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Sample records for splice donor site

  1. HIV-1 splicing at the major splice donor site is restricted by RNA structure.

    PubMed

    Mueller, Nancy; van Bel, Nikki; Berkhout, Ben; Das, Atze T

    2014-11-01

    The 5' leader region of the HIV-1 RNA contains the major 5' splice site (ss) that is used in the production of all spliced viral RNAs. This splice-donor (SD) region can fold a stem-loop structure. We demonstrate that whereas stabilization of this SD hairpin reduces splicing efficiency, destabilization increases splicing. Both stabilization and destabilization reduce viral fitness. These results demonstrate that the stability of the SD hairpin can modulate the level of splicing, most likely by controlling the accessibility of the 5'ss for the splicing machinery. The natural stability of the SD hairpin restricts splicing and this stability seems to be fine-tuned to reach the optimal balance between unspliced and spliced RNAs for efficient virus replication. The 5'ss region of different HIV-1 isolates and the related SIVmac239 can fold a similar structure. This evolutionary conservation supports the importance of this structure in viral replication. PMID:25305540

  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. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    PubMed

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects. PMID:26435450

  5. Impact of RNA structure on the prediction of donor and acceptor splice sites

    PubMed Central

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

    2006-01-01

    Background gene identification in genomic DNA sequences by computational methods has become an important task in bioinformatics and computational gene prediction tools are now essential components of every genome sequencing project. Prediction of splice sites is a key step of all gene structural prediction algorithms. Results we sought the role of mRNA secondary structures and their information contents for five vertebrate and plant splice site datasets. We selected 900-nucleotide sequences centered at each (real or decoy) donor and acceptor sites, and predicted their corresponding RNA structures by Vienna software. Then, based on whether the nucleotide is in a stem or not, the conventional four-letter nucleotide alphabet was translated into an eight-letter alphabet. Zero-, first- and second-order Markov models were selected as the signal detection methods. It is shown that applying the eight-letter alphabet compared to the four-letter alphabet considerably increases the accuracy of both donor and acceptor site predictions in case of higher order Markov models. Conclusion Our results imply that RNA structure contains important data and future gene prediction programs can take advantage of such information. PMID:16772025

  6. Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

    PubMed Central

    Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

    2015-01-01

    Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975

  7. Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome

    SciTech Connect

    Godfrey, M.; Vandemark, N.; Wang, M.; Han, J.; Rao, V.H. ); Velinov, M.; Tsipouras, P. ); Wargowski, D.; Becker, J.; Robertson, W.; Droste, S. )

    1993-08-01

    The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastic-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin-specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription-PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor-like motif. Examination of genomic DNA showed a G[yields]C transversion at the +1 consensus donor splice site. 45 refs., 7 figs.

  8. 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. PMID:24055421

  9. 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.

  10. The donor splice site mutation in NFkappaB-inducing kinase of alymphoplasia (aly/aly) mice.

    PubMed

    Macpherson, Andrew J; Uhr, Therese

    2003-01-01

    The alymphoplasia (aly/aly) mouse has a spontaneous mutation maintained on a C57BL/6xAEJ ( H-2(b)) background that results in an absence of extrasplenic secondary lymphoid tissues. The cDNA defect has previously been shown to reside in a point mutation causing a G855R substitution in NFkappaB-inducing kinase (NIK). Since the aly/aly female cannot lactate, the strain must be bred by intercrossing heterozygous females with homozygous males and the offspring typed by serum IgA levels at the age of 4-6 weeks. We originally determined the genomic location of the alymphoplasia mutation by sequencing boundaries of regions homologous to human NIK exons, although recently the entire genomic sequence of murine C57BL/6 NIK has become available through the mouse genome project. The aly mutation is at position -1 of an intron donor consensus splice site. Exon-connexion PCR confirmed that splicing does occur across this site. Using the genomic information, we also developed a method of PCR typing of aly/aly mice from tail clips, and used this to derive an aly/aly muMT double-mutant strain in which antibody independent typing is essential. Genetic typing should considerably simplify husbandry and manipulation of the aly/aly genetic background, which is widely used as a recipient in lymphocyte transfer experiments to permit examination of the relative role of secondary lymphoid structures in immune responses. PMID:12557055

  11. Donor splice-site mutation in CUL4B is likely cause of X-linked intellectual disability.

    PubMed

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

    2014-09-01

    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

  12. Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements.

    PubMed

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

    2015-11-01

    Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 59 splice site (59ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 59ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication. PMID:26385834

  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. PMID:8704236

  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.

    PubMed

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

    2004-12-20

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

  16. 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.

  17. 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

  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. PMID:1346483

  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. Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

    SciTech Connect

    Wadelius, C.; Lagerkvist, A. Uppsala Univ. ); Molin, A.K.; Larsson, A. ); Von Doebeln, U. )

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

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

    PubMed Central

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

    2011-01-01

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

  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. U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites.

    PubMed

    Uchikawa, Hideki; Fujii, Katsunori; Kohno, Yoichi; Katsumata, Noriyuki; Nagao, Kazuaki; Yamada, Masao; Miyashita, Toshiyuki

    2007-01-01

    A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy. PMID:17851636

  4. Severe Hb S-beta zero-thalassaemia with a T----C substitution in the donor splice site of the first intron of the beta-globin gene.

    PubMed

    Gonzalez-Redondo, J M; Stoming, T A; Kutlar, F; Kutlar, A; McKie, V C; McKie, K M; Huisman, T H

    1989-01-01

    Through direct sequencing and dot-blot analyses with synthetic oligonucleotide probes of amplified DNA, a new nucleotide substitution was discovered in a Black teenager with severe Hb S-beta zero-thalassaemia. The substitution involved a T----C replacement at the second position of the donor splice site of the first intervening sequence of the beta-globin gene. The clinical and haematological observations made in Black subjects with Hb S-beta zero-thalassaemia, with different types of thalassaemia, suggest severe disease resembling sickle cell anaemia. Only an occasional patient had a milder clinical course, perhaps because of a greatly increased production of fetal haemoglobin. PMID:2917118

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

    SciTech Connect

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

    1992-02-01

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

  6. Prediction of human mRNA donor and acceptor sites from the DNA sequence.

    PubMed

    Brunak, S; Engelbrecht, J; Knudsen, S

    1991-07-01

    Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding. PMID:2067018

  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-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

    PubMed Central

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

    1995-01-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. Images Figure 3 PMID:7534040

  9. 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.

  10. Gaucher disease: A G[sup +1][yields]A[sup +1] IVS2 splice donor site mutation causing exon 2 skipping in the acid [beta]-glucosidase mRNA

    SciTech Connect

    He, Guo-Shun ); Grabowski, G.A. )

    1992-10-01

    Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid [beta]-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-years-old, enzyme-deficient, 1226G (Asn[sup 370][yields]Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 ([Delta] EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 ([Delta] EX2-3), or a completely normal sequence. About 50% of the cDNAs were the [Delta] EX2, the [Delta] EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5[prime] and 3[prime] intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G[sup +1][yields]A[sup +1] transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed [open quotes]IVS2 G[sup +1],[close quotes] is the first in the Ashkenazi Jewish population. The occurrence of this [open quotes]pseudogene[close quotes]-type mutation in the structural gene indicates the role of acid [beta]-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease. 33 refs., 8 figs., 1 tab.

  11. 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

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

    PubMed Central

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

    2002-01-01

    XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82882 bp) and 15 introns (0.085.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcriptionpolymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in ?0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in ?0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (?2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility. PMID:12177305

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

    PubMed Central

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

    2015-01-01

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

  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. iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition

    PubMed Central

    Feng, Peng-Mian; Chou, Kuo-Chen

    2014-01-01

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

  16. Comparative analysis of sequence features involved in the recognition of tandem splice sites

    PubMed Central

    Bortfeldt, Ralf; Schindler, Stefanie; Szafranski, Karol; Schuster, Stefan; Holste, Dirk

    2008-01-01

    Background The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation. Results We analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for cis-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in H. sapiens and M. musculus. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information. Conclusion This comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell. PMID:18447903

  17. Characterization of the spliced pol transcript of feline foamy virus: the splice acceptor site of the pol transcript is located in gag of foamy viruses.

    PubMed Central

    Bodem, J; Löchelt, M; Winkler, I; Flower, R P; Delius, H; Flügel, R M

    1996-01-01

    Foamy viruses, or spumaviruses, are distinct members of the Retroviridae. Here we have characterized the long terminal repeat of the feline, or cat, foamy virus by determining the locations of the transcriptional start site and the poly(A) addition site. The splice donor and splice acceptor sites of the subgenomic mRNA responsible for Pro-Pol protein expression were identified by nucleotide sequencing of the corresponding cDNAs. The leader exon of the feline foamy virus is 57 nucleotides long. The splice acceptor of the subgenomic pol mRNA was found to be located in gag. The location of the splice acceptor of the human foamy virus pol mRNA was confirmed to map in gag. The pol splice acceptor site in gag of the cat foamy virus is located further downstream than that of human foamy virus. PMID:8971036

  18. Alternative splicing at GYNNGY 5′ splice sites: more noise, less regulation

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1995-01-01

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

  20. Comprehensive splice-site analysis using comparative genomics

    PubMed Central

    Sheth, Nihar; Roca, Xavier; Hastings, Michelle L.; Roeder, Ted; Krainer, Adrian R.; Sachidanandam, Ravi

    2006-01-01

    We have collected over half a million splice sites from five species—Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana—and classified them into four subtypes: U2-type GT–AG and GC–AG and U12-type GT–AG and AT–AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT–AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3′ splice sites (3′ss) and (iv) distinct evolutionary histories of 5′ and 3′ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing. PMID:16914448

  1. 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

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

    PubMed

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

    2014-01-01

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

  3. A Splicing Reporter Tuned to Non-AG Acceptor Sites Reveals that Luteolin Enhances the Recognition of Non-canonical Acceptor Sites.

    PubMed

    Chiba, Masanori; Ariga, Hiroyoshi; Maita, Hiroshi

    2016-02-01

    Removal of an intron requires precise recognition of the splice donor and acceptor sites located at the 5' and 3' termini of introns. Although the roles of these sequences differ, mutations in both sites easily block normal splicing and produce an aberrant mRNA. For example, many splice-site mutations occur in patients with inherited diseases. Several approaches have been evaluated to restore expression of a functional protein; however, because of the strict requirement for an AG dinucleotide at the 3' terminus of a U2-type intron, no method is available to correct splicing at a mutated sequence. To identify compounds that allow splicing at the non-AG acceptor site, in the present study we constructed a reporter gene with a modified polypyrimidine tract. However, the modified polypyrimidine tract mediated splicing at adjacent non-canonical acceptor sites, including the original mutated site. Further, we show that certain flavones such as luteolin and apigenin enhanced aberrant splicing at the non-canonical acceptor site of the reporter gene. These results suggest that the reporter gene and luteolin may be useful for further screening to identify molecules that correct aberrant splicing caused by a disease-associated splice acceptor site mutation. PMID:26348996

  4. Modification of the Creator recombination system for proteomics applications improved expression by addition of splice sites

    PubMed Central

    Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

    2006-01-01

    Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. PMID:16519801

  5. 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

  6. Splice site identification using probabilistic parameters and SVM classification

    PubMed Central

    Baten, AKMA; Chang, BCH; Halgamuge, SK; Li, Jason

    2006-01-01

    Background Recent advances and automation in DNA sequencing technology has created a vast amount of DNA sequence data. This increasing growth of sequence data demands better and efficient analysis methods. Identifying genes in this newly accumulated data is an important issue in bioinformatics, and it requires the prediction of the complete gene structure. Accurate identification of splice sites in DNA sequences plays one of the central roles of gene structural prediction in eukaryotes. Effective detection of splice sites requires the knowledge of characteristics, dependencies, and relationship of nucleotides in the splice site surrounding region. A higher-order Markov model is generally regarded as a useful technique for modeling higher-order dependencies. However, their implementation requires estimating a large number of parameters, which is computationally expensive. Results The proposed method for splice site detection consists of two stages: a first order Markov model (MM1) is used in the first stage and a support vector machine (SVM) with polynomial kernel is used in the second stage. The MM1 serves as a pre-processing step for the SVM and takes DNA sequences as its input. It models the compositional features and dependencies of nucleotides in terms of probabilistic parameters around splice site regions. The probabilistic parameters are then fed into the SVM, which combines them nonlinearly to predict splice sites. When the proposed MM1-SVM model is compared with other existing standard splice site detection methods, it shows a superior performance in all the cases. Conclusion We proposed an effective pre-processing scheme for the SVM and applied it for the identification of splice sites. This is a simple yet effective splice site detection method, which shows a better classification accuracy and computational speed than some other more complex methods. PMID:17254299

  7. Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation

    PubMed Central

    Razanau, Aleh; Zock-Emmenthal, Susanne; Lützelberger, Martin; Plath, Susann; Schmidt, Henning; Guerra-Moreno, Angel; Cozzuto, Luca; Ayté, José; Käufer, Norbert F.

    2016-01-01

    The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences. PMID:26730850

  8. 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; Aguil, Sira; Fernndez-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, Addisons 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

  9. 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.

  10. The theoretically ideal donor site dressing.

    PubMed

    Birdsell, D C; Hein, K S; Lindsay, R L

    1979-06-01

    Many of the choices for managing split-thickness skin graft donor sites are satisfactory, but none is ideal. Epidermal regeneration in a donor site is readily available for clinical study. We have reviewed experimental studies of epidermal regeneration and applied those results to the clinical study of a new donor site dressing. This dressing is a vapor-permeable, transparent, polyurethane film with a polyvinyl ether adhesive. Used on 100 patients, it was found to be safe and effective in allowing rapid and painless healing. Although the dressing is occlusive and theoretically could promote infection in a contaminated wound, no infections were encountered. Comparison was made with 15 patients managed by other methods. No marked difference in healing time was noted clinically. The striking advantage of the new dressing was painless healing. PMID:396845

  11. A splice site mutation confirms the role of LPIN2 in Majeed syndrome.

    PubMed

    Al-Mosawi, Zakiya S; Al-Saad, Khulood K; Ijadi-Maghsoodi, Roya; El-Shanti, Hatem I; Ferguson, Polly J

    2007-03-01

    Majeed syndrome is an autoinflammatory disorder consisting of chronic recurrent multifocal osteomyelitis, congenital dyserythropoietic anemia, and neutrophilic dermatosis. To date, 2 unrelated families with Majeed syndrome have been reported. Mutations in LPIN2 have been found in both families. Here we report a third consanguineous family with Majeed syndrome with a novel mutation. The patient, a 3-year-old Arabic girl, had hepatosplenomegaly and anemia as a neonate. At age 15 months, she developed recurrent episodes of fever and multifocal osteomyelitis. In addition, bone marrow aspiration demonstrated significant dyserythropoiesis, suggesting Majeed syndrome. Coding sequences and splice sites of LPIN2 were sequenced in the patient and her mother. A homozygous single-basepair change was detected in the donor splice site of exon 17 (c.2327+1G>C) in the patient; her mother was heterozygous at this site. These data confirm the role of LPIN2 mutations in the etiology of Majeed syndrome. PMID:17330256

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

    PubMed Central

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

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human exons. Direct Sanger sequencing of all PHEX exons in a sporadic case with HR from the Indian subcontinent revealed an additional novel PHEX mutation (c.1211_1215delACAAAinsTTTACAT, p.Asp404Valfs*5, de novo) located in exon 11. Conclusions Mutation analyses revealed two novel mutations and helped to confirm the clinical diagnoses of XLH in two families from India. WES helped to analyze all genes implicated in the underlying disease complex. Mutations at splice positions other than the canonical key sites need further functional investigation to support the assertion of pathogenicity. PMID:26107949

  13. Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information.

    PubMed Central

    Hebsgaard, S M; Korning, P G; Tolstrup, N; Engelbrecht, J; Rouzé, P; Brunak, S

    1996-01-01

    Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local prediction of splice sites, is refined by rules based on splice site confidence values, prediction scores, coding context and distances between potential splice sites. In this approach, the prediction of splice sites mutually affect each other in a non-local manner. The combined approach drastically reduces the large amount of false positive splice sites normally haunting splice site prediction. An analysis of the errors made by the networks in the first step of the method revealed a previously unknown feature, a frequent T-tract prolongation containing cryptic acceptor sites in the 5' end of exons. The method presented here has been compared with three other approaches, GeneFinder, Gene-Mark and Grail. Overall the method presented here is an order of magnitude better. We show that the new method is able to find a donor site in the coding sequence for the jelly fish Green Fluorescent Protein, exactly at the position that was experimentally observed in A.thaliana transformants. Predictions for alternatively spliced genes are also presented, together with examples of genes from other dicots, monocots and algae. The method has been made available through electronic mail (NetPlantGene@cbs.dtu.dk), or the WWW at http://www.cbs.dtu.dk/NetPlantGene.html PMID:8811101

  14. Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

    PubMed Central

    Carothers, A M; Urlaub, G; Grunberger, D; Chasin, L A

    1993-01-01

    Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence. Images PMID:8336736

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

    PubMed Central

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

    2015-01-01

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

  16. Coordinated tissue-specific regulation of adjacent alternative 3' splice sites in C. elegans.

    PubMed

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

    2015-07-01

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

  17. Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5? or 3? Splice Site Activation

    PubMed Central

    Bourgeois, Cyril F.; Popielarz, Michel; Hildwein, Georges; Stevenin, James

    1999-01-01

    The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5? splice sites and of one major or one minor 3? splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5? splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5? splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3? splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5? splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity. PMID:10523623

  18. A statistical analytical approach to decipher information from biological sequences: application to murine splice-site analysis and prediction.

    PubMed

    Reddy, B V; Pandit, M W

    1995-02-01

    A simple statistical approach for the analysis of biological sequences, such as splice-sites, promoter regions, helices and extended structure forming regions or any other sequence dependent functional entities in proteins, is presented. The approach has been proved useful to develop a method for prediction of such entities in newly available sequences. We first search for invariant sequence features of each functional entity from the experimentally available sequences and identify a set of 'like' sequences with similar sequence features. In the next step, concrete features of sequence entities in terms of occurrences of smaller subsequences are identified at various positions which are used as a knowledge base to select potential functional entities from the identified 'like' sequences. The third step consists of refinement of this pattern learning, statistical improvements of the knowledge base weight matrices, and finally its application to predict functional entities in newly available sequences. Such an analysis is operationally described for murine splice-site predictions. Regions comprising -30 to +30 nucleotides from the splice-junction at the murine splice-sites (donors and acceptors), reported earlier, were analyzed. Invariant sequence-specific features in terms of monomer frequency average were used to identify splice-site-like sequences in the EMBL murine DNA sequence data base. The frequencies of occurrence of mono-, di-, tri- and tetranucleotides in the known splice-sites were studied in comparison with the splice-site-like sequences; the significant differences in their occurrences were extracted as statistical knowledge coded in weight matrices for computer to identify potential splice-sites. The algorithm was refined and a method was developed to predict potential splice-sites in a given murine DNA; the analysis was also extended to human DNA. The success rate of the method to predict correct splice-sites in these species is found to be 80% and 85%, respectively. The major strength of this method lies in reducing significantly the number of false positives which are normally picked up in such analysis. PMID:7779300

  19. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    PubMed

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells. PMID:15363843

  20. 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.

