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

    PubMed

    Zou, Minjing; Bulu?, Derya; Al-Rijjal, Roua A; And?ran, Nesibe; BinEssa, Huda; Kattan, Walaa E; Meyer, Brian; Shi, Yufei

    2015-01-01

    X-linked hypophosphatemic rickets (XLH) is the most common inherited form of rickets. XLH is caused by inactivating mutations in the PHEX gene and is transmitted as an X-linked dominant disorder. We investigated PHEX mutation in a sporadic Turkish girl with hypophosphatemic rickets. The patient was 2 years of age with a complaint of inability to walk. She had bowing of legs and growth retardation. Laboratory data showed normal calcium, low phosphate with markedly elevated ALP, and low phosphate renal tubular reabsorption. She was treated with Calcitriol 0.5 mg/kg/day and oral phosphate supplement with good response. The entire coding region of PHEX gene was sequenced from patient's peripheral leukocyte DNA and a novel 13 bp deletion at the donor splice site of exon5 was found (c.663+12del). Instead of using the donor splice site of intron 4 to splice out exon 5 and intron 5, the spliceosome utilized two nearby cryptic donor splice sites (5' splice site) to splice out intron 4, resulting in two smaller transcripts. Both of them could not translate into functional proteins due to frameshift. Her parents did not carry the mutation, indicating that this is a de novo PHEX mutation likely resulting from mutagenesis of X chromosome in paternal germ cells. We conclude that c.663+12del is a novel mutation that can activate nearby cryptic 5' splice sites. The selection of cryptic 5' splice sites adds the complexity of cell's splicing mechanisms. The current study extends the database of PHEX mutation and cryptic 5' splice sites. PMID:25153221

  3. 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.

  4. 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.

  5. 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

  6. Validation of three splice donor and three splice acceptor sites for regulating four novel low-abundance spliced transcripts of human cytomegalovirus UL21.5 gene locus.

    PubMed

    Gao, Shuang; Ruan, Qiang; Ma, Yanping; Li, Mali; Wang, Lin; Zheng, Bo; Qi, Ying; Ji, Yaohua; Sun, Zhengrong; Huang, Yujing

    2015-01-01

    In a previous study, one spliced transcript of human cytomegalovirus (HCMV), named UL21.5 was identified. UL21.5 has been found to be one of the viral transcripts packaged within HCMV particles. The UL21.5 mRNA is translated into a secreted glycoprotein, which is a viral chemokine decoy receptor specifically interacting with regulated upon activation normal T cell expressed and secreted (RANTES). In the present study, four novel low-abundance 3'-coterminal spliced transcripts were identified to be transcribed from the UL21.5 gene region of a low-passage HCMV strain during the late infection phase by cDNA library screening, northern blot hybridization, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR. Three splicing donor and three splicing acceptor sites found in the UL21.5 gene region were validated to be functional in an in vitro expression system. In addition, the determinant regulatory region that is necessary for the splice donor site at nucleotide (nt) 25533 was located in a 9-bp sequence around the site; the regulatory regions for the splice acceptor sites at nt 26597 and nt 26633 were located in a 20-bp sequence upstream of the site at nt 26597 and in a 10-bp sequence from nt 26641 to nt 26650 downstream of the site at nt 26633, respectively. PMID:25370414

  7. 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

  8. 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.

  9. 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

  10. 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.

  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. 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.

  13. 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

  14. 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

  15. A novel deletion in the splice donor site of MLH1 exon 6 in a Japanese colon cancer patient with Lynch syndrome.

    PubMed

    Yamaguchi, Junya; Sato, Yuri; Kita, Mizuho; Nomura, Sachio; Yamamoto, Noriko; Kato, Yo; Ishikawa, Yuichi; Arai, Masami

    2015-10-01

    Lynch syndrome is an autosomal dominantly inherited disease that is characterized by a predisposition to cancers, mainly colorectal cancer. Germline mutations of DNA mismatch repair genes such as MLH1, MSH2, MSH6 and PMS2 have been described in patients with Lynch syndrome. Here, we report deletion of 2 bp in the splice donor site of the MLH1 exon 6 (c.545+4_545+5delCA) in a 48-year-old Japanese woman with Lynch syndrome. RT-PCR direct sequencing analysis revealed that this mutation led to an increase in the level of an MLH1 transcript in which exon 6 was skipped, and may cause a frameshift (p.E153FfsX8). Therefore, this mutation appears to be pathogenic and is responsible for Lynch syndrome. Additionally, analysis of the patient's tumor cells indicated microsatellite instability high phenotype and loss of the MLH1 and PMS2 proteins. To our knowledge, this is a germline splice site mutation of MLH1 that has not been reported previously. PMID:26185136

  16. 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; Magalln, 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

  17. 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; Magalln, 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

  18. 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.

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

    PubMed

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

    1997-12-01

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

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

    PubMed Central

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

    1997-01-01

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

  1. 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.

  2. Identification of alternative 5?/3? splice sites based on the mechanism of splice site competition

    PubMed Central

    Xia, Huiyu; Bi, Jianning; Li, Yanda

    2006-01-01

    Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5?/3? splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified ?70% of the splice sites into alternative and constitutive, as well as ?80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology. PMID:17098928

  3. 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.

