression profiles of spliced HBV RNAs remains unidentified. Moreover, the biological role of splicing in the life cycle of HBV is not yet understood. In this study, spliced RNAs generated from genotypes A and D were carefully characterized in transfected HepG2 cells. The species and frequency of the spliced RNAs were dramatically different in the two genotypes. Of note, a population of multiply spliced RNAs with intron 2067-2350 excision was identified in HBV genotype A-transfected HepG2 cells, but not in genotype D transfected HepG2 cells. Further, we found a single nucleotide difference (2335) located within the polypyrimidine tract of the splice acceptor site 2350 between the two genotypes, and a single base substitution at 2335 was able to convert the splicing pattern of genotype D (or genotype A) to that of genotype A (or genotype D). These findings suggest that different unique splice sites may be preferentially used in different HBV genotypes resulting in distinct populations of spliced RNAs. The possible significance of the distinct spliced RNAs generated from the different HBV genotypes in HBV infection is discussed.

  20. Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies.

    PubMed

    Xu, Dong-Qing; Mattox, William

    2006-01-15

    Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.

  1. 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.

  2. 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

  3. 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

  4. 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.

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

    PubMed Central

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

    2008-01-01

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

  6. 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

  7. 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

  8. Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

    PubMed

    Mameli, Eva; Lepori, Maria Barbara; Chiappe, Francesca; Ranucci, Giusy; Di Dato, Fabiola; Iorio, Raffaele; Loudianos, Georgios

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease.

  9. 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.

  10. 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

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

    PubMed

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

    2007-01-01

    We have greatly expanded the Alternative Splicing Annotation Project (ASAP) database: (i) its human alternative splicing data are expanded approximately 3-fold over the previous ASAP database, to nearly 90,000 distinct alternative splicing events; (ii) it now provides genome-wide alternative splicing analyses for 15 vertebrate, insect and other animal species; (iii) it provides comprehensive comparative genomics information for comparing alternative splicing and splice site conservation across 17 aligned genomes, based on UCSC multigenome alignments; (iv) it provides an approximately 2- to 3-fold expansion in detection of tissue-specific alternative splicing events, and of cancer versus normal specific alternative splicing events. We have also constructed a novel database linking orthologous exons and orthologous introns between genomes, based on multigenome alignment of 17 animal species. It can be a valuable resource for studies of gene structure evolution. ASAP II provides a new web interface enabling more detailed exploration of the data, and integrating comparative genomics information with alternative splicing data. We provide a set of tools for advanced data-mining of ASAP II with Pygr (the Python Graph Database Framework for Bioinformatics) including powerful features such as graph query, multigenome alignment query, etc. ASAP II is available at http://www.bioinformatics.ucla.edu/ASAP2.

  12. Functional analysis and in vitro correction of splicing FAH mutations causing tyrosinemia type I.

    PubMed

    Pérez-Carro, R; Sánchez-Alcudia, R; Pérez, B; Navarrete, R; Pérez-Cerdá, C; Ugarte, M; Desviat, L R

    2014-08-01

    Hereditary tyrosinemia type I (HT1) is a rare disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH) in the tyrosine catabolic pathway, resulting mainly in hepatic alterations due to accumulation of the toxic metabolites fumarylacetoacetate, maleylacetoacetate and succinylacetone. We have characterized using minigenes four splicing mutations affecting exonic or intronic nucleotides of the FAH gene identified in two HT1 patients. Two of the mutations are novel, c.82-1G>A and c.913G>C and the other two have been previously associated with a splicing defect (c.836A>G and c.1062+5G>A). All mutations were confirmed to affect splicing in minigenes, resulting in exon skipping or activation of a cryptic splice site. We have analyzed the effect of different compounds known to modulate splicing (valproic acid, phenyl butyrate, M344, EIPA, and resveratrol) and the overexpression of splice factors of the SR protein family on the transcriptional profile of the mutant minigenes. For the c.836A>G mutation, a partial recovery of the correctly spliced transcript was observed. These results confirm the relevance of performing functional studies for mutations potentially affecting the splicing process and open the possibility of supplementary therapeutic approaches to diseases caused by splicing defects.