  1. Features generated for computational splice-site prediction correspond to functional elements

    PubMed Central

    Dogan, Rezarta Islamaj; Getoor, Lise; Wilbur, W John; Mount, Stephen M

    2007-01-01

    Background Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA) that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals. Results We present examples from the observed features that correspond to known signals, both core signals (including the branch site and pyrimidine tract) and auxiliary signals (including GGG triplets and exon splicing enhancers). We present evidence that features identified by FGA include splicing signals not found by other methods. Conclusion Our generated features capture known biological signals in the expected sequence interval flanking splice sites. The method can be easily applied to other species and to similar classification problems, such as tissue-specific regulatory elements, polyadenylation sites, promoters, etc. PMID:17958908

  2. Analyzing alternative splicing data of splice junction arrays from Parkinson patients' leukocytes before and after deep brain stimulation as compared with control donors.

    PubMed

    Soreq, Lilach; Salomonis, Nathan; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2015-09-01

    Few studies so far examined alternative splicing alterations in blood cells of neurodegenerative disease patients, particularly Parkinson's disease (PD). Prototype junction microarrays interrogate known human genome junctions and enable characterization of alternative splicing events; however, the analysis is not straightforward and different methods can be used to estimate junction-specific alternative splicing events (some of which can also be applied for analyzing RNA sequencing junction-level data). In this study, we characterized alternative splicing changes in blood leukocyte samples from Parkinson's patients prior to, and following deep brain stimulation (DBS) treatment; both on stimulation and following 1 h off electrical stimulation. Here, we describe in detail analysis approaches for junction microarrays and provide suggestions for further analyses to delineate transcript level effects of the observed alterations as well as detection of microRNA binding sites and protein domains in the alternatively spliced target regions spanning across both untranslated and the coding regions of the targets. The raw expression data files are publically available in the Gene Expression Omnibus (GEO) database (accession number: GSE37591) and in Synapse, and can be re-analyzed. The results may be useful for designing of future experiments and cross correlations with other datasets from PD or patients having other neurodegenerative diseases. PMID:26484282

  3. Analyzing alternative splicing data of splice junction arrays from Parkinson patients' leukocytes before and after deep brain stimulation as compared with control donors

    PubMed Central

    Soreq, Lilach; Salomonis, Nathan; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2015-01-01

    Few studies so far examined alternative splicing alterations in blood cells of neurodegenerative disease patients, particularly Parkinson's disease (PD). Prototype junction microarrays interrogate known human genome junctions and enable characterization of alternative splicing events; however, the analysis is not straightforward and different methods can be used to estimate junction-specific alternative splicing events (some of which can also be applied for analyzing RNA sequencing junction-level data). In this study, we characterized alternative splicing changes in blood leukocyte samples from Parkinson's patients prior to, and following deep brain stimulation (DBS) treatment; both on stimulation and following 1 h off electrical stimulation. Here, we describe in detail analysis approaches for junction microarrays and provide suggestions for further analyses to delineate transcript level effects of the observed alterations as well as detection of microRNA binding sites and protein domains in the alternatively spliced target regions spanning across both untranslated and the coding regions of the targets. The raw expression data files are publically available in the Gene Expression Omnibus (GEO) database (accession number: GSE37591) and in Synapse, and can be re-analyzed. The results may be useful for designing of future experiments and cross correlations with other datasets from PD or patients having other neurodegenerative diseases. PMID:26484282

  4. Splice Site, Frameshift and Chimeric GFAP Mutations in Alexander Disease

    PubMed Central

    Flint, Daniel; Li, Rong; Webster, Lital S.; Naidu, Sakkubai; Kolodny, Edwin; Percy, Alan; van der Knaap, Marjo; Powers, James M.; Mantovani, John F.; Ekstein, Josef; Goldman, James E.; Messing, Albee; Brenner, Michael

    2012-01-01

    Alexander disease (AxD) is a usually fatal astrogliopathy primarily caused by mutations in the gene encoding GFAP, an intermediate filament protein expressed in astrocytes. We describe three patients with unique characteristics, and whose mutations have implications for AxD diagnosis and studies of intermediate filaments. Patient 1 is the first reported case with a non-coding mutation. The patient has a splice site change producing an in-frame deletion of exon 4 in about 10% of the transcripts. Patient 2 has an insertion and deletion at the extreme end of the coding region, resulting in a short frameshift. In addition, the mutation was found in buccal DNA but not in blood DNA, making this patient the first reported chimera. Patient 3 has a single base deletion near the C-terminal end of the protein, producing a short frameshift. These findings recommend inclusion of intronic splice site regions in genetic testing for AxD, indicate that alteration of only a small fraction of GFAP can produce disease, and provide caution against tagging intermediate filaments at their C-terminal end for cell biological investigations. PMID:22488673

  5. A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.

    PubMed

    Nasim, F H; Spears, P A; Hoffmann, H M; Kuo, H C; Grabowski, P J

    1990-07-01

    To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed. PMID:2210374

  6. 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. PMID:2647021

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

    PubMed

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

    2016-04-01

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

  8. U2AF1 mutations alter splice site recognition in hematological malignancies

    PubMed Central

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

    2015-01-01

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

  9. DBASS3 and DBASS5: databases of aberrant 3'- and 5'-splice sites.

    PubMed

    Buratti, Emanuele; Chivers, Martin; Hwang, Gyulin; Vorechovsky, Igor

    2011-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Spatial Organization of Protein-RNA Interactions in the Branch Site-3′ Splice Site Region during pre-mRNA Splicing in Yeast

    PubMed Central

    McPheeters, David S.; Muhlenkamp, Peggy

    2003-01-01

    A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3′ splice site-3′ exon region during yeast pre-mRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3′ splice site-3′ exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3′ splice site and the adjacent 3′ exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified ∼80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3′ splice site region during spliceosome assembly and catalysis. PMID:12773561

  13. pKa Coupling at the Intein Active Site: Implications for the Coordination Mechanism of Protein Splicing with a Conserved Aspartate

    PubMed Central

    Du, Zhenming; Zheng, Yuchuan; Patterson, Melissa; Liu, Yangzhong; Wang, Chunyu

    2011-01-01

    Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pKa of 6.1, two units above the normal pKa of aspartate. The elevated pKa of D422 is coupled to the depressed pKa of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pKa to normal, while a D422G mutation increases the C1 pKa from 7.5 to 8.5. The pKa coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N–S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing. PMID:21604815

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

    PubMed Central

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

    2010-01-01

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

  15. 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.

  16. 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

  17. Retinitis pigmentosa mutations of SNRNP200 enhance cryptic splice-site recognition.

    PubMed

    Cva?kov, Zuzana; Mat?j?, Daniel; Stan?k, David

    2014-03-01

    Mutations in SNRP200 gene cause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a ?-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function. PMID:24302620

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

    PubMed Central

    Luukkonen, B G; Séraphin, B

    1998-01-01

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

  19. 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.

  20. Identifying potential kidney donors using social networking web sites.

    PubMed

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

    2013-01-01

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

  1. 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

  2. Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site.

    PubMed

    Tacke, R; Goridis, C

    1991-08-01

    Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5'-splice site is an important element for regulation. Replacing it with a corresponding sequence from the alpha-globin gene resulted in constitutive use of the optional exon. When placed in the alpha-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5'-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells. PMID:1869048

  3. Improving the donor site cosmesis of the latissimus dorsi flap.

    PubMed

    Kelly, M B; Searle, A

    1998-12-01

    A modification in the design of the pedicled latissimus dorsi musculocutaneous flap is described that aims to minimize the cosmetic morbidity of its donor site. The implications of this variation are discussed with particular reference to use of the flap in postmastectomy reconstruction. PMID:9869136

  4. 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

  5. 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

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

    PubMed

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

    2015-10-01

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

  7. 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

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

    PubMed Central

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

    2011-01-01

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

  9. Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome

    PubMed Central

    Rice, Gillian I.; Reijns, Martin A.M.; Coffin, Stephanie R.; Forte, Gabriella M.A.; Anderson, Beverley H.; Szynkiewicz, Marcin; Gornall, Hannah; Gent, David; Leitch, Andrea; Botella, Maria P.; Fazzi, Elisa; Gener, Blanca; Lagae, Lieven; Olivieri, Ivana; Orcesi, Simona; Swoboda, Kathryn J.; Perrino, Fred W.; Jackson, Andrew P.; Crow, Yanick J.

    2013-01-01

    Aicardi-Goutières syndrome (AGS) is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1 or ADAR1. Here we provide molecular, biochemical and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families. PMID:23592335

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

    SciTech Connect

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

    1993-03-01

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

  11. Cross-kingdom patterns of alternative splicing and splice recognition

    PubMed Central

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

    2008-01-01

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

  12. Achieving ideal donor site aesthetics with autologous breast reconstruction

    PubMed Central

    2015-01-01

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

  13. An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

    PubMed

    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 U2AF(65) 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 U2AF(65)-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 U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) 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 U2AF(65) 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

  14. 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.

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

    SciTech Connect

    Hirschhorn, R.; Yang, D.R.; Israni, A.; Huie, M.L. ); Ownby, D.R. )

    1994-07-01

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

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

    PubMed

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

    2015-02-01

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

  18. 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

  19. 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.

  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. PMID:25802408

  2. A novel splice-site mutation in the GJB2 gene causing mild postlingual hearing impairment.

    PubMed

    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 (connexin-26) [corrected] 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 connexin-26 [corrected] encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

  3. 3′ Splice Site Sequences of Spinal Muscular Atrophy Related SMN2 Pre-mRNA Include Enhancers for Nearby Exons

    PubMed Central

    Loh, Tiing Jen; Kim, Hey-Ran; Shin, Myung-Geun; Liao, D. Joshua; Zheng, Xuexiu; Shen, Haihong

    2014-01-01

    Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5′ splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3′ splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3′ splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites. PMID:24616638

  4. Homologous SV40 RNA trans-splicing

    PubMed Central

    Eul, Joachim; Patzel, Volker

    2013-01-01

    Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5? donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5?ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5?ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes. PMID:24178438

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

    SciTech Connect

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

    1994-05-01

    Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. The authors have characterized the mutations responsible for ADA deficiency in siblings with disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G[sup +1][yields]A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings. 66 refs., 6 figs., 1 tab.

  6. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

    PubMed Central

    Lou, H; Yang, Y; Cote, G J; Berget, S M; Gagel, R F

    1995-01-01

    Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing. PMID:8524281

  7. Mutagenesis of the Yeast Gene Prp8 Reveals Domains Governing the Specificity and Fidelity of 3' Splice Site Selection

    PubMed Central

    Umen, J. G.; Guthrie, C.

    1996-01-01

    PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the uridine tract that precedes most yeast 3' slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3' splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3' splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the prp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3' splice site selection and fidelity of 3' splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing. PMID:8725222

  8. A laboratory study of fracture in the presence of lap splice multiple site damage

    NASA Astrophysics Data System (ADS)

    Mayville, Ronald A.; Warren, Thomas J.

    Flat coupons were tested in the laboratory to determine a fracture criterion for link-up of fuselage lap splice multiple site damage at adjacent rivet holes. Experiments were performed on 0.040 inch (1 mm) thick 2024-T3 clad aluminum sheet. Continuous and riveted lap splice coupons were tested with simulated uniform (equal crack lengths) and nonuniform MSD, and the effects of notch sharpness were also studied. A net section stress criterion was found to provide excellent predictions of fracture for uniform MSD and uniform stress distributions. This same criterion provides conservative predictions for nonuniform MSD in uniform stress fields. An overload/cyclic stress experiment was also conducted to explore the pressurized proof test scenario of ensuring structural integrity.

  9. Interaction of the yeast DExH-box RNA helicase Prp22p with the 3′ splice site during the second step of nuclear pre-mRNA splicing

    PubMed Central

    McPheeters, David S.; Schwer, Beate; Muhlenkamp, Peggy

    2000-01-01

    Using site-specific incorporation of the photochemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3′ splice sites. One of these is an unidentified ~122 kDa protein that cross-links to 3′ splice sites during formation of the pre-spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3′ splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3′ splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3′ splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3′ splice site of yeast introns is contacted much earlier than previously envisioned by a protein of ~120 kDa, and that a transient association of Prp22p with the 3′ splice site occurs between the first and second catalytic steps. PMID:10684925

  10. Biased exon/intron distribution of cryptic and de novo 3' splice sites.

    PubMed

    Královicová, Jana; Christensen, Mikkel B; Vorechovský, Igor

    2005-01-01

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

  11. 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

  12. BAP1 missense mutation c.2054 A>T (p.E685V) completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    PubMed

    Morrison, Arianne; Chekaluk, Yvonne; Bacares, Ruben; Ladanyi, Marc; Zhang, Liying

    2015-01-01

    BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V) variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing. PMID:25830670

  13. The U1 snRNP Base Pairs with the 5′ Splice Site within a Penta-snRNP Complex

    PubMed Central

    Malca, Hadar; Shomron, Noam; Ast, Gil

    2003-01-01

    Recognition of the 5′ splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5′ splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5′ splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5′ splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5′ splice site through base pairing with the 5′ end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5′ splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5′ splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5′ splice site in a mammalian preassembled penta-snRNP complex. PMID:12724403

  14. Similar Splice-Site Mutations of the ATP7A Gene Lead to Different Phenotypes: Classical Menkes Disease or Occipital Horn Syndrome

    PubMed Central

    Møller, Lisbeth Birk; Tümer, Zeynep; Lund, Connie; Petersen, Carsten; Cole, Trevor; Hanusch, Ralf; Seidel, Jürg; Jensen, Lars Riff; Horn, Nina

    2000-01-01

    More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)–PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6–specific primer, we were able to amplify exon 6–containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6–containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%–5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD. PMID:10739752

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

    PubMed Central

    2014-01-01

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

  16. 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.

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

    PubMed

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

    2012-08-01

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

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

    PubMed Central

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

    2005-01-01

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

  19. 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.

  20. 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

  1. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2009-08-01

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

  5. 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

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

    PubMed Central

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

    2015-01-01

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

  7. [Wound management with split skin flaps--donor sites. Covering with the moist gel Geliperm].

    PubMed

    Sattler, G; Hagedorn, M

    1990-02-20

    In 23 patients, donor sites of split-thickness skin grafts were treated with Geliperm Hydrogel, a swellable polyacrylamide agar. Healing duration, toleration, exudation and pain were all noted during the daily change of dressing. In 22 of the 23 cases, good healing was obtained after an average of 12.3 days. We feel that Geliperm is excellently suitable for covering the donor sites of split-thickness skin grafts. PMID:2328933

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

    PubMed

    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

  9. 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

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

    PubMed

    Finley, Jahahreeh

    2014-11-01

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

  11. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing.

    PubMed

    Krawczak, Michael; Thomas, Nick S T; Hundrieser, Bernd; Mort, Matthew; Wittig, Michael; Hampe, Jochen; Cooper, David N

    2007-02-01

    Although single base-pair substitutions in splice junctions constitute at least 10% of all mutations causing human inherited disease, the factors that determine their phenotypic consequences at the RNA level remain to be fully elucidated. Employing a neural network for splice-site recognition, we performed a meta-analysis of 478 disease-associated splicing mutations, in 38 different genes, for which detailed laboratory-based mRNA phenotype assessment had been performed. Inspection of the +/-50-bp DNA sequence context of the mutations revealed that exon skipping was the preferred phenotype when the immediate vicinity of the affected exon-intron junctions was devoid of alternative splice-sites. By contrast, in the presence of at least one such motif, cryptic splice-site utilization, became more prevalent. This association was, however, confined to donor splice-sites. Outside the obligate dinucleotide, the spatial distribution of pathological mutations was found to differ significantly from that of SNPs. Whereas disease-associated lesions clustered at positions -1 and +3 to +6 for donor sites and -3 for acceptor sites, SNPs were found to be almost evenly distributed over all sequence positions considered. When all putative missense mutations in the vicinity of splice-sites were extracted from the Human Gene Mutation Database for the 38 studied genes, a significantly higher proportion of changes at donor sites (37/152; 24.3%) than at acceptor splice-sites (1/142; 0.7%) was found to reduce the neural network signal emitted by the respective splice-site. Based upon these findings, we estimate that some 1.6% of disease-causing missense substitutions in human genes are likely to affect the mRNA splicing phenotype. Taken together, our results are consistent with correct donor splice-site recognition being a key step in exon recognition. PMID:17001642

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

    PubMed Central

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

    2015-01-01

    Purpose This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin. Methods Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1. Results A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028–1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408–2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein. Conclusions we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who manifested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well. PMID:26321862

  13. The Peculiarities of Large Intron Splicing in Animals

    PubMed Central

    Fedorov, Alexei

    2009-01-01

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

  14. Donor-Site Morbidity after Partial Second Toe Pulp Free Flap for Fingertip Reconstruction

    PubMed Central

    Kim, Hyung Su; Kim, Jin Soo; Roh, Si Young; Lee, Kyung Jin; Yang, Jae Won; Ki, Sae Hwi; Harijan, Aram

    2016-01-01

    Background In this study, we characterize the morbidity at the donor-site of partial second toe pulp free flaps in terms of wound management as well as long-term outcomes. Methods A single-institutional retrospective review was performed for patients who had undergone partial second toe pulp free flap transfer to the fingertip. Patient charts were reviewed for infection, skin necrosis, wound dehiscence, and hematoma for the donor site. Additionally, a questionnaire survey was given to patients who had a follow-up of longer than 1 year to characterize long-term postoperative pain and appearance. Results The review identified a total of 246 cases. Early wound complications were significant for wound dehiscence (n=8) and hematoma (n=5) for a wound complication rate of 5.3%. The questionnaire was distributed to 109 patients, and 54 patients completed the survey. Out of these 54 patients, 15 patients continued to have donor-site pain (28%) at a mean follow-up period of 32.4 months. However, the pain intensity was relatively low in the range between 2 to 5, on a 0–10 scale. None of these patients felt this donor-site pain interfered significantly with daily activity, nor did any patient require pain medications of any type. Donor-site appearance was satisfactory to most patients. Conclusions The partial second toe pulp flap was associated with low rates of wound complications and favorable long-term outcomes. Given the functional and aesthetic gain in the recipient finger, donor-site morbidities appear acceptable in this patient population. This study can be helpful in counseling patients regarding donor-site morbidity during the informed consent process. PMID:26848448

  15. Evaluation of splicing efficiency in lymphoblastoid cell lines from patients with splicing-factor retinitis pigmentosa

    PubMed Central

    Ivings, Lenka; Towns, Katherine V.; Matin, M.A.; Taylor, Charles; Ponchel, Frederique; Grainger, Richard J.; Ramesar, Rajkumar S.; Mackey, David A.