  4. 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

  5. The organization of nucleosomes around splice sites.

    PubMed

    Chen, Wei; Luo, Liaofu; Zhang, Lirong

    2010-05-01

    The occupancy of nucleosomes along chromosome is a key factor for gene regulation. However, except promoter regions, genome-wide properties and functions of nucleosome organization remain unclear in mammalian genomes. Using the computational model of Increment of Diversity with Quadratic Discriminant (IDQD) trained from the microarray data, the nucleosome occupancy score (NOScore) was defined and applied to splice junction regions of constitutive, cassette exon, alternative 3' and 5' splicing events in the human genome. We found an interesting relation between NOScore and RNA splicing: exon regions have higher NOScores compared with their flanking intron sequences in both constitutive and alternative splicing events, indicating the stronger nucleosome occupation potential of exon regions. In addition, NOScore valleys present at approximately 25 bp upstream of the acceptor site in all splicing events. By defining folding diversity-to-energy ratio to describe RNA structural flexibility, we demonstrated that primary RNA transcripts from nucleosome occupancy regions are relatively rigid and those from nucleosome depleted regions are relatively flexible. The negative correlation between nucleosome occupation/depletion of DNA sequence and structural flexibility/rigidity of its primary transcript around splice junctions may provide clues to the deeper understanding of the unexpected role for nucleosome organization in the regulation of RNA splicing. PMID:20097656

  6. 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.

  7. 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.

  8. An Intronic Splicing Enhancer Binds U1 snRNPs To Enhance Splicing and Select 5? Splice Sites

    PubMed Central

    McCullough, Andrew J.; Berget, Susan M.

    2000-01-01

    Intronic G triplets are frequently located adjacent to 5? splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5? splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5? splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5? splice sites promotes accurate splice site recognition by recruiting the U1 snRNP. PMID:11094074

  9. 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

  10. Enhancer-dependent 5?-Splice Site Control of fruitless Pre-mRNA Splicing*

    PubMed Central

    Lam, Bianca J.; Bakshi, Arati; Ekinci, Fatma Y.; Webb, Jenny; Graveley, Brenton R.; Hertel, Klemens J.

    2008-01-01

    The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5?-splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3?-splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3?-splice site choice. However, little is known about enhancer-dependent regulation of alternative 5?-splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5?-splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5?-splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5?- and 3?-splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5?- and 3?-splice sites. PMID:12646561

  11. 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

  12. 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 intronsare 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

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

    PubMed

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

    2015-01-01

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

  14. 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

  15. 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 sitesGYNNGYs (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

  16. Diverse regulation of 3' splice site usage.

    PubMed

    Sohail, Muhammad; Xie, Jiuyong

    2015-12-01

    The regulation of splice site (SS) usage is important for alternative pre-mRNA splicing and thus proper expression of protein isoforms in cells; its disruption causes diseases. In recent years, an increasing number of novel regulatory elements have been found within or nearby the 3'SS in mammalian genes. The diverse elements recruit a repertoire of trans-acting factors or form secondary structures to regulate 3'SS usage, mostly at the early steps of spliceosome assembly. Their mechanisms of action mainly include: (1) competition between the factors for RNA elements, (2) steric hindrance between the factors, (3) direct interaction between the factors, (4) competition between two splice sites, or (5) local RNA secondary structures or longer range loops, according to the mode of protein/RNA interactions. Beyond the 3'SS, chromatin remodeling/transcription, posttranslational modifications of trans-acting factors and upstream signaling provide further layers of regulation. Evolutionarily, some of the 3'SS elements seem to have emerged in mammalian ancestors. Moreover, other possibilities of regulation such as that by non-coding RNA remain to be explored. It is thus likely that there are more diverse elements/factors and mechanisms that influence the choice of an intron end. The diverse regulation likely contributes to a more complex but refined transcriptome and proteome in mammals. PMID:26370726

  17. Gene splice sites correlate with nucleosome positions.

    PubMed

    Kogan, Simon; Trifonov, Edward N

    2005-06-01

    Gene sequences in the vicinity of splice sites are found to possess dinucleotide periodicities, especially RR and YY, with the period close to the pitch of nucleosome DNA. This confirms previously reported findings about preferential positioning of splice junctions within the nucleosomes. The RR and YY dinucleotides oscillate counter-phase, i.e., their respective preferred positions are shifted about half-period from one another, as it was observed earlier for AA and TT dinucleotides. Species specificity of nucleosome positioning DNA pattern is indicated by the predominant use of the periodical GG(CC) dinucleotides in human and mouse genes, as opposed to predominant AA(TT) dinucleotides in Arabidopsis and C. elegans. PMID:15862762

  18. 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

  19. 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

  20. 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

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

    PubMed Central

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

    2015-01-01

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

  2. 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

  3. 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

  4. 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

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

    PubMed

    Eckert, Daniela; Andre, Nicole; Razanau, Aleh; Zock-Emmenthal, Susanne; Ltzelberger, Martin; Plath, Susann; Schmidt, Henning; Guerra-Moreno, Angel; Cozzuto, Luca; Ayt, Jos; Kufer, 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

  6. 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

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

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

    Human ? -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.

  8. Splice-site pairing is an intrinsically high fidelity process

    PubMed Central

    Fox-Walsh, Kristi L.; Hertel, Klemens J.

    2009-01-01

    The extensive alternative splicing in higher eukaryotes has initiated a debate whether alternative mRNA isoforms are generated by an inaccurate spliceosome or are the consequence of highly degenerate splice sites within the human genome. Here, we established a quantitative assay to evaluate the accuracy of splice-site pairing by determining the number of incorrect exon-skipping events made from constitutively spliced pre-mRNA transcripts. We demonstrate that the spliceosome pairs exons with an astonishingly high degree of accuracy that may be limited by the quality of pre-mRNAs generated by RNA pol II. The error rate of exon pairing is increased by the effects of the neurodegenerative disorder spinal muscular atrophy because of reduced levels of Survival of Motor Neuron, a master assembler of spliceosomal components. We conclude that all multi-intron-containing genes are alternatively spliced and that the reduction of SMN results in a general splicing defect that is mediated through alterations in the fidelity of splice-site pairing. PMID:19179398

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

    NASA Astrophysics Data System (ADS)

    Zuo, Ping; Manley, James L.