  13. Regulation of neurexin 1beta tertiary structure and ligand binding through alternative splicing.

    PubMed

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

    2008-03-01

    Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.

  14. 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

  15. Alternative splicing regulation and cell lineage differentiation.

    PubMed

    Liu, Huan; He, Ling; Tang, Liling

    2012-11-01

    The alternative splicing of precursor mRNA is an essential mechanism for protein diversity. It plays important biological roles, such as proliferation, differentiation and development of cells. Furthermore, alternative splicing participates in the pathogenesis of diseases, including cancer. Thus, in-depth understanding of splicing regulation is of great significance. Regulation of alternative splicing is an extraordinary complicated process in which several signal molecules are at work. Besides the cis-elements and trans-factors, several lines of evidences suggest that other molecules, structures or process also regulate splicing, such as RNA structures, transcription and transcription factors, chromatin and protein. Meanwhile, increasing body of evidence shows that alternative splicing correlated closely to stem cell lineage differentiation. It means that there is a fundamental role for splicing in controlling regulatory program required for cell lineage differentiation. This review systematically sums up the regulation of alternative splicing and summarizes the splicing events during cell lineage differentiation of stem cells.

  16. Targeting RNA splicing for disease therapy.

    PubMed

    Havens, Mallory A; Duelli, Dominik M; Hastings, Michelle L

    2013-01-01

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

  17. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    PubMed

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcáRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

  18. Splicing controls the ubiquitin response during DNA double-strand break repair

    PubMed Central

    Pederiva, C; Böhm, S; Julner, A; Farnebo, M

    2016-01-01

    Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer. PMID:27315300

  19. 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.

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

    PubMed

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

    2010-04-01

    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.

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2015-03-01

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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.

    PubMed

    Schor, Ignacio E; Llères, David; Risso, Guillermo J; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I; Kornblihtt, Alberto R

    2012-01-01

    Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Some Characterizations in Splicing Systems

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  16. 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

  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. 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

  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. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    PubMed

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  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. Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

    PubMed Central

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-01-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  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. PTBP1-dependent regulation of USP5 alternative RNA splicing plays a role in glioblastoma tumorigenesis.

    PubMed

    Izaguirre, Daisy I; Zhu, Wen; Hai, Tao; Cheung, Hannah C; Krahe, Ralf; Cote, Gilbert J

    2012-11-01

    Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line data sets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined polypyrimidine tract-binding protein 1 (PTBP1) levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each data set. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5' splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.

  5. 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

  6. Influenza Viruses and mRNA Splicing: Doing More with Less

    PubMed Central

    Dubois, Julia

    2014-01-01

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

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

    PubMed Central

    2014-01-01

    Background Alternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes. Results We used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event. Conclusions As described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures. PMID:24739459

  8. Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum.

    PubMed

    Reitter, Julie N; Cousin, Christopher E; Nicastri, Michael C; Jaramillo, Mario V; Mills, Kenneth V

    2016-03-01

    An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

  9. Splicing mosaic of the myophosphorylase gene due to a silent mutation in McArdle disease.

    PubMed

    Fernandez-Cadenas, I; Andreu, A L; Gamez, J; Gonzalo, R; Martín, M A; Rubio, J C; Arenas, J

    2003-11-25

    The authors report the molecular findings in a patient with McArdle disease who harbored a silent polymorphism (K608K) in the myophosphorylase gene. cDNA studies demonstrated that this polymorphism leads to a severe mosaic alteration in mRNA splicing, including exon skipping, activation of cryptic splice-sites, and exon-intron reorganizations. These findings suggest that, in patients with McArdle disease in whom no pathogenic mutation has been found, any a priori silent polymorphism should be re-evaluated as a putative splicing mutation.

  10. Trans-splicing with the group I intron ribozyme from Azoarcus

    PubMed Central

    Dolan, Gregory F.; Müller, Ulrich F.

    2014-01-01

    Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNAIle group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5′ terminus. In contrast to the previously optimized 5′-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5′ portion and the ribozyme 3′ exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme. PMID:24344321