    2008-01-01

    Purpose Retinitis pigmentosa (RP) is caused by mutations in a variety of genes, most of which have known functions in the retina. However, one of the most perplexing findings of recent retinal genetics research was the discovery of mutations causing dominant RP in four ubiquitously expressed splicing factors. The aim of this study was to use lymphoblast cell lines derived from RP patients to determine whether mutations in two of these splicing factors, PRPF8 and PRPF31, cause measurable deficiencies in pre-mRNA splicing. Methods cDNA was prepared from lymphoblastoid cell lines derived from RP patients bearing mutations in the splicing factor genes and controls, grown under a variety of conditions. Introns representing the U2 and U12 intron classes, with both canonical and noncanonical donor and acceptor sequences, were analyzed by real-time PCR to measure the ratio of spliced versus unspliced transcripts for these introns. In addition, plasmids encoding the retinal outer segment membrane protein-1 (ROM-1; exon 1 to exon 2) gene, both in the wild-type form and with mutations introduced into the splice donor sites, were transfected into cell lines. The spliced versus unspliced cDNA ratios were measured by real-time RT–PCR. Results Splicing of four canonical U2 introns in the actin beta (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PRPF8, and retinitis pigmentosa GTPase regulator (RPGR) genes was unaffected in PRPF8 mutant cells. However, the splicing efficiency of RPGR intron 9 was significantly decreased in PRPF31 mutant cell lines. In contrast, a consistent decrease in the splicing efficiency of all U12 and noncanonical U2 introns was seen in PRPF8, but not in PRPF31, mutant cells, with statistical significance for STK11 intron 3. Conclusions In spite of the ubiquitous expression patterns of the genes implicated in splicing factor RP, no pathology has yet been documented outside the retina. The observed differences in splicing efficiency described herein favor the hypothesis that these mutations may have a subpathological effect outside the retina. These observations argue against a defect in some yet to be discovered additional function of these proteins and support the alternative hypothesis that this form of RP does indeed result from aberrant splicing of retinal transcripts. PMID:19096719

  16. 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

  17. A specific GFP expression assay, penetrance estimate, and histological assessment for a putative splice site mutation in BRCA1.

    PubMed

    Southey, M C; Tesoriero, A; Young, M A; Holloway, A J; Jenkins, M A; Whitty, J; Misfud, S; kConFab; McLachlan, S A; Venter, D J; Armes, J E

    2003-07-01

    Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer. PMID:12815598

  18. Donor-site morbidity of the segmental rectus abdominis muscle flap.

    PubMed

    Geishauser, M; Staudenmaier, R W; Biemer, E

    1998-12-01

    The donor-site morbidity of the segmental rectus abdominis muscle flap was evaluated in 20 patients with an average follow-up time of 47 months. Our criteria were based on static and dynamic functional results including relaxation and hernia of the abdominal wall, aesthetic outcome and patient satisfaction. The dynamic functional tests of the abdominal wall showed good results corresponding to the reported minimal impairment of quality of life. There was one abdominal hernia after wound infection and secondary healing. There was no evidence of abdominal wall instability in any of the other patients. The aesthetic outcome was excellent when a transverse lower abdominal incision, asymmetrically elongated to the donor site, was used and moderate in the case of a paramedian vertical incision. Ninety-five per cent of the patients were completely satisfied or satisfied with the result at the donor site. In the segmental use of the free rectus abdominis flap a high degree of subjective patient satisfaction reflects the favourable outcome of our examinations. On the other hand there is a clinically significant functional donor-site defect of this flap. As this procedure is still widely used, and as its indication is closely linked to its absolute and relative donor-site defect, comparisons with the alternatives, e.g. the partial latissimus dorsi muscle flap, the extended gracilis muscle flap or the serratus anterior muscle flap will have to be made. PMID:10209463

  19. The 3? Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin

    PubMed Central

    Kierzek, Elzbieta; Kierzek, Ryszard; Priore, Salvatore F.; Turner, Douglas H.

    2012-01-01

    The 3? splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3? splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site. PMID:22685560

  20. 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 examples for which there is experimental evidence of polymorphisms affecting splicing in the literature. We also present a set of novel allele-specific splicing candidates and discuss the strengths and weaknesses of alternative technologies for inferring the effect of sequence variants on mRNA splicing. PMID:18518984

  1. 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

  2. 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.

  3. Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

    PubMed Central

    Steingrimsdottir, H; Rowley, G; Dorado, G; Cole, J; Lehmann, A R

    1992-01-01

    A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA. Images PMID:1373235

  4. ASSESSMENT OF DONOR SITE MORBIDITY FOR FREE RADIAL FOREARM OSTEOCUTANEOUS FLAPS

    PubMed Central

    Sinclair, Catherine F.; Gleysteen, John P.; Zimmermann, Terence M.; Wax, Mark K.; Givi, Babak; Schneider, Daniel; Rosenthal, Eben L.

    2014-01-01

    Purpose Assessment of donor site morbidity and recipient site complications following free radial forearm osteocutaneous flap (FRFOCF) harvest and evaluation of patient perceived upper limb disability for free radial forearm osteocutaneous versus fasciocutaneous flaps (FRFF). Methods First a case series was undertaken of 218 patients who underwent an FRFOCF at two tertiary referral centers between February 1998 and November 2010. Outcomes included forearm donor site morbidity and recipient site complications. Second, the disability of the arm, shoulder, and hand (DASH) questionnaire assessing patient perceived arm disability was administered by phone to 60 consecutive patients who underwent an FRFOCF or FRFF. Results Mean patient age was 63 years with male predominance (62.8%). Median bone length harvested was 8 cm (range, 3–12 cm) with prophylactic plating of the radius following harvest. Donor site morbidity included fracture (1 patient, 0.5%) and sensory neuropathy (5 patients, 2.3%). Mean DASH scores were comparative between groups and to established normative values. Mandibular malunion rate was 3.2% and hardware extrusion at the recipient site occurred in 15.6%. Conclusion Reluctance to perform FRFOCF by surgeons usually centers on concerns regarding potential donor site morbidity and adequacy of available bone stock; however, we identified minimal objective or patient perceived donor site morbidity or recipient site complications following harvest of FRFOCFs. Mild wrist weakness and stiffness are common but do not impede ability to perform activities of daily living. Data from this and other reports suggest this flap is particularly useful for midfacial and short segment mandibular reconstruction. PMID:22473601

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

    PubMed Central

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. Early-onset encephalopathy with epilepsy associated with a novel splice site mutation in SMC1A.

    PubMed

    Lebrun, Nicolas; Lebon, Sébastien; Jeannet, Pierre-Yves; Jacquemont, Sébastien; Billuart, Pierre; Bienvenu, Thierry

    2015-12-01

    We report on the clinical and molecular characterization of a female patient with early-onset epileptic encephalopathy, who was found to carry a de novo novel splice site mutation in SMC1A. This girl shared some morphologic and anthropometric traits described in patients with clinical diagnosis of Cornelia de Lange syndrome and with SMC1A mutation but also has severe encephalopathy with early-onset epilepsy. In addition, she had midline hand stereotypies and scoliosis leading to the misdiagnosis of a Rett overlap syndrome. Molecular studies found a novel de novo splice site mutation (c.1911 + 1G > T) in SMC1A. This novel splice mutation was associated with an aberrantly processed mRNA that included intron 11 of the gene. Moreover, quantitative approach by RT-PCR showed a severe reduction of the SMC1A transcript suggesting that this aberrant transcript may be unstable and degraded. Taken together, our data suggest that the phenotype may be due to a loss-of-function of SMC1A in this patient. Our findings suggest that loss-of-function mutations of SMC1A may be associated with early-onset encephalopathy with epilepsy. © 2015 Wiley Periodicals, Inc. PMID:26358754

  9. Functional VEGFA knockdown with artificial 3′-tailed mirtrons defined by 5′ splice site and branch point

    PubMed Central

    Kock, Kian Hong; Kong, Kiat Whye; Hoon, Shawn; Seow, Yiqi

    2015-01-01

    Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons – 3′-tailed mirtrons – has hairpins precisely defined on the 5′ end by the 5′ splice site and 3′ end by the branch point. Here, we present design principles for artificial 3′-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy. PMID:26089392

  10. 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 promoter alleles. PMID:26716871

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

    PubMed Central

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

    2006-01-01

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

  12. Donor-Site Morbidity Following Minimally Invasive Costal Cartilage Harvest Technique

    PubMed Central

    Yang, Hyung Chae; Jo, Si Young; Jang, Chul Ho

    2015-01-01

    Objectives Autologous costal cartilage is a promising alternative for mastoid obliteration. However, donor-site morbidities of the chest wall limit the use of this graft. To address this issue, we have developed a minimally-invasive technique of harvesting costal cartilage and report donor site morbidity associated with the procedure. Methods Donor site morbidities were evaluated for 151 patients who underwent costal cartilage harvest, canal wall down mastoidectomy, and mastoid obliteration. Pain and cosmetic concern were evaluated via visual analogue scale (VAS). Scars were evaluated via the modified Vancouver Scar Scale (VSS) and the Patient and Observer Scar Assessment Scale (POSAS). Postoperative complications were assessed during the follow-up period. Results The mean duration of noticeable pain was 5.3 days post operation. The mean VAS score for pain was 3.0 of 10 on the first day after the operation and gradually declined. At the 6 months post operation, the mean VAS cosmetic score at the costal cartilage harvest site was 0.6 of 10. The mean VSS score was 9.5 out of 10 total, and the mean POSAS score was 23.27 out of 110 total. Conclusion The minimally-invasive chopped costal cartilage harvest technique resulted in acceptable pain, cosmetic concern, and postoperative complications for most patients. There were no major postoperative complications. Costal cartilage is an acceptable donor for mastoid obliteration in canal wall down mastoidectomy, especially in the context of the extremely low donor site morbidity of the minimally-invasive technique presented in the study. PMID:25729490

  13. 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

  14. Conformational dynamics of single pre–mRNA molecules during in vitro splicing

    PubMed Central

    Abelson, John; Blanco, Mario; Ditzler, Mark A.; Fuller, Franklin; Aravamudhan, Pavithra; Wood, Mona; Villa, Tommaso; Ryan, Daniel E.; Pleiss, Jeffrey A.; Maeder, Corina; Guthrie, Christine; Walter, Nils G.

    2010-01-01

    The spliceosome is a complex small nuclear (sn)RNA–protein machine that removes introns from pre–mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA–dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre–mRNA conformational dynamics, we developed a strategy for single molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre–mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro we observe a multitude of generally reversible, time– and ATP–dependent conformational transitions of individual pre–mRNAs. The conformational dynamics of branchpoint and 3′ splice site mutants differ from one another and from wild–type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium. PMID:20305654

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  17. 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 IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects. PMID:26506010

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

    PubMed Central

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

    2001-01-01

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

  19. 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. PMID:23056301

  20. A Novel ERCC6 Splicing Variant Associated with a Mild Cockayne Syndrome Phenotype

    PubMed Central

    Swartz, Jonathan M; Akinci, Aysehan; Andrew, Shayne F.; Siğirci, Ahmet; Hirschhorn, Joel N; Rosenfeld, Ron G; Dauber, Andrew; Hwa, Vivian

    2014-01-01

    Background Cockayne syndrome is an autosomal recessive, heterogeneous syndrome with classic features, including short stature, microcephaly, developmental delay, neuropathy, and photosensitivity. New genomic approaches offer improved molecular diagnostic potential. Methods Whole-exome sequencing was employed to study a consanguineous extended family with severe short stature and variable presentations of peripheral neuropathy, lipoatrophy, photosensitivity, webbed neck, and hirsutism. Results We identified a novel homozygous ERCC6 variant at the donor splice site of intron 9 (c.1992+3A>G), which was predicted to only slightly perturb splicing efficiencies. Assessment of primary fibroblast-derived mRNAs, however, revealed a dominant splicing species that utilized an unsuspected putative donor splice site within exon 9, resulting in predicted early protein termination (p.Arg637Serfs*34). Conclusions We describe a new splicing ERCC6 defect causal of Cockayne syndrome. The application of exome sequence analysis was integral to diagnosis, given the complexity of phenotypic presentation in affected family members. The novel splicing defect, furthermore, illustrates how a seemingly minor change in the relative strength of a splice site can have significant biological consequences. PMID:25376329

  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. PMID:24563639

  2. A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron.

    PubMed

    Isshiki, M; Morino, K; Nakajima, M; Okagaki, R J; Wessler, S R; Izawa, T; Shimamoto, K

    1998-07-01

    In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation. PMID:9744101

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

    PubMed Central

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

    2006-01-01

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

  4. Structural basis for recognition of the intron branch site RNA by splicing factor 1.

    PubMed

    Liu, Z; Luyten, I; Bottomley, M J; Messias, A C; Houngninou-Molango, S; Sprangers, R; Zanier, K; Krämer, A; Sattler, M

    2001-11-01

    During spliceosome assembly, splicing factor 1 (SF1) specifically recognizes the intron branch point sequence (BPS) UACUAAC in the pre-mRNA transcripts. We show that the KH-QUA2 region of SF1 defines an enlarged KH (hn RNP K) fold which is necessary and sufficient for BPS binding. The 3' part of the BPS (UAAC), including the conserved branch point adenosine (underlined), is specifically recognized in a hydrophobic cleft formed by the Gly-Pro-Arg-Gly motif and the variable loop of the KH domain. The QUA2 region recognizes the 5' nucleotides of the BPS (ACU). The branch point adenosine acting as the nucleophile in the first biochemical step of splicing is deeply buried. BPS RNA recognition suggests how SF1 may facilitate subsequent formation of the prespliceosomal complex A. PMID:11691992

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

    PubMed

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

    2016-02-01

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

  6. Neural network detects errors in the assignment of mRNA splice sites.

    PubMed

    Brunak, S; Engelbrecht, J; Knudsen, S

    1990-08-25

    The use of databanks in genetic research assumes reliability of the information they contain. Currently, error-detection in the manually or electronically entered data contained in the nucleotide sequence databanks at EMBL, Heidelberg and GenBank at Los Alamos is limited. We have used a subset of sequences from these databanks to train neural networks to recognize pre-mRNA splicing signals in human genes. During the training on 33 human genes from the EMBL databank seven genes appeared to disturb the learning process. Subsequent investigation revealed discrepancies from the original published papers, for three genes. In four genes, we found wrongly assigned splicing frames of introns. We believe this to be a reflection of the fact that splicing frames cannot always be unambiguously assigned on the basis of experimental data. Thus incorrect assignment appear both due to mere typographical misprints as well as erroneous interpretation of experiments. Training on 241 human sequences from GenBank revealed nine new errors. We propose that such errors could be detected by computer algorithms designed to check the consistency of data prior to their incorporation in databanks. PMID:2395643

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

    PubMed

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

    2016-05-10

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

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

    PubMed Central

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

    2015-01-01

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

  9. Use of cultured human epithelium for coverage: a defect of radial forearm free flap donor site.

    PubMed

    Gallego, Lorena; Junquera, Luis; Villarreal, Pedro; Peña, Ignacio; Meana, Alvaro

    2010-01-01

    The radial forearm free flap has been popular in many areas of reconstructive surgery. Despite the many attributes of this flap in maxillofacial reconstruction, one of the disadvantages has been the morbidity of the donor site. Allogeneic cultured epidermis has been successfully applied on large second degree burns and on chronic leg ulcers. Autologous human keratinocytes and fibroblast equivalents can be cultured in-vitro from a small skin sample in order to produce a sufficient amount of epithelial autografts to cover the large defects of third-degree burn wounds. Interestingly, transplanted cultured epidermis retains characteristics of the original donor site. We report a case of a patient who underwent skin replacement by cultured epithelial autograft after wound breakdown occurred in the forearm donor site during the early postoperative period. This method could represent an auspicious alternative to conventional grafting methods for forearm free flap reconstruction. To the best of our knowledge, skin replacement by cultured epithelial autografts in this region has not been extensively described in the literature. PMID:19680169

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

    PubMed Central

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

    1995-01-01

    BACKGROUND: Human growth hormone is an anabolic agent that attenuates injury-induced catabolism and stimulates protein synthesis. Recombinant human growth hormone (rhGH) administered therapeutically to patients with massive burns has been shown to increase the rate of skin graft donor site healing. It has been postulated that growth hormone affects wound healing and tissue repair by stimulating the production of insulin-like growth factor-1 (IGF-1) by the liver to increase circulating IGF-1 concentrations. The mechanism by which it improves wound healing, however, remains in question. The authors hypothesize that rhGH up-regulates IGF-1 receptors and IGF-1 levels both systemically and locally in the wound site to stimulate cell mitosis and increase synthesis of laminin, collagen types IV and VII, and cytokeratin. This hypothesis was tested in nine patients with burns covering > 40% of total body surface area. OBJECTIVE: The authors assessed the efficacy of rhGH in promoting several major building materials in the donor site of patients with massive burns. METHODS: Ten massively burned patients with full-thickness burns covering more than 40% of total body surface area were participants in a placebo-controlled prospective study to determine the efficacy of 0.2 mg/kg/day rhGH on donor site wound healing and to identify some of the major components involved in wound healing and its integrity. RESULTS: Donor sites in burn patients receiving rhGH showed an increased coverage by the basal lamina of 26% for placebo to 68% coverage of the dermal-epidermal junction. Insulin-like growth factor-1 receptors and laminin, types IV and VII collagen, and cytokeratin-14 all increased significantly. Healing times of the donor sites were significantly decreased compared with patients receiving placebo. CONCLUSION: Results indicate that growth hormone or its secondary mediators may directly stimulate the cells of the epidermis and dermis during wound healing to produce the structural proteins and other components needed to rebuild the junctional structures. Images Figure 2. Figure 3. PMID:7794069

  11. 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

  12. The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C{alpha} intron

    SciTech Connect

    Brown, W.R.; Kacskovics, I.; Amendt, B.A.