    1994-04-01

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

  10. Alginate dressing as a donor site haemostat.

    PubMed Central

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

    1986-01-01

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

  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. 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 WatsonCrick/WatsonCrick) 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 WatsonCrick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair. PMID:25909229

  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. 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

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

    PubMed

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

    2016-02-25

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

  16. Sequencing of lariat termini in S. cerevisiae reveals 5' splice sites, branch points, and novel splicing events.

    PubMed

    Qin, Daoming; Huang, Lei; Wlodaver, Alissa; Andrade, Jorge; Staley, Jonathan P

    2016-02-01

    Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimal-especially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5' splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3' termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3' to 5' RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution. PMID:26647463

  17. A comprehensive survey of non-canonical splice sites in the human transcriptome

    PubMed Central

    Parada, Guillermo E.; Munita, Roberto; Cerda, Cledi A.; Gysling, Katia

    2014-01-01

    We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non-canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation. Our analysis provides a high-confidence group of U2/U12-like non-canonical splice sites, which exhibit distinctive features among the total human splice sites. PMID:25123659

  18. Prp40 and early events in splice site definition.

    PubMed

    Becerra, Soraya; Andrs-Len, Eduardo; Prieto-Snchez, Silvia; Hernndez-Munain, Cristina; Su, Carlos

    2016-01-01

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

  19. 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

  20. 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

  1. Successful Treatment of Chronic Donor Site Pain

    PubMed Central

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

    2015-01-01

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

  2. 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.

  3. Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.

    PubMed

    Bourbon, M; Duarte, M A; Alves, A C; Medeiros, A M; Marques, L; Soutar, A K

    2009-05-01

    Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH. PMID:19411563

  4. Human epigenome data reveal increased CpG methylation in alternatively spliced sites and putative exonic splicing enhancers.

    PubMed

    Anastasiadou, Christina; Malousi, Andigoni; Maglaveras, Nicos; Kouidou, Sofia

    2011-05-01

    The role of gene body methylation, which represents a major part of methylation in DNA, remains mostly unknown. Evidence based on the CpG distribution associates its presence with nucleosome positioning and alternative splicing. Recently, it was also shown that cytosine methylation influences splicing. However, to date, there is no methylation-based data on the association of methylation with alternative splicing and the distribution in exonic splicing enhancers (ESEs). We presently report that, based on the computational analysis of the Human Epigenome Project data, CpG hypermethylation (>80%) is frequent in alternatively spliced sites (particularly in noncanonical) but not in alternate promoters. The methylation frequency increases in sequences containing multiple putative ESEs. However, significant differences in the extent of methylation are observed among different ESEs. Specifically, moderate levels of methylation, ranging from 20% to 80%, are frequent in SRp55-binding elements, which are associated with response to extracellular conditions, but not in SF2/ASF, primarily responsible for alternative splicing, or in CpG islands. Finally, methylation is more frequent in the presence of AT repeats and CpGs separated by 10 nucleotides and lower in adjacent CpGs, probably indicating its dependence on helical formations and on the presence of nucleosome positioning-related sequences. In conclusion, our results show the regulation of methylation in ESEs and support its involvement in alternative splicing. PMID:21545276

  5. 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

  6. 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

  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. The in vivo use of alternate 3'-splice sites in group I introns.

    PubMed Central

    Sellem, C H; Belcour, L

    1994-01-01

    Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used. Images PMID:8165125

  10. Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

    PubMed Central

    Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

    1995-01-01

    We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly. Images PMID:7835348

  11. 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

  12. 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

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

    SciTech Connect

    Purandare, S.M.; Lanyon, W.G.; Arngrimsson, R.

    1994-09-01

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

  14. 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.

  15. 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

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

    PubMed Central

    Luukkonen, B G; Sraphin, 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

  17. Healing of Donor-site Buccal Mucosa Urethroplasty.

    PubMed

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

    2015-09-01

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

  18. Molecular diagnosis of hereditary inclusion body myopathy by linkage analysis and identification of a novel splice site mutation in GNE

    PubMed Central

    2011-01-01

    Background Many myopathies share clinical features in common, and diagnosis often requires genetic testing. We ascertained a family in which five siblings presented with distal muscle weakness of unknown etiology. Methods We performed high-density genomewide linkage analysis and mutation screening of candidate genes to identify the genetic defect in the family. Preserved clinical biopsy material was reviewed to confirm the diagnosis, and reverse transcriptase PCR was used to determine the molecular effect of a splice site mutation. Results The linkage scan excluded the majority of known myopathy genes, but one linkage peak included the gene GNE, in which mutations cause autosomal recessive hereditary inclusion body myopathy type 2 (HIBM2). Muscle biopsy tissue from a patient showed myopathic features, including small basophilic fibers with vacuoles. Sequence analysis of GNE revealed affected individuals were compound heterozygous for a novel mutation in the 5' splice donor site of intron 10 (c.1816+5G>A) and a previously reported missense mutation (c.2086G>A, p.V696M), confirming the diagnosis as HIBM2. The splice site mutation correlated with exclusion of exon 10 from the transcript, which is predicted to produce an in-frame deletion (p.G545_D605del) of 61 amino acids in the kinase domain of the GNE protein. The father of the proband was heterozygous for the splice site mutation and exhibited mild distal weakness late in life. Conclusions Our study expands on the extensive allelic heterogeneity of HIBM2 and demonstrates the value of linkage analysis in resolving ambiguous clinical findings to achieve a molecular diagnosis. PMID:21708040

  19. Polypyrimidine Tract Binding Protein Blocks the 5? Splice Site Dependent Assembly of U2AF and the Prespliceosomal E Complex

    PubMed Central

    Sharma, Shalini; Falick, Arnold M.; Black, Douglas L.