    1995-04-15

    Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C{alpha}1-C{alpha}2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C{alpha}2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C{alpha}1 nucleotide to nucleotide 532 in C{alpha}2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgA{sup a} and IgA{sup b}, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgA{sup b}) variant, in which the downstream cryptic splice is used, as well as a {open_quotes}normal-hinged{close_quotes} (six amino acids, IgA{sup a}) variant. Evidence that IgA{sup a} and IgA{sup b} are allelic was confirmed by genotypic analyses of progeny from matings of IgA{sup a}/IgA{sup b} heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant. 25 refs., 5 figs., 1 tab.

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

    PubMed

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

    2016-05-01

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

  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. 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.

  16. Effects of human recombinant growth hormone on donor-site healing in burned adults.

    PubMed

    Losada, Fernando; García-Luna, Pedro P; Gómez-Cía, Tomás; Garrido, Manuela; Pereira, Jose L; Marín, Fernando; Astorga, Ricardo

    2002-01-01

    Patients suffering severe burns have an accelerated catabolism with a highly negative nitrogen balance that may worsen their prognosis. Somatropin treatment has been shown to improve this balance in different hypercatabolic situations. Moreover, in children with extensive burns it also reduces the healing time of the skin graft donor site and shortens the hospital stay. In the existing literature there are no controlled prospective clinical trials in adult patients that confirm these data. Our aim was to demonstrate the efficacy of recombinant growth hormone (somatropin) in reducing the healing time of the skin graft donor sites and the length of stay in the burn unit in adult patients with severe burns. A randomized, double-blind, placebo-controlled clinical trial was carried out in 24 adult patients with severe burns (more than 40% of the total body surface burned or more than 15% full-thickness burns). Patients received placebo (n = 11) or somatropin (n = 13) at a dosage of 0.15 mg/kg/day divided into two equal doses (every 12 hours) via intramuscular injection. Treatment was initiated the day the first autograft was performed and terminated the day the patient was discharged from the burn unit. The mean number (+/- SD) of skin grafts per patient was similar between the two groups (4.2 +/- 1.8 vs 3.4 +/- 1.8 in the placebo and somatropin groups, respectively). No reduction in the healing time of the skin graft donor site was observed in the somatropin group compared to the placebo group. Likewise, the time admitted to the burn unit was not significantly different, either in the absolute number of days (36.2 +/- 19.7 vs 30.1 +/- 16.8 days in the placebo and somatropin groups, respectively) or in relation to the percentage of the total body surface burned or the body surface with full-thickness burns. Growth hormone and insulin-like growth factor I (IGF-I) levels were three and five times higher, respectively, in the somatropin group than in the placebo group. Ten of the patients treated with somatropin experienced hyperglycemia, and seven of them required insulin treatment. No other adverse side effect was observed. One patient in the placebo group died as a result of sepsis and multiple organ failure. Somatropin, with the treatment regimen and dosage used in these studies, did not reduce the healing time of the skin graft donor sites or the length of hospitalization in the burn unit in adult patients with severe burns. PMID:11898025

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

    PubMed

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

    2013-04-01

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

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

    PubMed

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

    2015-04-15

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2015-03-15

    Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones. PMID:25633667

  2. 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.

  3. Regulation of Alternative Splicing by Histone Modifications

    PubMed Central

    Luco, Reini F.; Pan, Qun; Tominaga, Kaoru; Blencowe, Benjamin J.; Pereira-Smith, Olivia M.; Misteli, Tom

    2010-01-01

    Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery. PMID:20133523

  4. Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

    PubMed Central

    Purcell, D F; Martin, M A

    1993-01-01

    Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity. Images PMID:8411338

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

    PubMed Central

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

    2015-01-01

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

  6. 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.

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

    SciTech Connect

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

    1996-01-01

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

  8. IVS8+1 DelG, a Novel Splice Site Mutation Causing DFNA5 Deafness in a Chinese Family

    PubMed Central

    Li-Yang, Mei-Na; Shen, Xiao-Fei; Wei, Qin-Jun; Yao, Jun; Lu, Ya-Jie; Cao, Xin; Xing, Guang-Qian

    2015-01-01

    Background: Nonsyndromic hearing loss (NSHL) is highly heterogeneous, in which more than 90 causative genes have currently been identified. DFNA5 is one of the deafness genes that known to cause autosomal dominant NSHL. Until date, only five DFNA5 mutations have been described in eight families worldwide. In this study, we reported the identification of a novel pathogenic mutation causing DFNA5 deafness in a five-generation Chinese family. Methods: After detailed clinical evaluations of this family, the genomic DNA of three affected individuals was selected for targeted exome sequencing of 101 known deafness genes, as well as mitochondrial DNA and microRNA regions. Co-segregation analysis between the hearing loss and the candidate variant was confirmed in available family members by direct polymerase chain reaction (PCR)-Sanger sequencing. Real-time PCR (RT-PCR) was performed to investigate the potential effect of the pathogenic mutation on messenger RNA splicing. Results: Clinical evaluations revealed a similar deafness phenotype in this family to that of previously reported DFNA5 families with autosomal dominant, late-onset hearing loss. Molecular analysis identified a novel splice site mutation in DFNA5 intron 8 (IVS8+1 delG). The mutation segregated with the hearing loss of the family and was absent in 120 unrelated control DNA samples of Chinese origin. RT-PCR showed skipping of exon 8 in the mutant transcript. Conclusions: We identified a novel DFNA5 mutation IVS8+1 delG in a Chinese family which led to skipping of exon 8. This is the sixth DFNA5 mutation relates to hearing loss and the second one in DFNA5 intron 8. Our findings provide further support to the hypothesis that the DFNA5-associated hearing loss represents a mechanism of gain-of-function. PMID:26365971

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

    PubMed

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

    2015-01-15

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

  11. 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

  12. 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

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

    2016-02-01

    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

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

    PubMed

    Slavov, D; Gardiner, K

    2002-10-16

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

  14. Use of lyophilized bovine collagen for split-thickness skin graft donor site management.

    PubMed

    Uygur, Fatih; Evinc, Rahmi; Ulkur, Ersin; Celikoz, Bahattin

    2008-11-01

    Donor site management after split-thickness skin graft applications can have problems such as late healing and pain. Many dressing methods and medical applications are reported to solve these problems but none of them were ideal. In this study we aimed to promote epithelisation and remove pain earlier with using lyophilized bovine collagen (gelfix spray). According to our results, epithelisation time for the gelfix group was earlier than control group (9.09 days mean and 11.2 days mean for control group (p<0.05)). Pain relief was determined by visual analogue pain scale. In the gelfix group, there was pain relief up to 40 h from the operation. There were no differences between groups for scarring 30 and 90 days after surgery. PMID:18407418

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. 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

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

    PubMed

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

    2013-05-01

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

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

    PubMed

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

    2016-06-01

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

  20. [Functional characterization of two novel splicing mutations in glucokinase gene in monogenic diabetes MODY2].

    PubMed

    Igudin, E L; Spirin, P V; Prasolov, V S; Zubkov, N A; Petria?kina, E E; Tiul'pakov, A N; Rubtsov, P M

    2014-01-01

    Two novel mutations in glucokinase (GCK) gene-G to C substitution at -1 position of intron 7 acceptor splice site (c. 864-1G>C) and synonymous substitution c. 666C>G (GTC>GTG, p.V222V) in exon 6--were identified in patients with monogenic diabetes MODY2 (Maturity Onset Diabetes of Young). GCK minigenes with these mutations were constructed. Analysis of splicing products upon transfection of minigenes into human embryonic cell line HEK293 has shown that each of these nucleotide substitutions impair normal splicing. Mutation c.864-1G>C blocks the usage of normal acceptor site which activates cryptic acceptor splice sites within intron 7 and generates aberrant RNAs containing the portions ofintron 7. Synonymous substitution c.666C>G creates novel donor splice site in exon 6 that leads to formation of defective GCK mRNA with deletion of 16 nucleotides of exon 6. Analysis of in vitro splicing of minigenes confirms the inactivating action of novel mutations on glucokinase expression. PMID:25850297

  1. 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

  2. Novel splice site mutation in the fumarate hydratase (FH) gene is associated with multiple cutaneous leiomyomas in a Japanese patient.

    PubMed

    Yoshinaga, Yukina; Nakai, Hiroyuki; Hayashi, Ryota; Ito, Akiko; Kariya, Naoyuki; Ito, Masaaki; Shimomura, Yutaka

    2016-01-01

    Cutaneous leiomyoma is a benign skin tumor that originates from the smooth muscle, such as the arrector pili muscle of the hair follicles. Familial cases with multiple cutaneous leiomyomas exist, which typically show an autosomal dominant inheritance trait. Most patients with the disease are known to carry heterozygous germ line mutations in the fumarate hydratase (FH) gene and can be complicated by tumors in internal organs, especially uterine leiomyoma and renal cell cancer in high frequency. In this study, we identified a Japanese male patient with multiple cutaneous leiomyomas and found a novel heterozygous splice site mutation, c.738 + 2T>A, in the FH gene of the patient, which was unexpectedly inherited from his unaffected father. Further analysis demonstrated loss of heterozygosity in the tumor tissue, which resulted in a hemizygote state of the mutant allele. Expression studies with the tumor tissue showed that the mutation led to skipping of exon 5 at mRNA levels, which was predicted to cause an in-frame deletion of FH protein (p.Ser186_Gln246del). The protein structure analysis strongly suggested that the deletion would severely disrupt the conformation of the FH protein including the substrate-binding domain, and thus would severely affect the expression and the function. Our findings further disclose the molecular basis of multiple cutaneous leiomyomas and also provide precious information to the mutation carriers in the family for an early diagnosis of renal cell cancer in the future. PMID:26173633

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

    PubMed

    Vanlander, Arnaud V; Menten, Bjrn; Smet, Jol; 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

  4. 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

  5. Design, synthesis, and testing of antisickling agents. 2. Proline derivatives designed for the donor site.

    PubMed

    Abraham, D J; Mokotoff, M; Sheh, L; Simmons, J E

    1983-04-01

    We have used a three-dimensional model of deoxyhemoglobin to design potential antisickling agents with an intended binding site in the vicinity of the beta-6 mutation (donor site). Two proline derivatives, (4S)-1-butyryl-4-[(carboxymethyl)amino]-L-proline (9a) and its 1-benzoyl analogue (9b), were designed to interact, via ionic or hydrogen bonds, with polar residues beta His-2, beta Thr-4, and beta Lys-132 of hemoglobin S (HbS). Two other proline derivatives containing a salicylate leaving group, (4S)-1-butyryl-4-[(carboxymethyl)methylamino]-L-proline, 2-ester with salicyclic acid (14a), and its 1-benzoyl analogue (14b), were designed to bind covalently to beta Lys-132, as well as to interact with beta His-2 and beta Thr-4 via ionic and hydrogen bonds. This paper describes the synthesis of these agents, beginning with natural L-hydroxyproline methyl ester, and the testing of their ability to increase or decrease the solubility of dHbS by using a standard solubility assay. The covalent derivatives 14a,b were found to be inactive, while the noncovalent compounds 9a,b showed weak antigelling activity, below that observed for phenylalanine. The presence of only weak activity does not invalidate this approach, since only one structural parameter has been investigated. PMID:6834388

  6. Early Clinical Diagnosis of PC1/3 Deficiency in a Patient With a Novel Homozygous PCSK1 Splice-Site Mutation.

    PubMed

    Härter, Bettina; Fuchs, Irene; Müller, Thomas; Akbulut, Ulas Emre; Cakir, Murat; Janecke, Andreas R

    2016-04-01

    Autosomal recessive proprotein convertase 1/3 (PC1/3) deficiency, caused by mutations in the PCSK1 gene, is characterized by severe congenital malabsorptive diarrhea, early-onset obesity, and certain endocrine abnormalities. We suspected PC1/3 deficiency in a 4-month-old girl based on the presence of congenital diarrhea and polyuria. Sequencing the whole coding region and splice sites detected a novel homozygous PCSK1 splice-site mutation, c.544-2A>G, in the patient. The mutation resulted in the skipping of exon 5, the generation of a premature termination codon, and nonsense-mediated PCSK1 messenger ribonucleic acid decay, which was demonstrated in complementary DNA derived from fibroblasts. PMID:26488123

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

    PubMed Central

    Chen, I T; Chasin, L A

    1994-01-01

    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. Images PMID:8114744

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

    PubMed Central

    2014-01-01

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

  9. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

    PubMed Central

    Burd, C G; Dreyfuss, G

    1994-01-01

    Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing. Images PMID:7510636

  10. 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.

  11. Aesthetic closure of the donor site of a radial forearm free flap with two local curved skin grafts.

    PubMed

    Kim, Soung Min; Park, Jung Min; Yang, Hoon Joo; Myoung, Hoon; Lee, Suk Keun; Lee, Jong Ho

    2016-06-01

    The authors have designed an aesthetic and effective coverage technique using local curved skin grafts along with vascular pedicles without additional skin incisions to solve the disadvantages of skin coverage problem of donor site defect after radial forearm free flap (RFFF) harvesting. This has, to the authors' knowledge, not been previously described. PMID:26999429

  12. 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/.

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

    PubMed Central

    2014-01-01

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

  14. 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

  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 Central

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

    2014-01-01

    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. PMID:25111964

  17. 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

  18. Ca2+ -dependent splicing of neurexin IIalpha.

    PubMed

    Rozic-Kotliroff, G; Zisapel, N

    2007-01-01

    Neurexins are synaptic adhesion proteins encoded by 3 genes (NRXN1, NRXN2, and NRXN3) each transcribed from 2 promoters to yield longer (alpha) and shorter (beta) forms. The primary gene transcripts undergo extensive alternative splicing leading to products that may differ in synaptic coupling properties. Here we show that depolarization of neurons modulates splicing of NRXN2alpha, particularly at splice sites 1 and 3. Furthermore, we demonstrate that exclusion of exon 11 at splice site 3 is calcium-dependent. These data indicate neuronal activity-dependent splicing of NRXN2alpha. This dynamic process may be important for maintenance of mature neuronal circuits. PMID:17107668

  19. In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects

    PubMed Central

    Lastella, Patrizia; Surdo, Nicoletta Concetta; Resta, Nicoletta; Guanti, Ginevra; Stella, Alessandro

    2006-01-01

    Background Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay. Results We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion. Conclusion Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers. PMID:16995940

  20. Recursive splicing in long vertebrate genes

    PubMed Central

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

    2015-01-01

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

  1. Recursive splicing in long vertebrate genes.

    PubMed

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

    2015-05-21

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

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

    PubMed

    Drake, David B; Wong, Lesley G

    2003-04-01

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

  3. 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

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

    PubMed Central

    Hodges, P E; Rosenberg, L E

    1989-01-01

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

  5. 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

  6. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  8. The U11-48K Protein Contacts the 5′ Splice Site of U12-Type Introns and the U11-59K Protein▿ †

    PubMed Central

    Turunen, Janne J.; Will, Cindy L.; Grote, Michael; Lührmann, Reinhard; Frilander, Mikko J.

    2008-01-01

    Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5′ splice sites (5′ss). This interaction did not require branch point recognition and was sensitive to 5′ss mutations, suggesting that 48K interacts with the 5′ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5′ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5′ss, as well as functionally important interactions within the U11/U12 di-snRNP. PMID:18347052

  9. [Application of Opsite at donor sites, burns and postoperative wounds (author's transl)].

    PubMed

    Cavlak, Y

    1980-12-01

    The article reports on the Opsite foil and its properties. This is a film consisting of polyurethane which is permeable to gas and water vapour. Prolonged skin contact does not produce an allergic reaction. The wound becomes rapidly covered with an epithelium. Opsite is an extremely successful dressing for covering donor areas and wounds from burns, as well as postoperative wounds. PMID:6116406

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

    PubMed Central

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

    2007-01-01

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

  11. 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

  12. 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. PMID:25979191

  13. Complete nucleotide sequence of the fast skeletal troponin T gene. Alternatively spliced exons exhibit unusual interspecies divergence.

    PubMed

    Breitbart, R E; Nadal-Ginard, B

    1986-04-01

    The continuous nucleotide sequence of the rat fast skeletal muscle troponin T gene is reported, complementing the previous determinations of its structural organization and its capacity to encode multiple isoforms via alternative RNA splicing. Canonical promoter elements, as well as consensus sequences that may be involved in the 3' processing of the primary transcript, are present. All exons are flanked by conventional donor and acceptor splice sites, which can hybridize to U1 RNA. Extensive computer-assisted analyses of the genomic sequence do not reveal cis elements that unambiguously distinguish alternative from constitutive exons. Local RNA secondary structures can be predicted, however, that sequester exons or their splice sites in stem-and-loop formations, and which may also pair with small nuclear RNAs. These interactions might, in theory, contribute to differential exon usage. The structural features of exon organization that characterize this rat skeletal gene are closely conserved in the chicken cardiac troponin T gene, but the former exhibits a more diversified capacity for differential splicing. Implications for the mechanisms of alternative RNA splicing are considered. Comparisons of troponin T amino acid sequences among several species reveal striking dissimilarities, in contrast to the otherwise highly conserved contractile proteins. These divergences involve entire peptide subsegments and are concentrated in the same domains as are encoded by alternatively spliced exons, suggesting that exon shuffling may have contributed to the evolution of troponin T. PMID:3735424

  14. Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron.

    PubMed Central

    Zhao, Q; Schoborg, R V; Pintel, D J

    1994-01-01

    mRNAs R1 and R2 of the parvovirus minute virus of mice encode the two essential viral regulatory proteins NS1 and NS2. Both RNAs are spliced between map units 44 and 46 (nucleotides 2280 and 2399); R2 RNAs are additionally spliced upstream between map units 10 and 39 (nucleotides 514 and 1989), using a nonconsensus donor and poor 3' splice site. The relative accumulation of R1 and R2 is determined by alternative splicing: there is twice the steady-state accumulation of R2 relative to that of R1 throughout viral infection, though they are generated from the same promoter and have indistinguishable stabilities. Here we demonstrate that efficient excision of the large intron to generate R2 is dependent on at least the initial presence, in P4-generated pre-mRNAs, of sequences within the downstream small intron. This effect is orientation dependent and related to the size of the intervening exon. Prior splicing of the small intron is unnecessary. Excision of the large intron is enhanced by changing its donor site to consensus, but only in the presence of the small intron sequences. Excision of the large intron is also enhanced by improving the polypyrimidine tract within its 3' splice site; however, in contrast, this change renders excision of the large intron independent of the downstream small intron. We suggest that sequences within the small intron play a primary role in efficient excision of the upstream large intron, perhaps as the initial entry site(s) for an element(s) of the splicesome, which stabilizes the binding of required factors to the polypyrimidine tract within the 3' splice site of the large intron. Images PMID:8151756

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

    PubMed

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

    2012-04-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2006-07-01

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

  18. Group VIA Phospholipase A2 (iPLA2?) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in ?-Cells.

    PubMed

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

    2015-04-24

    Diabetes is a consequence of reduced ?-cell function and mass, due to ?-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during ?-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2? (iPLA2?) 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 ?-cells. However, chemical inactivation or knockdown of iPLA2? augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2? transgenic mice, whereas islets from global iPLA2?(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2? 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 iPLA2?/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 ?-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2?. PMID:25762722

  19. 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. PMID:26800692

  20. 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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  3. The role of gene polymorphism in HLA class I splicing.