    2006-01-01

    SUMMARY Polypyrimidine tract binding protein (PTB) represses the splicing of many alternatively spliced exons. This repression can sometimes involve the direct occlusion of splice sites by PTB. We show here that PTB prevents splicing of the c-src N1 exon to downstream exon 4 by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5? splice site, but instead prevents formation of the pre-spliceosomal Early (E) complex across the intervening intron. If only the repressed 5? splice site of the N1 exon is present, the splicing factor U2AF does not assemble on the downstream 3? splice site of exon 4. When the unregulated 5? splice site of the upstream exon 3 is included in the RNA, U2AF binding is restored and splicing between exons 3 and 4 proceeds, in spite of the presence of the PTB bound across the N1 exon. Rather than directly blocking the N1 splice sites, PTB is blocking the 5? splice site dependent assembly of U2AF into the E complex, presumably through an interaction with the U1 snRNP. This mechanism of repression is likely to also occur in many other alternative exons. PMID:16109373

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

    PubMed Central

    Kralovicova, Jana

    2010-01-01

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

  2. 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

  3. 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

  4. Graft harvesting and management of the donor site.

    PubMed

    Buchwach, Kenneth A

    2013-08-01

    The strip technique remains the most popular method of harvesting grafts. The challenge in every patient is to maximize the number of grafts while minimizing the scar. Fortunately, there are many ways to ensure that the donor site will be inconspicuous. This article reviews the details of planning for follicle graft harvesting, including formulae for assessing scalp laxity and calculating strip dimensions. The procedure is discussed in detail, from preparation of the donor site and estimation of graft total through closure of the incision. The author presents his preferences for the technique with rationale and surgical tips. PMID:24017978

  5. 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

  6. DNA splice site detection: a comparison of specific and general methods.

    PubMed Central

    Kim, Won; Wilbur, W. John

    2002-01-01

    In an era when whole organism genomes are being routinely sequenced, the problem of gene finding has become a key issue on the road to understanding. For eukaryotic organisms a large part of locating the genes is accomplished by predicting the likely location of splice sites on a DNA strand. This problem of splice site location has been ap- proached using a number of machine learning or statistical methods tailored more or less specifically to the nature of the problem. Recently large margin classifiers and boosting methods have been found to give improvements over more traditional methods in a number of areas. Here we compare large margin classifiers (SVM and CMLS) and boosted decision trees with the three most common models used for splice site detection (WMM, WAM, and MDT). We find that the newer methods compare favorably in all cases and can yield significant improvement in some cases. PMID:12463853

  7. 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

  8. 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

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

    PubMed Central

    Jacob, M; Gallinaro, H

    1989-01-01

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

  10. Clinical heterogeneity and molecular findings in five Polish patients with glycerol kinase deficiency: investigation of two splice site mutations with computerized splice junction analysis and Xp21 gene-specific mRNA analysis.

    PubMed

    Hellerud, Christina; Adamowicz, Maciej; Jurkiewicz, Dorota; Taybert, Joanna; Kubalska, Jolanta; Ciara, Elzbieta; Popowska, Ewa; Ellis, James R; Lindstedt, Sven; Pronicka, Ewa

    2003-07-01

    Five cases of glycerol kinase deficiency are presented with clinical, biochemical, and genetic results. Two had the glycerol kinase deficiency as part of an Xp21 contiguous gene deletion syndrome-complex form-and three had an isolated form of the enzyme deficiency. In these we found two splice site mutations (IVS1+4A>G, IVS9-1G>T) and one insertion (1393_1394insG). In patients with the complex form, a deletion of the DAX1, GK genes and the distal part of the DMD gene was found. A computerized study was performed to predict the effects of the splice site mutations. It showed that the IVS9-1G>T mutation substantially altered and removed the wild-type site and enhanced a cryptic site seven nucleotides downstream, and that the IVS1+4A>G diminished the strength of the wild-type donor site from strong to leaky. To verify these predictions, we developed an RT-PCR system with gene-specific primers that exclusively amplifies the Xp21 glycerol kinase gene transcript. Identification of individuals at risk is motivated by a need to avoid delay in a correct diagnosis. For reliable identification of heterozygotes for isolated glycerol kinase deficiency, knowledge of the specific mutation in the proband is required. This is easily obtained with the RT-PCR analyses developed in this study. PMID:12855219

  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. hnRNP A1 and hnRNP H can collaborate to modulate 5? splice site selection

    PubMed Central

    Fisette, Jean-Franois; Toutant, Johanne; Dugr-Brisson, Samuel; Desgroseillers, Luc; Chabot, Benoit

    2010-01-01

    The mammalian proteins hnRNP A1 and hnRNP H control many splicing decisions in viral and cellular primary transcripts. To explain some of these activities, we have proposed that self-interactions between bound proteins create an RNA loop that represses internal splice sites while simultaneously activating the external sites that are brought in closer proximity. Here we show that a variety of hnRNP H binding sites can affect 5? splice site selection. The addition of two sets of hnRNP H sites in a model pre-mRNA modulates 5? splice site selection cooperatively, consistent with the looping model. Notably, binding sites for hnRNP A1 and H on the same pre-mRNA can similarly collaborate to modulate 5? splice site selection. The C-terminal portion of hnRNP H that contains the glycine-rich domains (GRD) is essential for splicing activity, and it can be functionally replaced by the GRD of hnRNP A1. Finally, we used the bioluminescence resonance energy transfer (BRET) technology to document the existence of homotypic and heterotypic interactions between hnRNP H and hnRNP A1 in live cells. Overall, our study suggests that interactions between different hnRNP proteins bound to distinct locations on a pre-mRNA can change its conformation to affect splicing decisions. PMID:19926721

  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. Compound heterozygosity for new splice site mutations in the plakophilin 1 gene (PKP1) in a Chinese case of ectodermal dysplasia-skin fragility syndrome.