    PubMed

    Voorter, C E M; Gerritsen, K E H; Groeneweg, M; Wieten, L; Tilanus, M G J

    2016-04-01

    Among the large number of human leucocyte antigen (HLA) alleles, only a few have been identified with a nucleotide polymorphism impairing correct splicing. Those alleles show aberrant expression levels, due to either a direct effect of the polymorphism on the normal splice site or to the creation of an alternative splice site. Furthermore, in several studies, the presence of alternatively spliced HLA transcripts co-expressed with the mature spliced transcripts was reported. We evaluated the splice site sequences of all known HLA class I alleles and found that, beside the consensus GT and AG sequences at the intron borders, there were some other highly conserved nucleotides for the different class I genes. In this review, we summarize the splicing mechanism and evaluate what is known today about alternative splicing of HLA class I genes. PMID:26920492

  4. A novel homozygous splice site mutation in NALCN identified in siblings with cachexia, strabismus, severe intellectual disability, epilepsy and abnormal respiratory rhythm.

    PubMed

    Gal, Moran; Magen, Daniella; Zahran, Younan; Ravid, Sarit; Eran, Ayelet; Khayat, Morad; Gafni, Chen; Levanon, Erez Y; Mandel, Hanna

    2016-04-01

    We studied three siblings, born to consanguineous parents who presented with severe intellectual disability, cachexia, strabismus, seizures and episodes of abnormal respiratory rhythm. Whole exome sequencing led to identification of a novel homozygous splice site mutation, IVS29-1G > A in the NALCN gene, that resulted in aberrant transcript in the patients. NALCN encodes a voltage-independent cation channel, involved in regulation of neuronal excitability. Three homozygous mutations in the NALCN gene were previously identified in only eight patients with severe hypotonia, speech impairment, cognitive delay, constipation and Infantile-Neuroaxonal-dystrophy- like symptoms. Our patients broaden the clinical spectrum associated with recessive mutations in NALCN, featuring also disrupted respiratory rhythm mimicking homozygous Nalcn knockout mice. PMID:26923739

  5. Identification of a unique splice site variant in SLC39A4 in bovine hereditary zinc deficiency, lethal trait A46: An animal model of acrodermatitis enteropathica.

    PubMed

    Yuzbasiyan-Gurkan, Vilma; Bartlett, Elizabeth

    2006-10-01

    Lethal trait A46, also known as bovine hereditary zinc deficiency, Adema disease, and hereditary parakeratosis, is an autosomal recessive disorder first described in 1964, with a clinical presentation similar to that of acrodermatitis enteropathica (AE) in humans. The molecular basis of the defect has not been previously identified. Recently, the basic defect in AE was found to lie in SLC39A4. We report the characterization of the bovine ortholog of SLC39A4 and identification of a unique splice site variant within this gene in affected animals. The mutation leads to exon skipping, leaving the coding region in frame. The gene product is predicted to lack two critical motifs, which lie in adjacent transmembrane domains implicated in the formation of a pore responsible for the transport of zinc. While further functional studies are warranted, this unique variant is likely to be responsible for the impaired zinc absorption in this disease. PMID:16714095

  6. RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts

    PubMed Central

    Sorber, Katherine; Dimon, Michelle T.; DeRisi, Joseph L.

    2011-01-01

    Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5′ GU-AG 3′ and 5 new 5′ GC-AG 3′ junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite’s transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite’s transcriptome. PMID:21245033

  7. 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 0.710.23 mL (0.27-1.96 mL) and the cancellous volume was 2.160.76 mL (0.86-6.28 mL). The minimal cortical vestibular thickness was 1.540.41 mm (0.61-3.25 mm), and the maximal cortical vestibular thickness was 3.140.75mm(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

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

    PubMed

    Nakabayashi, Keita; Nishio, Masaki; Miyasaka, Hitoshi

    2016-03-01

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

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

    PubMed

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

    2016-02-01

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

  10. 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

  11. Generation of spirotricyclic site-differentiated cyclotriphosphazenes: a solvent-free approach to multidentate N/O donor ligand systems.

    PubMed

    Harmjanz, Michael; Piglosiewicz, Ingmar M; Scott, Brian L; Burns, Carol J

    2004-01-26

    Cyclotriphosphazene-based ligand systems are valuable materials to model the metal-binding event on the structurally and electronically related functionalized high molecular weight polyphosphazenes. We here report the facile synthesis of novel spirotricyclic cyclotriphosphazenes N(3)P(3)(MeNC(2)H(4)NMe)(2)L(2), N(3)P(3)(iPrNC(2)H(4)NiPr)(2)L(2), and N(3)P(3)(o-O(2)C(12)H(8))(2)L(2) that enables different substituents to be incorporated into the ligand system. This synthetic approach allows for control over the solubility and steric requirements of the exocyclic bidentate substituents, as well as the donor type and denticity of the coordination sites. A mononuclear lanthanum complex ([La(NO(3))(3)[N(3)P(3)(pzpy)(2)(MeNC(2)H(4)NMe)(2)

  12. Human Splicing Finder: an online bioinformatics tool to predict splicing signals

    PubMed Central

    Desmet, Franois-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Broud, Gwenalle; Claustres, Mireille; Broud, Christophe

    2009-01-01

    Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-? Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5? and 3? splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project. PMID:19339519

  13. 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.

  14. 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.

  15. An Aberrant Splice Acceptor Site Due to a Novel Intronic Nucleotide Substitution in MSX1 Gene Is the Cause of Congenital Tooth Agenesis in a Japanese Family

    PubMed Central

    Tatematsu, Tadashi; Kimura, Masashi; Nakashima, Mitsuko; Machida, Junichiro; Yamaguchi, Seishi; Shibata, Akio; Goto, Hiroki; Nakayama, Atsuo; Higashi, Yujiro; Miyachi, Hitoshi; Shimozato, Kazuo; Matsumoto, Naomichi; Tokita, Yoshihito

    2015-01-01

    Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency. PMID:26030286

  16. A suppressive effect of Sp1 recruitment to the first leader 5' splice site region on L4-22K-mediated activation of the adenovirus major late promoter.

    PubMed

    Lan, Susan; Östberg, Sara; Punga, Tanel; Akusjärvi, Göran

    2015-12-01

    Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5' splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5' splice site. Binding of Sp1 to the 5' splice site region had an inhibitory effect on L4-22K-activated MLP transcription. PMID:26247419

  17. Mechanical reliability of fiber optic splices

    NASA Astrophysics Data System (ADS)

    Reith, Leslie A.; Yuce, Hakan H.; Grimado, Philip B.

    1993-11-01

    Today's feeder applications and future distribution applications call for shorter-length, higher fiber count cables, more splices per kilometer, and increased connectorization. Whapham estimates that five to eight splices per subscriber will be required for a branched distribution and loop network. In addition, splices and connectors in the loop will experience harsher environments than the controlled environment of a telephone central office or typical remote site. In the distribution portion, between the remote site and the optical network unit (ONU), the splices can be subjected to a wide range of temperature and humidity extremes, as can the ONU itself. The increased handling and the harsher environments in the local loop place significant new demands on the performance of optical splices.

  18. A novel splice mutation in PAK3 gene underlying mental retardation with neuropsychiatric features.

    PubMed

    Rejeb, Imen; Saillour, Yoann; Castelnau, Laetitia; Julien, Cdric; Bienvenu, Thierry; Taga, Patricia; Chaabouni, Habiba; Chelly, Jamel; Ben Jemaa, Lamia; Bahi-Buisson, Nadia

    2008-11-01

    PAK3-related mental retardation represents a rare cause of X-linked mental retardation associated with behavioural symptoms. So far, four families carrying PAK3 mutations have been reported, and in most cases PAK3 dysfunction resulted from missense mutations thought to affect either the catalytic or the N-terminal regulatory domain activity. Here, we report on a Tunisian family of X-linked moderate mental retardation with behavioural symptoms, common dysmorphic features, oro-motor impairment and secondary microcephaly. Linkage analysis showed that affected male subjects and obligate carrier female subjects share a common haplotype in the Xp21.31 - Xq23 region that contains the PAK3 gene. Direct sequencing of PAK3 coding exons and flanking intronic sequences allowed us to identify the first splice mutation in PAK3 gene located at the 5' end of intron 6 (c.276+4A>G), which results in a complete switch-off of the genuine donor splice site and an activation of a cryptic donor splice site (GTAAG) located four nucleotides downstream to the genuine one. RT-PCR experiments using the RNA from the patient's lymphoblasts showed that PAK3 transcripts contain four additional nucleotides that lead to a disruption of reading frame with a premature stop codon at position 128. Together with previously reported observations, our data further confirm that PAK3 mutations result in a specific form of X-linked mental retardation with fairly constant clinical features. PMID:18523455

  19. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

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

    2015-01-01

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

  20. A synonymous change, p.Gly16Gly in MECP2 Exon 1, causes a cryptic splice event in a Rett syndrome patient

    PubMed Central

    2013-01-01

    Background Mutations in MECP2 are the main cause of Rett Syndrome. To date, no pathogenic synonymous MECP2 mutation has yet been identified. Here, we investigated a de novo synonymous variant c.48C>T (p.Gly16Gly) identified in a girl presenting with a typical RTT phenotype. Methods In silico analyses to predict the effects of sequence variation on mRNA splicing were employed, followed by sequencing and quantification of lymphocyte mRNAs from the subject for splice variants MECP2_E1 and MECP2_E2. Results Analysis of mRNA confirmed predictions that this synonymous mutation activates a splice-donor site at an early position in exon 1, leading to a deletion (r.[=, 48_63del]), codon frameshift and premature stop codon (p.Glu17Lysfs*16) for MECP2_E1. For MECP2_E2, the same premature splice site is used, but as this is located in the 5′untranslated region, no effect on the amino acid sequence is predicted. Quantitative analysis that specifically measured this cryptic splice variant also revealed a significant decrease in the quantity of the correct MECP2_E1 transcript, which indicates that this is the etiologically significant mutation in this patient. Conclusion These findings suggest that synonymous variants of MECP2 as well as other known disease genes—and de novo variants in particular— should be re-evaluated for potential effects on splicing. PMID:23866855

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    van Santen, V L; Spritz, R A

    1986-01-01

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

  3. 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.

  4. 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

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

    PubMed

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

    2013-06-01

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

  6. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. 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; Berg, 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. PMID:19959335

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  12. 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.

  13. 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 N? of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N? in other quinone-processing sites. The experiments with 15N showed that the N? 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 N? 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 0.13. 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

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

    PubMed Central

    2013-01-01

    Background Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC + K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. Results Healing time was reduced from 13.9 ± 0.5 days (mean ± SEM) in the control group to 7.2 ± 0.2 days in the PC group (P < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01). Conclusion The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. Clinical trial registry information Protocol Record Identification Number: 132/03 Registry URL: http://www.clinicaltrials.gov PMID:23570605

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

    PubMed

    Thomassen, Mads; Blanco, Ana; Montagna, Marco; Hansen, Thomas V O; Pedersen, Inge S; Gutirrez-Enrquez, Sara; Menndez, Mireia; Fachal, Laura; Santamaria, Marta; Steffensen, Ane Y; Jnson, Lars; Agata, Simona; Whiley, Phillip; Tognazzo, Silvia; Tornero, Eva; Jensen, Uffe B; Balmaa, Judith; Kruse, Torben A; Goldgar, David E; Lzaro, Conxi; Diez, Orland; Spurdle, Amanda B; Vega, Ana

    2012-04-01

    Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management. PMID:21769658

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

    PubMed Central

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

    1984-01-01

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

  17. 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.

  18. Minor splicing pathway is not minor any more: implications for the pathogenesis of motor neuron diseases.

    PubMed

    Onodera, Osamu; Ishihara, Tomohiko; Shiga, Atsushi; Ariizumi, Yuko; Yokoseki, Akio; Nishizawa, Masatoyo

    2014-02-01

    To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate-rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases. PMID:24112438

  19. 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

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

    PubMed Central

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

    1995-01-01

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

  1. 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

  2. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

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

  3. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  6. The role of U2AF35 and U2AF65 in enhancer-dependent splicing.

    PubMed Central

    Graveley, B R; Hertel, K J; Maniatis, T

    2001-01-01

    Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35. PMID:11421359

  7. Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

    PubMed Central

    Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M.

    2010-01-01

    CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. PMID:20558599

  8. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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. PMID:26578562

  11. 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

  12. Splicing shielded cables

    NASA Technical Reports Server (NTRS)

    Lind, W. P.; Mcgougan, W. R.

    1979-01-01

    Simple repair technique retains cable characteristic impedance. Shielded-Cable Splicing Technique retains cable characteristic impedance. Procedure involves splicing inner conductors in staggered pattern, installing jumper braid by heat-shrinking two solder sleeves, and placing insulation sleeve over repaired section and heat-shrinking it. Two possible insulation materials are modified polyvinylidene fluoride and polytetrafluoroethylene.

  13. Splicing code modeling.

    PubMed

    Barash, Yoseph; Vaquero-Garcia, Jorge

    2014-01-01

    How do cis and trans elements involved in pre-mRNA splicing come together to form a splicing "code"? This question has been a driver of much of the research involving RNA biogenesis. The variability of splicing outcome across developmental stages and between tissues coupled with association of splicing defects with numerous diseases highlights the importance of such a code. However, the sheer number of elements involved in splicing regulation and the context-specific manner of their operation have made the derivation of such a code challenging. Recently, machine learning-based methods have been developed to infer computational models for a splicing code. These methods use high-throughput experiments measuring mRNA expression at exonic resolution and binding locations of RNA-binding proteins (RBPs) to infer what the regulatory elements that control the inclusion of a given pre-mRNA segment are. The inferred regulatory models can then be applied to genomic sequences or experimental conditions that have not been measured to predict splicing outcome. Moreover, the models themselves can be interrogated to identify new regulatory mechanisms, which can be subsequently tested experimentally. In this chapter, we survey the current state of this technology, and illustrate how it can be applied by non-computational or RNA splicing experts to study regulation of specific exons by using the AVISPA web tool. PMID:25201114

  14. Micromachined multifiber optic splices

    NASA Astrophysics Data System (ADS)

    Sellers, Gregory J.; Roth, Richard F.

    1995-05-01

    A multifiber mechanical splice concept has been developed for simultaneous mechanical joining of optical fibers in cable. This splice uses a novel 'multifiber positioner' to secure and precisely position the mating pairs of individual optical fibers to achieve excellent light throughput characteristics. The multifiber positioner is a micromachined structure that includes multiple V-grooves in silicon created via anisotropic etching. This concept has been used to create a 4-channel optical splice suitable for aircraft application. The multifiber positioner is mounted on a flexible polymeric 'elevator' and housed within a protective metal shell. The splice and shell design have been developed to facilitate user-friendly operation. The shell assembly includes a novel method for cable attachment and provides an environmental seal. The splice design concept should allow practical field use in adverse conditions.

  15. 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.

  16. Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease.

    PubMed

    Cogn, M; Bakhshi, A; Korsmeyer, S J; Guglielmi, P

    1988-09-01

    The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells. PMID:3137264

  17. Multifunctional photopolymerized semiinterpenetrating network (sIPN) system containing bupivacaine and silver sulfadiazine is an effective donor site treatment in a swine model.

    PubMed

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

    2010-01-01

    Previously, we have shown in a cross-comparison study that multifunctional photopolymerized semiinterpenetrating 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 with the control site through qualitative clinical evaluation and to compare the rate and quality of donor site healing through histologic evaluation. Eight Yucatan swine of 40 lbs each were randomly divided into four groups of two pigs before surgery. Each animal had 5.6% TBSA 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 cm(2) 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 cm(2) of autograft, which was used to divide the test areas. Wound assessment and killing occurred at days 7, 9, 14, and 21. A full-thickness biopsy was taken from each of the study areas for histological analysis. By 14 days, all areas showed complete epidermal coverage histologically. The 30/1000th-inch site revealed a thicker, more irregular dermis compared with 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 clinical or histological evaluations of any treatment. The addition of bupivacaine and silver sulfadiazine to sIPN does not show any alterations in wound healing of a donor site in a swine model when compared with sIPN without loaded drugs and a standard control dressing. This efficacy may be coupled with established localized sIPN drug delivery profiles and allow further studies to evaluate the efficacy of these drugs to promote healing, eradicate and prevent infection, and manage pain. PMID:20061849

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

    PubMed

    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

    2016-02-01

    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 = 0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age. PMID:26913521

  19. 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 = 0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age. PMID:26913521

  20. Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

    Kim, Jeong Tae; Kim, Sang Wha; Youn, Seungki; Kim, Youn Hwan

    2015-07-01

    Microsurgical free tissue transfer is regarded as the best available method of tissue reconstruction for intractable defects. The ideal soft tissue flap is thought to be the anterolateral thigh flap. On the basis of 334 procedures involving the latissimus dorsi (LD) flap, we discuss the advantages of the LD flap over the current universal option, and we aimed to establish whether the LD could also gain universal status in all reconstructive fields.Three hundred thirty-four reconstructive procedures using the LD flap were performed in 322 patients between September 2002 and July 2012. In accordance with defect characteristics, we performed 334 procedures using flaps, which included the LD muscle flap with skin graft, the myocutaneous flap, the muscle-sparing flap, the perforator flap, the chimeric flap, and the 2-flap technique using the serratus anterior branch.Flap-related complications occurred in 21 patients (6.3%), including total and partial flap failure. In 253 cases, the donor site was closed primarily, and in the remaining cases, we used split-thickness skin grafts. Donor-site complications occurred in 20 cases (6%). In 11 of the 182 cases, no suitable perforators were identified during surgery.The advantages of the LD as a donor site include the possibility of various harvesting positions without position change, versatility of components, availability of muscle to fill extensive defects, and presence of thick fascia to enable full abdominal reconstruction. On the basis of our experience, we concluded that this flap has the potential to be used as widely as, or in preference to, the anterolateral thigh flap in most reconstructive areas. PMID:25785382

  3. 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

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

    PubMed

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

    2014-10-01

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

  5. Exon circularization requires canonical splice signals.

    PubMed

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

    2015-01-01

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

  6. Rapid splicing and stepwise processing of a transcript from the psbB operon in tobacco chloroplasts: determination of the intron sites in petB and petD.

    PubMed

    Tanaka, M; Obokata, J; Chunwongse, J; Shinozaki, K; Sugiura, M

    1987-10-01

    Expression of the psbB gene cluster in tobacco chloroplasts has been studied. This cluster contains the genes for the 51 kDa chlorophyll a apoprotein (psbB) and the 10 kDa phosphoprotein (psbH) of the photosystem II, and cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in this order. Northern blot hybridization and reverse transcription analyses have revealed that petB and petD contain single introns and the psbB gene cluster is transcribed as a single polycistronic unit. The primary transcript seems to be spliced very rapidly and then processed into several small RNA species. The exact splice sites have been located by cDNA sequencing. The transcriptional initiation site of the psbB operon has been determined by S1 mapping with in vitro capped chloroplast RNA. The stepwise processing of chloroplast RNA precursors is discussed. PMID:17193705

  7. RNA helicases in splicing

    PubMed Central

    Cordin, Olivier; Beggs, Jean D.

    2013-01-01

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

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

    PubMed Central

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

    1993-01-01

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

  9. Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3′ splice-site organization and activity of U2AF-related proteins

    PubMed Central

    Kralovicova, Jana; Knut, Marcin; Cross, Nicholas C. P.; Vorechovsky, Igor

    2015-01-01

    The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3′ splice site (3′ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3′ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3′UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing. PMID:25779042

  10. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    SciTech Connect

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. ); Schweikert, H.U. ); Zegers, N.D. ); Hodgins, M.B. )

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  11. A nonionic inhibitor with high specificity for the UDP-Gal donor binding site of human blood group B galactosyltransferase: design, synthesis, and characterization.

    PubMed

    Schaefer, Katrin; Sindhuwinata, Nora; Hackl, Thomas; Ktzler, Miriam P; Niemeyer, Felix C; Palcic, Monica M; Peters, Thomas; Meyer, Bernd

    2013-03-14

    9-(5-O-?-D-galactopyranosyl)-D-arabinityl-1,3,7-trihydropurine-2,6,8-trione (1) was designed and synthesized as a nonionic inhibitor for the donor binding site of human blood group B galactosyltransferase (GTB). Enzymatic characterization showed 1 to be extremely specific, as the highly homologous human N-acetylgalactosaminyltransferase (GTA) is not inhibited. The binding epitope of 1 demonstrates a high involvement of the arabinityl linker, whereas the galactose residue is only making contact to the protein via its C-2 site, which is very important for the discrimination between galactose and N-acetylgalactosamine, the substrate transferred by GTA. The approach can generate highly specific glycosyltransferase inhibitors. PMID:23406460

  12. Alternative splicing in C. elegans.

    PubMed Central

    Zahler, Alan M

    2005-01-01

    Alternative splicing is a common mechanism for the generation of multiple isoforms of proteins. It can function to expand the proteome of an organism and can serve as a way to turn off gene expression post-transcriptionally. This review focuses on splicing and its regulation in C. elegans. The fully-sequenced C. elegans genome combined with its elegant genetics offers unique advantages for exploring alternative splicing regulation in metazoans. The topics covered in this review include constitutive splicing factors, identification of alternatively spliced genes, examples of alternative splicing in C. elegans, and alternative splicing regulation. Key genes whose regulated alternative splicing are reviewed include let-2, unc-32, unc-52, egl-15 and xol-1. Factors involved in alternative splicing that are discussed include mec-8, smu-1, smu-2, fox-1, exc-7 and unc-75. PMID:18050427

  13. An Intronic Enhancer Regulates Splicing of the Twintron of Drosophila melanogaster prospero Pre-mRNA by Two Different Spliceosomes

    PubMed Central

    Scamborova, Petra; Wong, Anthony; Steitz, Joan A.

    2004-01-01

    We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5′ splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes. PMID:14966268

  14. Multifunctional in situ photopolymerized semi-interpenetrating network system is an effective donor site dressing: a cross comparison study in a swine model.

    PubMed

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

    2009-01-01

    Effective dressings for donor sites or other partial thickness wounds must promote removal of nonviable or necrotic tissue, eradication and prevention of microbial infiltrate, exudate absorbance, and regrowth of healthy epidermis and dermis. There are many commonly used products that facilitate these processes. Established properties of an in situ photopolymerizable semi-interpenetrating network (sIPN) suggest that it is also a viable treatment option. The widely varying material properties suggest that these dressing treatments may elicit different healing responses via different cellular mechanisms. In this study, we sought to resolve the differences in healing between Acticoat, sIPN, nonadherent dressing with Tisseel, and Xeroform dressing treatments in a porcine partial thickness wound model. Donor site wounds were produced on pigs at two cut depths and dressed with Acticoat, sIPN, nonadherent dressing with Tisseel, and Xeroform with alternatively placed autografts to provide a control area between each test site. Pigs were euthanized at 4, 7, 14, and 42 days for macroscopic examination and biopsy collection. Biopsies were analyzed histologically by two blinded observers for cellular densities and regional thicknesses within the tissue. sIPN- and Xeroform-treated wounds were healed by 7 days, and Acticoat- and nonadherent dressing with Tisseel-treated wounds were healed by 14 days. Inflammatory responses were between comparable treatment type across all time periods. Dermal granulation features increased with time but were not significantly different. All dressing treatments elicited wound healing without outstanding toxicity or pathology indicating that sIPN is a comparable and viable treatment for partial thickness wounds. PMID:19131760

  15. Novel splice-site mutation in WDR62 revealed by whole-exome sequencing in a Sudanese family with primary microcephaly.

    PubMed

    Bastaki, Fatma; Mohamed, Madiha; Nair, Pratibha; Saif, Fatima; Tawfiq, Nafisa; Aithala, Gururaj; El-Halik, Majdi; Al-Ali, Mahmoud; Hamzeh, Abdul Rezzak

    2016-05-01

    The WDR62 gene encodes a scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. It plays a critical role in laying out various cellular layers in the cerebral cortex during embryogenesis, and hence the dramatic clinical features resulting from WDR62 mutations. These mutations are associated with autosomal recessive primary microcephaly 2, with or without cortical malformations (MCPH2). Using whole exome sequencing we uncovered a novel WDR62 variant; c.390G > A, from two Sudanese siblings whose parents are first cousins. The patients suffered MCPH2 with incomplete lissencephaly and developmental delay. The mutation affects the last nucleotide of exon4, and probably leads to aberrant splicing, which may result in a truncated protein lacking all functional domains. PMID:26577670

  16. 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.

  17. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae

    PubMed Central

    Schreiber, Konrad; Csaba, Gergely; Haslbeck, Martin; Zimmer, Ralf

    2015-01-01

    mRNA splicing is required in about 4% of protein coding genes in Saccharomyces cerevisiae. The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. PMID:26469855

  18. 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

  19. A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571+1G>A) leading to excision of exon 3

    PubMed Central

    Frugier, Tony; Mitchell, Nadia L.; Tammen, Imke; Houweling, Peter J.; Arthur, Donald G.; Kay, Graham W.; van Diggelen, Otto P.; Jolly, Robert D.; Palmer, David N.

    2008-01-01

    Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To date eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571+G>A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies. PMID:17988881

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

    PubMed Central

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

    2008-01-01

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

  1. Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.

    PubMed

    Ramalho, Anabela S; Clarke, Luka A; Sousa, Marisa; Felicio, Verónica; Barreto, Celeste; Lopes, Carlos; Amaral, Margarida D

    2016-01-01

    The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18nts of exon 22 which produce a truncated CFTR protein with residual Cl(-) channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis. PMID:25735457

  2. 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

  3. Quantification of stochastic noise of splicing and polyadenylation in Entamoeba histolytica.

    PubMed

    Hon, Chung-Chau; Weber, Christian; Sismeiro, Odile; Proux, Caroline; Koutero, Mikael; Deloger, Marc; Das, Sarbashis; Agrahari, Mridula; Dillies, Marie-Agnes; Jagla, Bernd; Coppee, Jean-Yves; Bhattacharya, Alok; Guillen, Nancy

    2013-02-01

    Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited. Lastly, we revised the gene models and annotated their 3'UTR in AmoebaDB, providing valuable resources to the community. PMID:23258700

  4. Quantification of stochastic noise of splicing and polyadenylation in Entamoeba histolytica

    PubMed Central

    Hon, Chung-Chau; Weber, Christian; Sismeiro, Odile; Proux, Caroline; Koutero, Mikael; Deloger, Marc; Das, Sarbashis; Agrahari, Mridula; Dillies, Marie-Agnes; Jagla, Bernd; Coppee, Jean-Yves; Bhattacharya, Alok; Guillen, Nancy

    2013-01-01

    Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited. Lastly, we revised the gene models and annotated their 3′UTR in AmoebaDB, providing valuable resources to the community. PMID:23258700

  5. Effect of Schneiderian membrane perforation on sinus lift graft outcome using two different donor sites: a retrospective study of 105 maxillary sinus elevation procedures

    PubMed Central

    Sakkas, Andreas; Konstantinidis, Ioannis; Winter, Karsten; Schramm, Alexander; Wilde, Frank

    2016-01-01

    Background: Sinuslift is meanwhile an established method of bone augmentation in the posterior maxilla. Aim of the study was to evaluate the significance of intraoperative Schneiderian membrane perforations during maxillary sinus floor elevation surgery using autogenous bone harvested from two different donor sites using a Safescraper device on the success rate, graft survival and implant integration. Methods: The investigators conducted a retrospective cohort study at the Department of Oral and Maxillofacial Surgery of Military Hospital Ulm composed of patients with severe maxillary atrophy who underwent sinus augmentation from January 2011 until December 2011. Ninety-nine consecutive patients (89 men, 10 women) with a mean age of 43.1 years underwent sinus graft procedures in a 2-stage procedure using the lateral wall approach, as described by Tatum (1986). Data on patient age, smoking status, donor site and surgical complications were recorded and the relationship between Schneiderian membrane perforation and complication rate was evaluated. Dental implants were inserted 4 months after grafting. Results: A total of 105 sinus lift procedures were performed in 99 patients. Sixty-one patients (61.6%) underwent sinus elevation with autogenous bone from the buccal sinus wall, while 38 patients (38.4%) bone harvesting from the iliac crest. Intraoperative perforation of the Schneiderian membrane was observed in 11 of the 105 sinuses (10.4%). These perforations resulted in 4 (36.3%) of the cases in major postoperative complications accompanied by swelling and wound infection. Membrane perforations were slightly associated with the appearance of postoperative complications (p=0.0762). In 2.4% of all cases, regarding 2 patients the final rehabilitation with dental implants was not possible because of extensive bone resorption. Conclusion: Intraoperative complications performing sinus augmentation may lead to postoperative complications. With careful clinical and radiographic evaluation and appropriate treatment, the complications and risk for graft material displacement and implant loss can be eliminated. PMID:26955510

  6. Genome-wide discovery of human splicing branchpoints

    PubMed Central

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

    2015-01-01

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

  7. 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.

  8. 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.

  9. cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control.

    PubMed Central

    Katz, R A; Kotler, M; Skalka, A M

    1988-01-01

    The full-length retroviral RNA transcript serves as (i) mRNA for the gag and pol gene products, (ii) genomic RNA that is assembled into progeny virions, and (iii) a pre-mRNA for spliced subgenomic mRNAs. Therefore, a balance of spliced and unspliced RNA is required to generate the appropriate levels of protein and RNA products for virion production. We have introduced an insertion mutation near the avian sarcoma virus env splice acceptor site that results in a significant increase in splicing to form functional env mRNA. The mutant virus is replication defective, but phenotypic revertant viruses that have acquired second-site mutations near the splice acceptor site can be isolated readily. Detailed analysis of one of these viruses revealed that a single nucleotide change at -20 from the splice acceptor site, within the original mutagenic insert, was sufficient to restore viral growth and significantly decrease splicing efficiency compared with the original mutant and wild-type viruses. Thus, minor sequence alterations near the env splice acceptor site can produce major changes in the balance of spliced and unspliced RNAs. Our results suggest a mechanism of control in which splicing is modulated by cis-acting sequences at the env splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing control. Images PMID:2839694

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

    PubMed

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

    2015-06-01

    This prospective, randomised study compares a new transforming methacrylate dressing (TMD) with a silver-containing carboxymethylcellulose dressing (CMC-Ag) after application to split-thickness skin graft (STSG) donor sites. This was an unblinded, non-inferiority, between-patient, comparison study that involved patients admitted to a single-centre burn unit who required two skin graft donor sites. Each patient's donor sites were covered immediately after surgery: one donor site with TMD and the other with CMC-Ag. The donor sites were evaluated until healing or until 24 days post-application, whichever came first. Study endpoints were time to healing, daily pain scores, number of dressing changes, patient comfort and physicians' and patients' willingness to use the dressings in the future. Nineteen patients had both the dressings applied. No statistically significant difference was noted in time to healing between the two dressings (14·2 days using TMD compared with 13·2 days using CMC-Ag). When pain scores were compared, TMD resulted in statistically significantly less pain at three different time periods (2-5 days, 6-10 days and 11-15 days; P < 0·001 at all time periods). Patients also reported greater comfort with TMD (P < 0·001). Users rated TMD as being less easy to use because of the time and technique required for application. Reductions in pain and increased patient comfort with the use of the TMD dressing, compared with CMC-Ag, were seen as clinical benefits as these are the major issues in donor site management. PMID:23919667

  11. Comprehensive prediction of mRNA splicing effects of BRCA1 and BRCA2 variants.

    PubMed

    Mucaki, Eliseos J; Ainsworth, Peter; Rogan, Peter K

    2011-07-01

    Variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes potentially affecting coding sequence as well as normal splicing activity have confounded predisposition testing in breast cancer. Here, we apply information theory to analyze BRCA1/2 mRNA splicing mutations categorized as VUS. The method was validated for 31 of 36 mutations known to cause missplicing in BRCA1/2 and all 26 that do not alter splicing. All single-nucleotide variants in the Breast Cancer Information Resource (BIC; Breast Cancer Information Core Database; http://research.nhgri.nih.gov/bic; last access June 1, 2010) were then analyzed. Information analysis is similar in sensitivity to other predictive methods; however, the thermodynamic basis of the theory also enables splice-site affinity to be determined accurately, which is important for assessing mutations that render natural splice sites partially functional and competition between cryptic and natural splice sites. We report 299 of 2,071 single-nucleotide BIC mutations that are predicted to significantly weaken natural sites and/or strengthen cryptic splice sites, 171 of which are not designated as splicing mutations in the database. Splicing alterations are predicted for 68 of 690 BRCA1 and 60 of 958 BRCA2 mutations designated as VUS. These analyses should be useful in prioritizing suspected mutations for downstream expression studies and for predicting aberrantly spliced isoforms generated by these mutations. PMID:21523855

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

    PubMed Central

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

    2013-01-01

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

  13. Role of Acinus in regulating retinoic acid-responsive gene pre-mRNA splicing.

    PubMed

    Wang, Fang; Soprano, Kenneth J; Soprano, Dianne Robert

    2015-04-01

    Acinus-S' is a corepressor 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

  14. 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

  15. Functional studies on the ATM intronic splicing processing element

    PubMed Central

    Lewandowska, Marzena A.; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E.; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing. PMID:16030351

  16. 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.

  17. Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR

    PubMed Central

    Estrada, April D.; Freese, Nowlan H.; Blakley, Ivory C.

    2015-01-01

    Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results. PMID:25945312

  18. 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

  19. 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.

  20. Marshall syndrome associated with a splicing defect at the COL11A1 locus.

    PubMed Central

    Griffith, A J; Sprunger, L K; Sirko-Osadsa, D A; Tiller, G E; Meisler, M H; Warman, M L

    1998-01-01

    Marshall syndrome is a rare, autosomal dominant skeletal dysplasia that is phenotypically similar to the more common disorder Stickler syndrome. For a large kindred with Marshall syndrome, we demonstrate a splice-donor-site mutation in the COL11A1 gene that cosegregates with the phenotype. The G+1-->A transition causes in-frame skipping of a 54-bp exon and deletes amino acids 726-743 from the major triple-helical domain of the alpha1(XI) collagen polypeptide. The data support the hypothesis that the alpha1(XI) collagen polypeptide has an important role in skeletal morphogenesis that extends beyond its contribution to structural integrity of the cartilage extracellular matrix. Our results also demonstrate allelism of Marshall syndrome with the subset of Stickler syndrome families associated with COL11A1 mutations. PMID:9529347

  1. 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.

  2. Hydrogen bonds between nitrogen donors and the semiquinone in the QB-site of bacterial reaction centers

    PubMed Central

    Martin, Erik; Samoilova, Rimma I.; Narasimhulu, Kupala V.; Wraight, Colin A.; Dikanov, Sergei A.

    2010-01-01

    Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (QA) and secondary (QB) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the QB site semiquinone with nitrogens from the local protein environment in natural and 15N uniformly labeled reactions centers. 14N and 15N HYSCORE spectra of the QB semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from 14N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. 15N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen, and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the QB semiquinone forms hydrogen bonds with two nitrogens, and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the QB site. Based on the quadrupole coupling constant, one nitrogen can only be assigned to N? of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen. PMID:20672818

  3. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    PubMed Central

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  4. Recovery and safety profiles of marrow and PBSC donors: experience of the National Marrow Donor Program.

    PubMed

    Miller, John P; Perry, Elizabeth H; Price, Thomas H; Bolan, Charles D; Karanes, Chatchada; Boyd, Theresa M; Chitphakdithai, Pintip; King, Roberta J

    2008-09-01

    The National Marrow Donor Program (NMDP) has been facilitating hematopoietic cell transplants since 1987. Volunteer donors listed on the NMDP Registry may be asked to donate either bone marrow (BM) or peripheral blood stem cells (PBSC); however, since 2003, the majority of donors (72% in 2007) have been asked to donate PBSC. From the donor's perspective these stem cell sources carry different recovery and safety profiles. The majority of BM and PBSC donors experienced symptoms during the course of their donation experience. Pain is the number 1 symptom for both groups of donors. BM donors most often reported pain at the collection site (82% back or hip pain) and anesthesia-related pain sites (33% throat pain; 17% post-anesthesia headache), whereas PBSC donors most often reported bone pain (97%) at various sites during filgrastim administration. Fatigue was the second most reported symptom by both BM and PBSC donors (59% and 70%, respectively). PBSC donors reported a median time to recovery of 1 week compared to a median time to recovery of 3 weeks for BM donors. Both BM and PBSC donors experienced transient changes in their WBC, platelet, and hemoglobin counts during the donation process, with most counts returning to baseline values by 1 month post-donation and beyond. Serious adverse events are uncommon, but these events occurred more often in BM donors than PBSC donors (1.34% in BM donors, 0.6% in PBSC donors) and a few BM donors may have long-term complications. NMDP donors are currently participating in a randomized clinical trial that will formally compare the clinical and quality-of-life outcomes of BM and PBSC donors and their graft recipients. PMID:18721778

  5. BUILDING ROBUST TRANSCRIPTOMES WITH MASTER SPLICING FACTORS

    PubMed Central

    Jangi, Mohini; Sharp, Phillip A.