    PubMed

    Zheng, Rui; Bu, Ding-Fang; Zhu, Xue-Jun

    2005-01-01

    Ectodermal dysplasia-skin fragility syndrome is a rare autosomal recessive inherited disease characterized by skin fragility, nail dystrophy and hyperkeratosis of palms and soles. Skin biopsy shows the loss of cell adhesion and the decrease of desmosomes in number and size. Mutations in PKP1 have been found to be the underlying cause of the syndrome. We report here a Chinese case of ectodermal dysplasia-skin fragility syndrome. Mutation analysis revealed compound heterozygosity for mutations in PKP1 of the proband. A new splice site mutation (c.1053 T>A+c.1054+1 G>T) near the 3' end of exon 5 and at the donor end of intron 5 on one allele was transmitted from the proband's mother. Another new splice site mutation (c.1835-2 A>G) near the acceptor end of intron 10 originated from her father. The absence of the mutant mRNA and plakophilin 1 protein in the proband's skin may result from the mechanism of nonsense-mediated mRNA decay induced by premature stop codons in PKP1 transcripts due to the two splice site mutations. PMID:16159729

  15. The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites

    PubMed Central

    Hodson, Mark J.; Hudson, Andrew J.; Cherny, Dmitry; Eperon, Ian C.

    2012-01-01

    Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5? splice site, components recognizing the 3? splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5? splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5? splice sites. In complex E most transcripts with two alternative 5? splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3? splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5? splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex. PMID:22505580

  16. 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.

  17. 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 3terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5splice 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 3cleavage 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 3terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise. PMID:25826302

  18. 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

  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. 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

  1. Bioinformatics in the Thermodynamic Limit: Applications to pre-mRNA Splice Site Detection

    NASA Astrophysics Data System (ADS)

    Daub, Eric G.; Aalberts, Daniel P.

    2004-03-01

    In the thermodynamic limit, physical systems exhibit predictable behavior. Traditional bioinformatics methods of matching patterns from a few cases can be superseded by alternate techniques when the number of instances approaches the thermodynamic limit. By enhancing an already large dataset, we find that we can differentiate real signals from decoys with superior accuracy. We apply our framework to the problem of precursor mRNA splice site detection in human sequences.

  2. 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.1254T?>?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 participants 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

  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. 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

  5. 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

  6. 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.

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

    PubMed

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

    2016-02-01

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

  9. Cwc21p promotes the second step conformation of the spliceosome and modulates 3' splice site selection.

    PubMed

    Gautam, Amit; Grainger, Richard J; Vilardell, J; Barrass, J David; Beggs, Jean D

    2015-03-31

    Pre-mRNA splicing involves two transesterification steps catalyzed by the spliceosome. How RNA substrates are positioned in each step and the molecular rearrangements involved, remain obscure. Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, SRm300/SRRM2. Our microarray analysis shows changes in 3' splice site selection at elevated temperature in a subset of introns in cwc21? cells. Considering all the available data, we propose a role for Cwc21p positioning the 3' splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP. This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells. PMID:25740649

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

    PubMed Central

    Kiss-Lszl, Z; Blanc, S; Hohn, T

    1995-01-01

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

  11. 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

  12. Spontaneous deletions in Ig heavy chain genes: flanking sequences influence splice site selection.

    PubMed Central

    Ward, S B; Morrison, S L

    1991-01-01

    The cell line G403.4.7, isolated as a spontaneous variant of the MPC-11 derived myeloma G403.4, produces a truncated gamma 2b HC protein, but no light chain (LC), and a single gamma 2b specific transcript of 2.4kb. This gamma 2b transcript consists of the VDJ and CH1 exons, the CH1 to Hinge (Hi) intervening sequence (IVS) and HI exon, part of the IVS between the two membrane exons M1 and M2, and most of the membrane 3' untranslated (UT) region. Even though the mature mRNA contains intronic sequences, it is abundant in the cytoplasm. Analysis of the gamma 2b genomic organization reveals that this unusual transcript results in part from two genomic deletions of 2.5kb and 588bp and in part from an altered splicing pattern. This altered splicing pattern is probably a consequence of the sequence alterations resulting from the genomic deletions. Analysis of these events provides some interesting insights into the mechanism of splice site selection and the evolution of introns and exons. Images PMID:1754385

  13. 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

  14. 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 Central

    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 melanoma, 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 1416; introns 1516; 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

  15. 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

  16. Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases

    PubMed Central

    Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo

    2013-01-01

    Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC3 (NM_003786.3:c.1783-1G>A), KLHDC1 (NM_172193.1:c.568-2A>G), HOOK1 (NM_015888.4:c.1662-1G>A), SMAD9 (NM_001127217.2:c.1004-1C>T), and DNAH9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC3, HOOK1. In ABCC3 and HOOK1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 46 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis. PMID:24498620

  17. 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

  18. 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

  19. U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements.

    PubMed Central

    Jamison, S F; Pasman, Z; Wang, J; Will, C; Lhrmann, R; Manley, J L; Garcia-Blanco, M A

    1995-01-01

    Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition. Images PMID:7667103

  20. 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.

  1. 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.

  2. Using multiple donor sites for enhanced flood estimation in ungauged catchments

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  3. 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, Genevive; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; stergaard, 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 PelizaeusMerzbacher 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

  4. 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 ? 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

  5. 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

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

    PubMed

    Metzger, Julia; Whlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Kch, 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

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

    PubMed

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

    2015-01-01

    Congenital nephrotic syndrome (CNS) is defined as heavy proteinuria or nephrotic syndrome occurring before 3 months of age. It is characterized by early onset and progresses to end-stage renal disease. Recently, several genes associated with CNS have been identified, including NPHS1 and NPHS2. Mutations in the NPHS1 gene have been identified in patients with CNS in Finland with relatively high frequency. Thus far, only a few case reports about CNS have described an NPHS1 mutation in China. In this study, mutational analyses of NPHS1 and NPHS2 were performed in a Chinese child with CNS. Mutations were analyzed in all exons and exon/intron boundaries of NPHS1 and NPHS2 in the patient and his parents as well as in 50 unrelated controls using polymerase chain reaction and direct sequencing techniques. No mutations were detected in NPHS2. A novel splice site mutation (IVS11+1G>A) within intron 11 and a missense mutation within exon 8 (c.928G>A) in the NPHS1 gene were detected in the child. The child's mother had normal urinalysis and a c.928G>A (D310N) heterozygous mutation, and his father had normal urinalysis and IVS11+1G>A. These were not identified in the 50 unrelated controls. The novel splice site mutation of IVS11+1G>A and a missense mutation at c.928G>A in NPHS1 were found to cause CNS in this Chinese child. PMID:25729976

  8. 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

  9. Donor-site morbidity and anterior knee problems after anterior cruciate ligament reconstruction using autografts.

    PubMed

    Kartus, J; Movin, T; Karlsson, J

    2001-01-01

    The authors review the current knowledge on donor site-related problems after using different types of autografts for anterior cruciate ligament (ACL) reconstruction and make recommendations on minimizing late donor-site problems. Postoperative donor-site morbidity and anterior knee pain following ACL surgery may result in substantial impairment for patients. The selection of graft, surgical technique, and rehabilitation program can affect the severity of pain that patients experience. The loss or disturbance of anterior sensitivity caused by intraoperative injury to the infrapatellar nerve(s) in conjunction with patellar tendon harvest is correlated with donor-site discomfort and an inability to kneel and knee-walk. The patellar tendon at the donor site has significant clinical, radiographic, and histologic abnormalities 2 years after harvest of its central third. Donor-site discomfort correlates poorly with radiographic and histologic findings after the use of patellar tendon autografts. The use of hamstring tendon autografts appears to cause less postoperative donor-site morbidity and anterior knee problems than the use of patellar tendon autografts. There also appears to be a regrowth of the hamstring tendons within 2 years of the harvesting procedure. There is little known about the effect on the donor site of harvesting fascia lata and quadriceps tendon autografts. Efforts should be made to spare the infrapatellar nerve(s) during ACL reconstruction using patellar tendon autografts. Reharvesting the patellar tendon cannot be recommended due to significant clinical, radiographic, and histologic abnormalities 2 years after harvesting its central third. It is important to regain full range of motion and strength after the use of any type of autograft to avoid future anterior knee problems. If randomized controlled trials show that the long-term laxity measurements following ACL reconstruction using hamstring tendon autografts are equal to those of patellar tendon autografts, we recommend the use of hamstring tendon autografts because there are fewer donor-site problems. PMID:11694930

  10. 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 ?-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

  11. Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4.

    PubMed Central

    Dagher, S F; Fu, X D

    2001-01-01

    The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition. Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing. PMID:11565750

  12. 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

  13. Honey Dressing Accelerates Split-Thickness Skin Graft Donor Site Healing.

    PubMed

    Subrahmanyam, M

    2015-12-01

    The management of the donor site after harvesting a skin graft is an important issue, as patients often report more discomfort at the donor site than at the recipient site. There is, however, a plethora of dressings available for the treatment and management of donor sites, yet, there is no widely accepted method established for these partial thickness wounds. Honey has been found to be useful in the treatment of burns and other wounds, split-thickness skin graft donor sites are like partial thickness burn wounds and honey's healing effect on burn wound can also be expected on these types of wounds. Therefore, this study was undertaken to evaluate the effect of honey on skin graft donor sites. From 2002 to 2004, 100 patients who have undergone skin grafting for various reasons formed the material of the randomized study divided into two groups of 50 each in honey-treated group and Vaseline gauze-treated group. Graft donor site area ranged from 30 to 48cm(2), mean 32.6cm(2). In the group treated with honey, 90% of the patients had nil or only moderate pain, whereas in the group treated with Vaseline gauze,88% had nil or mild pain (p?>?0.001, not significant). There were no allergic reactions in any of the patients in either group. On opening of the dressing on the 7th day, epithelialization has occurred in 48 patients as compared to 39 in group 2, i.e., donor sites treated with Vaseline gauze (p?donor sites wounds faster with good cosmetic result. PMID:26730006

  14. Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein

    PubMed Central

    Valas, Stephen; Rolland, Morgane; Perrin, Ccile; Perrin, Grard; Mamoun, Robert Z

    2008-01-01

    Background Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression. Results We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein. Conclusion The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions. PMID:18312636

  15. 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 insilico 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.20281G>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.14082A>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

  16. Probabilistic simple splicing systems

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  17. 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

  18. [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

  19. 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

  20. Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632.

    PubMed

    Somberg, Monika; Zhao, Xiaomin; Frhlich, Monika; Evander, Magnus; Schwartz, Stefan

    2008-04-01

    We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5' splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle. PMID:18216120

  1. A Splice Site Mutation in Laminin-?2 Results in a Severe Muscular Dystrophy and Growth Abnormalities in Zebrafish

    PubMed Central

    Gupta, Vandana A.; Kawahara, Genri; Myers, Jennifer A.; Chen, Aye T.; Hall, Thomas E.; Manzini, M. Chiara; Currie, Peter D.; Zhou, Yi; Zon, Leonard I.; Kunkel, Louis M.; Beggs, Alan H.