    2014-01-01

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

  6. Purifying Selection on Exonic Splice Enhancers in Intronless Genes.

    PubMed

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  7. 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

  8. Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy

    PubMed Central

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

    2013-01-01

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

  9. Genome-wide survey of Alternative Splicing in Sorghum Bicolor.

    PubMed

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

    2014-07-01

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

  10. Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin

    PubMed Central

    Mallinjoud, Pierre; Villemin, Jean-Philippe; Mortada, Hussein; Polay Espinoza, Micaela; Desmet, François-Olivier; Samaan, Samaan; Chautard, Emilie; Tranchevent, Léon-Charles; Auboeuf, Didier

    2014-01-01

    Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type–specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution. PMID:24307554

  11. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels.

    PubMed

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

    2002-07-01

    Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels. PMID:12097481

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

    PubMed Central

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

    2011-01-01

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

  13. Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase.

    PubMed

    Morra, Simone; Giraudo, Alberto; Di Nardo, Giovanna; King, Paul W; Gilardi, Gianfranco; Valetti, Francesca

    2012-01-01

    [FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k(cat) only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O(2) diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site. PMID:23133586

  14. Management of Young Blood Donors

    PubMed Central

    Newman, Bruce H.

    2014-01-01

    Summary The emphasis on high-school blood drives and acceptance of 16-year-old blood donors led to more research on physiologic and psychological ways to decrease vasovagal reaction rates in young blood donors and to increase donor retention. Research on how to accomplish this has been advantageous for the blood collection industry and blood donors. This review discussed the current situation and what can be done psychologically, physiologically, and via process improvements to decrease vasovagal reaction rates and increase donor retention. The donation process can be significantly improved. Future interventions may include more dietary salt, a shorter muscle tension program to make it more feasible, recommendations for post-donation muscle tension / squatting / laying down for lightheadedness, more donor education by the staff at the collection site, more staff attention to donors with fear or higher risk for a vasovagal reaction (e.g. estimated blood volume near 3.5 l, first-time donor), and a more focused donation process to ensure a pleasant and safer procedure. PMID:25254024

  15. 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.

  16. Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA.

    PubMed

    Shaked, Hadassa; Wachtel, Chaim; Tulinski, Pawel; Yahia, Nasreen Hag; Barda, Omer; Darzynkiewicz, Edward; Nilsen, Timothy W; Michaeli, Shulamit

    2010-06-01

    In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome. PMID:20159996

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

    PubMed

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

    2014-04-28

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

  18. Unusual Phenotypic Features in a Patient with a Novel Splice Mutation in the GHRHR Gene

    PubMed Central

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (−5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  19. Unusual phenotypic features in a patient with a novel splice mutation in the GHRHR gene.

    PubMed

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (-5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  20. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  1. 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

  2. Anatomical study of the greater palatine artery and related structures of the palatal vault: considerations for palate as the subepithelial connective tissue graft donor site.

    PubMed

    Klosek, Sebastian Krystian; Rungruang, Thanaporn

    2009-04-01

    Palate is considered as a tissue graft donor site for dental surgical procedures. Therefore, the aim of this study was to investigate the anatomy of palatal structures, such as greater palatine artery, greater palatine foramen, and incisive fossa, in order to consider their topography at planning the graft dimensions and reduce the potential risk of injury of greater palatine artery. Direct inspection of 41 Thai cadavers was performed. The results showed the statistically significant differences as for the length of female and male palates (p = 0.017); however, vertical measurements were equally distributed in examined population. Main location of greater palatine foramen was palatal to the second molar (35.7%), as well as, interproximal to the second and third molars (35.7%) in women, and palatal to the second molar in men (65%). GPA was branching most frequently at the level of first premolar (38%) and at first and second molars together (43%) in women. In men, the branching on the alveolar process side was commonly observed at the level of first and second premolars together (56%), and at the level of second and third molars together (32%). In the area between maxillary first premolar and second molar, it appeared possible to harvest a connective tissue graft measuring at least 5 mm in height. The results of this research will provide the useful data for other comparative studies and for assisting periodontologists in planning the dimensions and harvesting the subepithelial connective tissue grafts from palate. PMID:19015806

  3. A Systematic Review of Outcomes of Contralateral C7 for the Treatment of Traumatic Brachial Plexus Injury: Part 2-Donor-site morbidity of contralateral C7 transfer for traumatic brachial plexus injury

    PubMed Central

    Yang, Guang; Chang, Kate W.-C.; Chung, Kevin C.

    2016-01-01

    Background Although contralateral C7 (CC7) transfer has been widely used for treating traumatic brachial plexus injury, the procedure safety is questionable. We performed a systematic review to investigate the donor-site morbidity including sensory abnormality and motor deficit to guide clinical decision-making. Methods A systematic review on CC7 transfer for traumatic brachial plexus injury was performed for the original articles in PubMed and EMBASE databases. Patient demographic data and donor-site morbidity of CC7 transfer including incidence, recovery rate and time were extracted from the included studies. The sensory abnormality areas and muscles involved in motor weakness were also summarized. Results A total 904 patients from 27 studies were reviewed. Overall, 74% of patients (668/897) experienced sensory abnormalities, and 98% of patients (618/633) recovered to normal; the mean recovery time was 3 months. For motor function, 20% of patients (118/592) had motor deficit after CC7 transfer and 91% (107/117) regained normal motor functions; mean recovery time was 6 months. Sensory abnormality mainly happened in the median innervated area of the hand, whereas motor deficit most often involved radial nerve innerved muscles. There were 2% of patients (19/904) with long-term morbidity of donor site in the studies. Conclusions The incidence of donor-site morbidity after CC7 transfer was relatively high, and severe and long-term defects occurred occasionally. CC7 transfer should be indicated only when other donor nerves are not available, and with a comprehensive knowledge of the potential risks. Level of evidence Level III PMID:26397267

  4. Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein.

    PubMed

    Guglielmi, P; Bakhshi, A; Cogne, M; Seligmann, M; Korsmeyer, S J

    1988-09-01

    Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein. PMID:3137265

  5. Reinforcement of a minor alternative splicing event in MYO7A due to a missense mutation results in a mild form of retinopathy and deafness

    PubMed Central

    Ben Rebeh, Imen; Morinière, Madeleine; Ayadi, Leila; Benzina, Zeineb; Charfedine, Ilhem; Feki, Jamel; Ayadi, Hammadi; Ghorbel, Abdelmonem; Baklouti, Faouzi

    2010-01-01

    Purpose Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf–blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the type and location of the MYO7A mutations corresponding to the USH1B and DFNB2 phenotype. We investigated the molecular determinant of a mild form of retinopathy in association with a subtle splicing modulation of MYO7A mRNA. Methods Affected members underwent detailed audiologic and ocular characterization. DNA samples from family members were genotyped with polymorphic microsatellite markers. Sequencing of MYO7A was performed. Endogenous lymphoid RNA analysis and a splicing minigene assay were used to study the effect of the c.1935G>A mutation. Results Funduscopy showed mild retinitis pigmentosa in adults with HL. Microsatellite analysis showed linkage to markers in the region on chromosome 11q13.5. Sequencing of MYO7A revealed a mutation in the last nucleotide of exon 16 (c.1935G>A), which corresponds to a substitution of a methionine to an isoleucine residue at amino acid 645 of the myosin VIIA. However, structural prediction of the molecular model of myosin VIIA shows that this amino acid replacement induces only minor structural changes in the immediate environment of the mutation and thus does not alter the overall native structure. We found that, although predominantly included in mature mRNA, exon 16 is in fact alternatively spliced in control cells and that the mutation at the very last position is associated with a switch toward a predominant exclusion of that exon. This observation was further supported using a splicing minigene transfection assay; the c.1935G>A mutation was found to trigger a partial impairment of the adjacent donor splice site, suggesting that the unique change at the last position of the exon is responsible for the enhanced exon exclusion in this family. Conclusions This study shows how an exonic mutation that weakens the 5′ splice site enhances a minor alternative splicing without abolishing a complete exclusion of the exon and therefore causes a less severe retinitis pigmentosa than the USH1B-associated alleles. It would be interesting to examine a possible correlation between intrafamilial phenotypic variability and the subtle variation in exon 16 inclusion, probably related to genetic background specificities. PMID:21031134

  6. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

  7. 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.

  8. Splicing of aged fibers

    NASA Astrophysics Data System (ADS)

    Volotinen, Tarja T.; Yuce, Hakan H.; Bonanno, Nicholas; Frantz, Rolf A.; Duffy, Sean

    1993-11-01

    The deployment of fiber in the subscriber loop will require that an optical fiber network maintain the highest possible level of reliability over time, despite being subjected to extremes of temperature, humidity, and other environmental and mechanical stresses imposed on the outside plant. At the same time, both the initial cost and the ongoing maintenance expenses for loop equipment must be kept low. Fiber in the Loop (FITL) applications will entail increased fiber handling. Cable lengths will be shorter, and fiber counts higher, than has been the case so far in long-distance applications. There will also be more cable sheath openings per unit length of cable and/or fiber, as well as more splicing and connectorization. It may become a common practice that a customer is connected to a cable installed many years earlier. In subscriber loops, cables and fibers will be installed in harsher and more varying environments. Fibers will be exposed to higher humidity and temperature, particularly in splice boxes mounted on building walls, in pedestal cabinets, and in other similar enclosures. Corrosive gases and/or liquids may also be present at some locations and will adversely affect the fibers. The combination of increased handling, greater exposure, and more stressful environments may give rise to a need for new, more stringent requirements for fiber mechanical reliability. These can include increaSed fiber strength, increased aging resistance, and increased fatigue resistance.

  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. SF3B1 association with chromatin determines splicing outcomes.

    PubMed

    Kfir, Nir; Lev-Maor, Galit; Glaich, Ohad; Alajem, Adi; Datta, Arnab; Sze, Siu K; Meshorer, Eran; Ast, Gil

    2015-04-28

    Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. PMID:25892229

  11. Modulation of Splicing by Single-Stranded Silencing RNAs.

    PubMed

    Liu, Jing; Hu, Jiaxin; Hicks, Jessica A; Prakash, Thazha P; Corey, David R

    2015-06-01

    Single-stranded silencing RNAs (ss-siRNAs) are chemically modified single-stranded oligonucleotides that can function through the cellular RNA interference (RNAi) machinery to modulate gene expression. Because their invention is recent, few studies have appeared describing their use and the potential of ss-siRNAs as a platform for controlling gene expression remains largely unknown. Using oligonucleotides to modulate splicing is an important area for therapeutic development and we tested the hypothesis that ss-siRNAs targeting splice sites might also be capable of directing increased production of therapeutically promising protein isoforms. Here we observe that ss-siRNAs alter splicing of dystrophin. Altered splicing requires a seed sequence complementarity to the target and expression of the RNAi factor argonaute 2. These results demonstrate that ss-siRNAs can be used to modulate splicing, providing another option for therapeutic development programs that aim to increase production of key protein isoforms. Splicing is a classical nuclear process and our data showing that it can be modulated through the action of RNA and RNAi factors offers further evidence that RNAi can take place in mammalian cell nuclei. PMID:25757055

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

    PubMed

    Papasaikas, Panagiotis; Tejedor, J Ramn; Vigevani, Luisa; Valcrcel, Juan

    2015-01-01

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

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

    PubMed

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

    2015-03-17

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

  15. 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

  16. Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...

  17. LDLR gene synonymous mutation c.1813C>T results in mRNA splicing variation in a kindred with familial hypercholesterolaemia.

    PubMed

    Ho, Clement K M; Musa, Fathel Rahman; Bell, Christine; Walker, Simon W

    2015-11-01

    Familial hypercholesterolaemia, one of the most common inherited diseases in the general population, is associated with mutations in at least three different genes including the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and protein convertase subtilisin/kexin type 9 (PCSK9) genes. In this report, we describe an unclassified DNA variant (c.1813C>T; p.Leu605Leu) within exon 12 of the LDLR gene in a kindred in which familial hypercholesterolaemia is associated with c.1813C>T heterozygosity. In silico analysis suggested that c.1813C>T might affect splicing of the LDLR gene by creating a cryptic donor splice site, which was confirmed by RT-PCR coupled with cDNA sequencing, to result in the loss of 34 base pairs in the coding sequence. The latter truncated mRNA is predicted to generate a frameshift leading to a premature stop at codon 652 and early termination of the low density lipoprotein receptor polypeptide chain, and thus provides a molecular basis for the hypercholesterolaemic phenotype. This case report highlights the emerging utility of RNA studies for the molecular diagnosis of familial hypercholesterolaemia in patients with potential mRNA splicing variants. PMID:25624525

  18. A new multimode optical fiber splicing technique

    NASA Astrophysics Data System (ADS)

    Ruello, Y.; Malavieille, F. L.

    1986-11-01

    For the French local cable network, it is necessary to perform a great number of multimode optical fiber splices. We present a low cost splicing technique which has been industrialized to satisfy the requirements of the videocommunication networks. In this splice, fibers are aligned in an elastomeric groove and then, bonded to a glass plate. Training time is very short and field installation is easy. It is a good quality, reliable splice and more than 100,000 splices has been already realized in the field. In this paper we present this splicing technique and the performances of the splice.

  19. 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.

  20. 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

  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 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

  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

    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. Hyperfine and Nuclear Quadrupole Tensors of Nitrogen Donors in the QA Site of Bacterial Reaction Centers: Correlation of the Histidine N? 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 N? 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 N? and a(14N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 N? 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, ? = 0.97 and e2Qq/4h = 0.74 MHz, ? = 0.59 for His-M219 N? 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 TownesDailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter ? to be a sensitive probe of the histidine N?semiquinone hydrogen bond strength. This is supported by a strong correlation observed between ? 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 ? will provide an important structural characterization tool in future studies of semiquinone-binding proteins. PMID:25026433

  4. Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures.

    PubMed Central

    Muranyi, W; Flügel, R M

    1991-01-01

    Mapping of transcripts of the human foamy virus genome was carried out in permissive human embryonic fibroblast cells by Northern blot hybridization and S1 nuclease analysis. Since several splice sites that are localized within a relatively narrow genomic region were detected, the polymerase chain reaction (PCR) was employed, and cloning and sequencing of the splice site junctions of the corresponding viral cDNAs were subsequently performed. All spumavirus transcripts have a common but relatively short leader RNA. Genomic, singly spliced env mRNAs and several singly and multiply spliced subgenomic transcripts were identified. The multiply spliced viral mRNAs consist of various exons located in the central or 3' part of the viral genome. At least four novel gene products, termed Bet, Bes, Beo, and Bel3, are predicted to exist. The poly(A) addition site that defines the boundary of the R and U5 region in the 3' long terminal repeat was determined. The pattern of spumavirus splicing is more complex than that of oncoviruses and more similar to that of lentiviruses. One of the characteristic features of spumavirus transcription is the existence of singly spliced bel1 and bel2 mRNAs that alternatively are multiply spliced, thereby generating a complexity comparable to, but different from, that of lentiviruses and from that of other known retroviruses. The complex spumavirus transcriptional pattern of human spumavirus and the coding potential of the 10 exons identified are discussed. Images PMID:1846194

  5. AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing

    PubMed Central

    Kamhi, Eyal; Yahalom, Galit; Kass, Gideon; Hacham, Yael; Sperling, Ruth; Sperling, Joseph

    2006-01-01

    More than 90% of human genes are rich in intronic latent 5′ splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5′ splice sites regulates alternative 5′ splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin—a drug known to block translation by its ability to recognize an RNA fold—but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism. PMID:16855285

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

    PubMed Central

    2014-01-01

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

  7. 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

  8. 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.

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

    PubMed

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

    2014-05-01

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

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

    PubMed

    Wachter, Andreas; Rühl, Christina; Stauffer, Eva

    2012-01-01

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

  11. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing. PMID:15972844

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25589247

  14. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing “minigene” in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  15. 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. PMID:25652415

  16. Structure of the branched intermediate in protein splicing

    PubMed Central

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

    2014-01-01

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

  17. Structure of the branched intermediate in protein splicing.

    PubMed

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

    2014-06-10

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

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

    NASA Astrophysics Data System (ADS)

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

    2000-06-01

    The properties of serum albumin obtained from donors and from paranoid schizophrenia patients were studied with the fluorescent probe K-35 (N-carboxyphenylimide of dimethylaminonaphthalic acid) and time-resolved fluorescence spectroscopy on the SR beam station of the S-60 synchrotron of the Lebedev Physical Institute. The mean fluorescence quantum yield of K-35 in patients serum was decreased significantly by 25-60% comparing with donors. The analysis of pre-exponential factors of fluorescence decay using "amplitude standard" method has shown that in patient sera the fraction of K-35 molecules bound with albumin and inaccessible to fluorescence quenchers ("bright" K-35 molecules with τ1=8.0±0.4 ns) is 1.2-3 times less than in the donor sera. The fraction of K-35 molecules with partly quenched fluorescence ( τ2=1.44±0.22 ns) was significantly increased in schizophrenia patients. The results obtained suggest that the properties of binding region in serum albumin molecules of acute paranoid schizophrenia patients change significantly.

  19. 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.

  20. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

    PubMed Central

    2005-01-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859

  1. Finding a Donor

    MedlinePlus

    ... our daughters life A single mother's transplant story Knowledge is power Donor stories Paul, marrow donor, explains donation process Nehal and Rohit donate their baby's cord blood to help others Parents Monique and Michael talk ...

  2. National Marrow Donor Program

    MedlinePlus

    ... our daughters life A single mother's transplant story Knowledge is power Donor stories Paul, marrow donor, explains donation process Nehal and Rohit donate their baby's cord blood to help others Parents Monique and Michael talk ...

  3. Donor cell myeloid sarcoma.

    PubMed

    Walshauser, Mark A; Go, Aileen; Sojitra, Payal; Venkataraman, Girish; Stiff, Patrick

    2014-01-01

    Donor cell derived malignancies are a rare and interesting complication of allogeneic bone marrow transplantation. We present a case of a 56-year-old male with donor cell myeloid sarcoma of the stomach and myocardium. PMID:24822132

  4. Donor Cell Myeloid Sarcoma

    PubMed Central

    Walshauser, Mark A.; Sojitra, Payal

    2014-01-01

    Donor cell derived malignancies are a rare and interesting complication of allogeneic bone marrow transplantation. We present a case of a 56-year-old male with donor cell myeloid sarcoma of the stomach and myocardium. PMID:24822132

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

    PubMed Central

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

    2014-01-01

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame. PMID:24918291

  6. Splice junctions are constrained by protein disorder

    PubMed Central

    Smithers, Ben; Oates, Matt E.; Gough, Julian

    2015-01-01

    We have discovered that positions of splice junctions in genes are constrained by the tolerance for disorder-promoting amino acids in the translated protein region. It is known that efficient splicing requires nucleotide bias at the splice junction; the preferred usage produces a distribution of amino acids that is disorder-promoting. We observe that efficiency of splicing, as seen in the amino-acid distribution, is not compromised to accommodate globular structure. Thus we infer that it is the positions of splice junctions in the gene that must be under constraint by the local protein environment. Examining exonic splicing enhancers found near the splice junction in the gene, reveals that these (short DNA motifs) are more prevalent in exons that encode disordered protein regions than exons encoding structured regions. Thus we also conclude that local protein features constrain efficient splicing more in structure than in disorder. PMID:25934802

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

    PubMed Central

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

    1999-01-01

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

  8. 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.

  9. Transition splices and cost comparison

    NASA Technical Reports Server (NTRS)

    Remedios, M. D.

    1972-01-01

    The development and testing of two designs of transition splices are reported. The design goal was to produce splice terminations that are electrically insulated to withstand the environmental conditions of commercial aircraft and are capable of being repaired and reworked on installed cables with the use of hand tools. In addition, a cost study comparison of FCC vs. RCC is reported. The comparison was made on a basis of 10 aircraft with each vehicle using approximately 100,000 feet of wiring and 2,000 connectors. The results are tabulated for seven different wiring configurations.

  10. Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

    PubMed Central

    Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B.; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

    2015-01-01

    Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

  11. Exon first nucleotide mutations in splicing: evaluation of in silico prediction tools.

    PubMed

    Grodecká, Lucie; Lockerová, Pavla; Ravčuková, Barbora; Buratti, Emanuele; Baralle, Francisco E; Dušek, Ladislav; Freiberger, Tomáš

    2014-01-01

    Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting. PMID:24586880

  12. Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.

    PubMed

    Sharma, Neeraj; Sosnay, Patrick R; Ramalho, Anabela S; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S; Amaral, Margarida D; Karchin, Rachel; Cutting, Garry R

    2014-10-01

    Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585-1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

  13. 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

  14. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes.

    PubMed

    Machinaga, Akihito; Ishihara, Syuhei; Shirai, Akiko; Takase-Yoden, Sayaka

    2016-01-01

    Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5' long terminal repeat (LTR), 5' leader sequence, gag, pol, env, and 3' LTR. Transcription from proviral DNA begins from the R region of the 5' LTR and ends at the polyadenylation signal located at the R region of the other end of the 3' LTR. There is a 5' splice site in the 5' leader sequence and a 3' splice site at the 3' end of the pol region. Both full-length unspliced mRNAs and a singly spliced mRNA (env-mRNA) are produced in MLV-infected cells. The MLV Env protein plays important roles both in viral adsorption to host cells and in neuropathogenic disease in MLV-infected mice and rats. Understanding the regulatory mechanisms controlling Env expression is important for determining the functions of the Env protein. We have previously shown that splicing increases env-mRNA stability and translation efficiency. Generally, mRNA polysome formation correlates with translation efficiency. Therefore, here we investigated the effects of env-mRNA splicing on polysome formation to identify mechanisms for Env up-regulation due to splicing. We performed polysome profile analyses using Env-expression plasmids producing spliced or unspliced env-mRNA and showed that the former formed polysomes more efficiently than the latter. Thus, splicing of env-mRNA facilitated polysome formation, suggesting that this contributes to up-regulation of Env expression. We replaced the env region of the expression plasmids with a luciferase (luc) gene, and found that in this case both unspliced and spliced luc-mRNA formed polysomes to a similar extent. Thus, we conclude that whether mRNA polysome formation is affected by splicing depends on the structure of gene in question. PMID:26909075

  15. 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

  16. Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy.

    PubMed

    Chen, Jieliang; Wu, Min; Wang, Fan; Zhang, Wen; Wang, Wei; Zhang, Xiaonan; Zhang, Jiming; Liu, Yinghui; Liu, Yi; Feng, Yanling; Zheng, Ye; Hu, Yunwen; Yuan, Zhenghong

    2015-01-01

    During hepatitis B virus (HBV) replication, spliced HBV genomes and splice-generated proteins have been widely described, however, their biological and clinical significance remains to be defined. Here, an elevation of the proportion of HBV spliced variants in the sera of patients with chronic hepatitis B (CHB) is shown to correlate with an impaired respond to interferon-α (IFN-α) therapy. Transfection of the constructs encoding the three most dominant species of spliced variants into cells or ectopic expression of the two major spliced protein including HBSP and N-terminal-truncated viral polymerase protein result in strong suppression of IFN-α signaling transduction, while mutation of the major splicing-related sites of HBV attenuates the viral anti-IFN activities in both cell and mouse models. These results have associated the productions of HBV spliced variants with the failure response to IFN therapy and illuminate a novel mechanism where spliced viral products are employed to resist IFN-mediated host defense. PMID:26585041

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

    PubMed

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

    2014-10-13

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

  18. An Integrated Regulatory Network Reveals Pervasive Cross-Regulation among Transcription and Splicing Factors

    PubMed Central

    Kosti, Idit; Radivojac, Predrag; Mandel-Gutfreund, Yael

    2012-01-01

    Traditionally the gene expression pathway has been regarded as being comprised of independent steps, from RNA transcription to protein translation. To date there is increasing evidence of coupling between the different processes of the pathway, specifically between transcription and splicing. To study the interplay between these processes we derived a transcription-splicing integrated network. The nodes of the network included experimentally verified human proteins belonging to three groups of regulators: transcription factors, splicing factors and kinases. The nodes were wired by instances of predicted transcriptional and alternative splicing regulation. Analysis of the network indicated a pervasive cross-regulation among the nodes; specifically, splicing factors are significantly more connected by alternative splicing regulatory edges relative to the two other subgroups, while transcription factors are more extensively controlled by transcriptional regulation. Furthermore, we found that splicing factors are the most regulated of the three regulatory groups and are subject to extensive combinatorial control by alternative splicing and transcriptional regulation. Consistent with the network results, our bioinformatics analyses showed that the subgroup of kinases have the highest density of predicted phosphorylation sites. Overall, our systematic study reveals that an organizing principle in the logic of integrated networks favor the regulation of regulatory proteins by the specific regulation they conduct. Based on these results, we propose a new regulatory paradigm postulating that gene expression regulation of the master regulators in the cell is predominantly achieved by cross-regulation. PMID:22844237

  19. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

    PubMed Central

    Caminsky, Natasha; Mucaki, Eliseos J.; Rogan, Peter K.

    2014-01-01

    The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368

  20. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis.

    PubMed

    Caminsky, Natasha; Mucaki, Eliseos J; Rogan, Peter K

    2014-01-01

    The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368

  1. VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance.

    PubMed

    Sargent, Kevin M; Clopton, Debra T; Lu, Ningxia; Pohlmeier, William E; Cupp, Andrea S

    2016-01-01

    Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo. PMID:26553653

  2. SRSF1-Regulated Alternative Splicing in Breast Cancer.

    PubMed

    Anczuków, Olga; Akerman, Martin; Cléry, Antoine; Wu, Jie; Shen, Chen; Shirole, Nitin H; Raimer, Amanda; Sun, Shuying; Jensen, Mads A; Hua, Yimin; Allain, Frédéric H-T; Krainer, Adrian R

    2015-10-01

    Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5' splice site generally promoted exon inclusion, whereas binding near the 3' splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development. PMID:26431027

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

    PubMed

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

    2014-01-01

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

  4. 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.

  5. A Carbohydrate-Derived Splice Modulator.

    PubMed

    Dhar, Sachin; La Clair, James J; León, Brian; Hammons, Justin C; Yu, Zhe; Kashyap, Manoj K; Castro, Januario E; Burkart, Michael D

    2016-04-20

    Small-molecule splice modulators have recently been recognized for their clinical potential for diverse cancers. This, combined with their use as tools to study the importance of splice-regulated events and their association with disease, continues to fuel the discovery of new splice modulators. One of the key challenges found in the current class of materials arises from their instability, where rapid metabolic degradation can lead to off-target responses. We now describe the preparation of bench-stable splice modulators by adapting carbohydrate motifs as a central scaffold to provide rapid access to potent splice modulators. PMID:27058259

  6. 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.

  7. Operational characteristics of trailing cable splices

    SciTech Connect

    Yenchek, M.R.; Schuster, K.C.; Hudson, A.J.

    1995-12-31

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

  8. Evolution of alternative splicing after gene duplication.

    PubMed

    Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

    2006-02-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates. PMID:16365379

  9. 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 hosts 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

  10. Influenza viruses and mRNA splicing: doing more with less.

    PubMed

    Dubois, Julia; Terrier, Olivier; Rosa-Calatrava, Manuel

    2014-01-01

    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

  11. Son maintains accurate splicing for a subset of human pre-mRNAs

    PubMed Central

    Sharma, Alok; Markey, Michael; Torres-Muñoz, Keshia; Varia, Sapna; Kadakia, Madhavi; Bubulya, Athanasios; Bubulya, Paula A.

    2011-01-01

    Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression. PMID:22193954

  12. A novel splicing regulator shares a nuclear import pathway with SR proteins.

    PubMed

    Lai, Ming-Chih; Kuo, Hao-Wei; Chang, Wen-Cheng; Tarn, Woan-Yuh

    2003-03-17

    Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus. PMID:12628928

  13. A novel splicing regulator shares a nuclear import pathway with SR proteins

    PubMed Central

    Lai, Ming-Chih; Kuo, Hao-Wei; Chang, Wen-Cheng; Tarn, Woan-Yuh

    2003-01-01

    Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin β-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus. PMID:12628928

  14. 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

  15. Electrochemical synthesis and characterisation of alternating tripyridyl-dipyrrole molecular strands with multiple nitrogen-based donor-acceptor binding sites.

    PubMed

    Tabatchnik-Rebillon, Alexandra; Aubé, Christophe; Bakkali, Hicham; Delaunay, Thierry; Manh, Gabriel Thia; Blot, Virginie; Thobie-Gautier, Christine; Renault, Eric; Soulard, Marine; Planchat, Aurélien; Le Questel, Jean-Yves; Le Guével, Rémy; Guguen-Guillouzo, Christiane; Kauffmann, Brice; Ferrand, Yann; Huc, Ivan; Urgin, Karène; Condon, Sylvie; Léonel, Eric; Evain, Michel; Lebreton, Jacques; Jacquemin, Denis; Pipelier, Muriel; Dubreuil, Didier

    2010-10-18

    Synthesis of alternating pyridine-pyrrole molecular strands composed of two electron-rich pyrrole units (donors) sandwiched between three pyridinic cores (acceptors) is described. The envisioned strategy was a smooth electrosynthesis process involving ring contraction of corresponding tripyridyl-dipyridazine precursors. 2,6-Bis[6-(pyridazin-3-yl)]pyridine ligands 2a-c bearing pyridine residues at the terminal positions were prepared in suitable quantities by a Negishi metal cross-coupling procedure. The yields of heterocyclic coupling between 2-pyridyl zinc bromide reagents 12a-c and 2,6-bis(6-trifluoromethanesulfonylpyridazin-3-yl)pyridine increased from 68 to 95% following introduction of electron-donating methyl groups on the metallated halogenopyridine units. Favorable conditions for preparative electrochemical reduction of tripyridyl-dipyridazines 2b,c were established in THF/acetate buffer (pH 4.6)/acetonitrile to give the targeted 2,6-bis[5-(pyridin-2-yl)pyrrol-2-yl]pyridines 1b and 1c in good yields. The absorption behavior of the donor-acceptor tripyridyl-dipyrrole ligands was evaluated and compared to theoretical calculations. Highly fluorescent properties of these chromophores were found (ν(em)≈2 × 10(4) cm(-1) in MeOH and CH(2)Cl(2)), and both pyrrolic ligands exhibit a remarkable quantum yield in CH(2)Cl(2) (φ(f)=0.10). Structural studies in the solid state established the preferred cis conformation of the dipyrrolic ligands, which adopting a planar arrangement with an embedded molecule of water having a complexation energy exceeding 10 kcal mol(-1). The ability of the tripyridyl-dipyrrole to complex two copper(II) ions in a pentacoordinate square was investigated. PMID:20839373

  16. 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. PMID:26913597

  17. 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

  18. Living Related Kidney Donors

    PubMed Central

    Lazarovits, Andrew I.

    1992-01-01

    A kidney transplant is the treatment of choice for suitable patients with end-stage renal disease. The living related kidney donor represents an important source of kidneys because graft survival is much better and there is a critical shortage of organ donors. This article reviews the risks to the donor and the means by which these individuals are assessed in order to minimize the risk. PMID:21221370

  19. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    PubMed

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein. PMID:26609938

  20. O-Glycosyl Donors

    NASA Astrophysics Data System (ADS)

    Lpez, J. Cristbal

    O-Glycosyl donors, despite being one of the last successful donors to appear, have developed themselves into a burgeoning class of glycosyl donors. They can be classified in two main types: O-alkyl and O-aryl (or hetaryl) glycosyl donors. They share, however, many characteristics, they can be (1) synthesized from aldoses, either by modified Fisher glycosidation (O-alkyl) or by nucleophilic aromatic substitution (O-aryl or O-hetaryl), (2) stable to diverse chemical manipulations, (3) directly used for saccharide coupling, and (4) chemoselectively activated. Among these, n-pentenyl glycosides stand apart. They were the first O-alkyl glycosyl donors to be described and have paved the way to many conceptual developments in oligosaccharide synthesis. The development of the chemoselectivity-based "armed-disarmed" approach for saccharide coupling, including its stereoelectronic or torsional variants, now extended to other kinds of glycosyl donors, was first recognized in n-pentenyl glycosides. The chemical manipulation of the anomeric substituent in the glycosyl donor to induce reactivity differences between related species (sidetracking) was also introduced in n-pentenyl glycosides. An evolution of this concept, the "latent-active" strategy for glycosyl couplings, first described in thioglycosyl donors (vide infra), has been elegantly applied to O-glycosyl donors. Thus, allyl and vinyl glycosides, 2-(benzyloxycarbonyl)benzyl (BCB) glycosides and 2'-carboxybenzyl (CB) glycosides are useful "latent-active" glycosyl pairs. Finally, unprotected 3-methoxy-2-pyridyl (MOP) glycosides have been used in glycosylation processes with moderate success.

  1. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  2. Spliced leader RNA trans-splicing discovered in copepods.

    PubMed

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A; Sturm, Nancy R; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3'-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  3. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  4. Extraordinary Separation of Acetylene-Containing Mixtures with Microporous Metal-Organic Frameworks with Open O Donor Sites and Tunable Robustness through Control of the Helical Chain Secondary Building Units.

    PubMed

    Yao, Zizhu; Zhang, Zhangjing; Liu, Lizhen; Li, Ziyin; Zhou, Wei; Zhao, Yunfeng; Han, Yu; Chen, Banglin; Krishna, Rajamani; Xiang, Shengchang

    2016-04-11

    Acetylene separation is a very important but challenging industrial separation task. Here, through the solvothermal reaction of CuI and 5-triazole isophthalic acid in different solvents, two metal-organic frameworks (MOFs, FJU-21 and FJU-22) with open O donor sites and controllable robustness have been obtained for acetylene separation. They contain the same paddle-wheel {Cu2 (COO2 )4 } nodes and metal-ligand connection modes, but with different helical chains as secondary building units (SBUs), leading to different structural robustness for the MOFs. FJU-21 and FJU-22 are the first examples in which the MOFs' robustness is controlled by adjusting the helical chain SBUs. Good robustness gives the activated FJU-22 a, which has higher surface area and gas uptakes than the flexible FJU-21 a. Importantly, FJU-22 a shows extraordinary separation of acetylene mixtures under ambient conditions. The separation capacity of FJU-22 a for 50:50 C2 H2 /CO2 mixtures is about twice that of the high-capacity HOF-3, and its actual separation selectivity for C2 H2 /C2 H4 mixtures containing 1 % acetylene is the highest among reported porous materials. Based on first-principles calculations, the extraordinary separation performance of C2 H2 for FJU-22 a was attributed to hydrogen-bonding interactions between the C2 H2 molecules with the open O donors on the wall, which provide better recognition ability for C2 H2 than other functional sites, including open metal sites and amino groups. PMID:26934040

  5. Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

    PubMed

    Zhang, Qiao; Kota, Krishna P; Alam, Samer G; Nickerson, Jeffrey A; Dickinson, Richard B; Lele, Tanmay P

    2016-06-01

    Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces. J. Cell. Physiol. 231: 1269-1275, 2016. © 2015 Wiley Periodicals, Inc. PMID:26496460

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

    PubMed Central

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

    2012-01-01

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

  7. Insertion of part of an intron into the 5[prime] untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene

    SciTech Connect

    Conrad, R.; Thomas, J.; Spieth, J.; Blumenthal, T. )

    1991-04-01

    In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, Sl1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. The authors demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5[prime] splice site into the 5[prime] untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3[prime] splice site, were inserted into the 5[prime] untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. They termed the region of a trans-spliced mRNA precursor between the 5[prime] end and the first 3[prime] splice site an 'outrun'. The results suggest that if a transcript begins with intronlike sequence followed by a 3[prime] splice site, this alone may constitute an outrun and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.

  8. Computational analysis reveals a correlation of exon-skipping events with splicing, transcription and epigenetic factors.

    PubMed

    Ye, Zhenqing; Chen, Zhong; Lan, Xun; Hara, Stephen; Sunkel, Benjamin; Huang, Tim H-M; Elnitski, Laura; Wang, Qianben; Jin, Victor X

    2014-03-01

    Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, 'skipping', exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon-intron boundary establishment leading to skipping events. PMID:24369421

  9. SpliceProt: a protein sequence repository of predicted human splice variants.

    PubMed

    Tavares, Raphael; de Miranda Scherer, Nicole; Pauletti, Bianca Alves; Araújo, Elói; Folador, Edson Luiz; Espindola, Gabriel; Ferreira, Carlos Gil; Paes Leme, Adriana Franco; de Oliveira, Paulo Sergio Lopes; Passetti, Fabio

    2014-02-01

    The mechanism of alternative splicing in the transcriptome may increase the proteome diversity in eukaryotes. In proteomics, several studies aim to use protein sequence repositories to annotate MS experiments or to detect differentially expressed proteins. However, the available protein sequence repositories are not designed to fully det