    2012-01-01

    Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-?2 gene (LAMA2). Laminin-?2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 815 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-?2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-?2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD. PMID:22952766

  2. 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

  3. 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

  4. 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

  5. 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

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

    PubMed Central

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

    2013-01-01

    Background In the treatment of burns, patients own skin is the preferred material to cover burn wounds, resulting in the need to create a donor site wound. Enhancement of healing of the donor site wound would be beneficial in burn patients. Insulin, an anabolic agent, is routinely used to treat hyperglycemia after injury. We investigated whether intensive insulin treatment (INS) increases fractional synthesis rate (FSR) of the donor site wound protein and decreases the length of hospitalization normalized for total body surface area burned (LOS/TBSA). Methods FSR of the donor site wound protein was measured in pediatric patients randomized to control (CNT) (n = 13) and INS (n = 10) treatments. Depending on the postoperative day when the tracer study was done studies were divided into Early (days < 5) and Late (days >=5) periods. Results FSR of the donor site wound protein was greater in the INS group at the Early period of wound healing (CNT vs. INS, 8.23.8 vs. 13.16.9 %/day, p: < 0.05); but not at the Late (CNT vs. INS, 19.74.6 vs. 16.64.0 %/day, p > 0.05). Despite these differences LOS/TBSA was not decreased in the INS group. Correlation analyses demonstrated that independently of the treatment regimen FSR positively correlated (p < 0.05) with time post creation of the donor site and negatively correlated (p < 0.05) with LOS/TBSA. Conclusions Insulin treatment increased FSR of the donor site wound protein in the early period of wound healing; FSR correlated with LOS/TBSA independently of the treatment regimen. PMID:21236451

  7. 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.

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

    PubMed

    Williams, Christopher; Hoppe, Hans-Jrgen; 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

  9. 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

  10. 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

  11. 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

  12. 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

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

    PubMed

    Catania, Francesco; Lynch, Michael

    2013-06-01

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

  14. 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

  15. Spliced leader RNA trans-splicing in dinoflagellates

    PubMed Central

    Zhang, Huan; Hou, Yubo; Miranda, Lilibeth; Campbell, David A.; Sturm, Nancy R.; Gaasterland, Terry; Lin, Senjie

    2007-01-01

    Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), to the 5? end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 5060 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter. PMID:17360573

  16. Evaluation of Amniotic Membrane Effectiveness in Skin Graft Donor Site Dressing in Burn Patients.

    PubMed

    Salehi, Seyed Hamid; As'adi, Kamran; Mousavi, Seyed Jaber; Shoar, Saeed

    2015-12-01

    Although the recipient site in burn wounds is dressed with universally accepted materials, the ideal management of split-thickness skin donor sites remains controversial. The aim of our study is to compare two methods of wound dressing in donor sites of split-thickness skin graft in patients undergoing burn wound reconstructive surgery. Forty-two consecutive patients with second- and third-degree burns with a total body surface area between 20 and 40% were enrolled in this randomized clinical trial conducted in Motahari Burn Hospital in Tehran, Iran. In each patient, two anatomic areas with similar features were randomly selected as intervention and control donor sites. The intervention site was dressed with amniotic membrane, whereas the control site was treated with Vaseline-impregnated gauze. Wounds were examined daily by expert surgeons to measure the clinical outcomes including duration of healing, severity of pain, and infection rate. The mean SD age of patients was 31.17??13.72years; furthermore, burn percentage had a mean SD of 31.19??10.56. The mean SD of patients' cooperation score was 1.6??0.79 in the intervention group compared with 2.93??0.71 in the control group, revealing a statistically significant difference (P?donor sites in the control and intervention groups (P?>?0.05). Amniotic membrane as an alternative for dressing of skin graft donor sites provides significant benefits by increasing patients' comfort via diminishing the number of dressing changes and facilitating the process of wound healing. PMID:26730039

  17. 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.; Set-Salvia, Nria; 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 iPSCneurons 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

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

    PubMed

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

    2015-09-15

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

  19. 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

  20. Necrosis of the donor site after hair restoration with follicular unit extraction (FUE): a case report.

    PubMed

    Karaal, Naci; Uralo?lu, Muhammet; Dindar, Tuba; Livao?lu, Murat

    2012-04-01

    Follicular unit extraction (FUE) has been developed as one type of follicular unit transplantation surgery, a widely accepted hair-restoration technique. FUE has many advantages, including a small donor area scar, less pain and a slender graft without extra surrounding tissue. Complications are uncommon in the literature. We describe a case of donor-site necrosis after hair restoration with FUE, leading to cicatricial alopecia in the left half of the occipital region. The surgical management of this complication is also discussed. Cicatricial alopecia was treated by two-stage surgery with tissue expanders. There were no complications in the postoperative period and healing was uneventful. We conclude that although FUE has many advantages, necrosis of the donor site may be a serious problem. PMID:21768010

  1. 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, Mara Cecilia; Sobrero, Gabriela; Miras, Mirta B; Targovnik, Hctor 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 169bp 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

  2. In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay.

    PubMed

    Ulzi, Gianna; Sansone, Valeria A; Magri, Francesca; Corti, Stefania; Bresolin, Nereo; Comi, Giacomo P; Lucchiari, Sabrina

    2014-05-01

    Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis. PMID:24452722

  3. 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

  4. 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

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

    PubMed

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

    2013-01-01

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

  6. 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

  7. 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

  8. RNA mis-splicing in disease.

    PubMed

    Scotti, Marina M; Swanson, Maurice S

    2016-01-01

    The human transcriptome is composed of a vast RNA population that undergoes further diversification by splicing. Detecting specific splice sites in this large sequence pool is the responsibility of the major and minor spliceosomes in collaboration with numerous splicing factors. This complexity makes splicing susceptible to sequence polymorphisms and deleterious mutations. Indeed, RNA mis-splicing underlies a growing number of human diseases with substantial societal consequences. Here, we provide an overview of RNA splicing mechanisms followed by a discussion of disease-associated errors, with an emphasis on recently described mutations that have provided new insights into splicing regulation. We also discuss emerging strategies for splicing-modulating therapy. PMID:26593421

  9. 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

  10. 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

  11. Splice junction mutations at the Menkes locus that maintain some proper splicing are associated with milder clinical phenotypes, including typical occipital horn syndrome

    SciTech Connect

    Kaler, S.G.; Gahl, W.A.

    1994-09-01

    Menkes disease is an X linked recessive disorder of copper metabolism produced by abnormalities in a gene that encodes a copper transporting ATPase. The clinical spectrum of Menkes disease includes a range of neurological severity from the classical type to the occipital horn syndrome (OHS) in which slightly subnormal intelligence or signs of autonomic dysfunction are the only neurologic abnormalities. We previously documented a distinctive, less severe Menkes phenotype associated with a +3 intronic splice donor mutation at the 3{prime} end of the gene in which exon skipping occurred but some normally spliced message was also detectable. We now report a similar splicing mutation in a patient with a typical OHS phenotype an A to G transition at the 2 exonic position of a splice donor site in the middle of the Menkes coding sequence. Some normally sized transcripts are evident by RT-PCR of lymphoblast mRNA from this individual, as well as 2 truncated fragments generated by exon skipping and activation of a cryptic splice acceptor site, respectively. The predicted effect of the mutation on the gene product involves a serine to glycine substitution in a noncritical region of the Menkes ATPase from the patient`s normally sized message, and premature termination due to translational frameshift in both truncated transcripts. The mutation eliminates a Dde 1 restriction site in the gene which provided a method to rapidly screen other family members, and revealed that the patient`s mother is a non-carrier. The mutational base change was not present in 25 normal X chromosomes studied. Preliminary analysis of the Menkes locus in 5 other Menkes disease families indicates aberrant mRNA splicing in 2. Our findings confirm allelism at the Menkes locus, indicate that splice mutations are relatively common mutational event in Menkes disease, and suggest that splice mutations in which some normal splicing is preserved may underlie milder Menkes disease variants, including OHS.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2015-01-01

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

  14. 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.

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

    PubMed

    Riecke, Bjrn; Kohlmeier, Carsten; Assaf, Alexandre T; Wikner, Johannes; Drabik, Anna; Catal-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

  16. Modeling the H bond donor strength of -OH, -NH, and -CH sites by local molecular parameters.

    PubMed

    Schwbel, Johannes; Ebert, Ralf-Uwe; Khne, Ralph; Schrmann, Gerrit

    2009-07-15

    A quantum chemical model is introduced to predict the H-bond donor strength of monofunctional organic compounds from their ground-state electronic properties. The model covers -OH, -NH, and -CH as H-bond donor sites and was calibrated with experimental values for the Abraham H-bond donor strength parameter A using the ab initio and density functional theory levels HF/6-31G** and B3LYP/6-31G**. Starting with the Morokuma analysis of hydrogen bonding, the electrostatic (ES), polarizability (PL), and charge transfer (CT) components were quantified employing local molecular parameters. With hydrogen net atomic charges calculated from both natural population analysis and the ES potential scheme, the ES term turned out to provide only marginal contributions to the Abraham parameter A, except for weak hydrogen bonds associated with acidic -CH sites. Accordingly, A is governed by PL and CT contributions. The PL component was characterized through a new measure of the local molecular hardness at hydrogen, eta(H), which in turn was quantified through empirically defined site-specific effective donor and acceptor energies, EE(occ) and EE(vac). The latter parameter was also used to address the CT contribution to A. With an initial training set of 77 compounds, HF/6-31G** yielded a squared correlation coefficient, r(2), of 0.91. Essentially identical statistics were achieved for a separate test set of 429 compounds and for the recalibrated model when using all 506 compounds. B3LYP/6-31G** yielded slightly inferior statistics. The discussion includes subset statistics for compounds containing -OH, -NH, and active -CH sites and a nonlinear model extension with slightly improved statistics (r(2) = 0.92). PMID:19037860

  17. ALTERNATIVE SPLICE VARIANTS OF PLASMA MEMBRANE CALCIUM-ATPases IN HUMAN CORNEAL EPITHELIUM

    PubMed Central

    Talarico, Ernest F.; Mangini, Nancy J.

    2007-01-01

    Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55 to 76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2(i). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2(i) variant. Finally, this study suggests that the molecular configuration of PMCA 1, 2 and 3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE. PMID:17931625

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  20. The Pathobiology of Splicing

    PubMed Central

    Ward, Amanda J.; Cooper, Thomas A.

    2010-01-01

    Ninety-four percent of human genes are discontinuous such that segments expressed as mRNA are contained within exons and separated by intervening segments, called introns. Following transcription, genes are expressed as precursor mRNAs (pre-mRNAs) which are spliced co-transcriptionally and the flanking exons are joined together to form a continuous mRNA. One advantage of this architecture is that it allows alternative splicing by differential use of exons to generate multiple mRNAs from individual genes. Regulatory elements located within introns and exons guide the splicing complex, the spliceosome, and auxiliary RNA binding proteins to the correct sites for intron removal and exon joining. Misregulation of splicing and alternative splicing can result from mutations in cis regulatory elements within the affected gene or from mutations that affect the activities of trans-acting factors that are components of the splicing machinery. Mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. An understanding of the role of splicing in disease expands potential opportunities for therapeutic intervention by either directly addressing the cause or by providing novel approaches to circumvent disease processes. PMID:19918805

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

    PubMed Central

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

    2013-01-01

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

  2. 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.

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

    PubMed Central

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

    2013-01-01

    Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF65 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 U2AF65 recognizes degenerate Py tracts, we determined six crystal structures of human U2AF65 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 U2AF65 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 U2AF65 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 U2AF65 conformations. Our results highlight both local and global conformational selection as a means for universal 3? splice site recognition by U2AF65. PMID:23376934

  4. An alternatively spliced region of the human hexabrachion contains a repeat of potential N-glycosylation sites.

    PubMed

    Gulcher, J R; Nies, D E; Marton, L S; Stefansson, K

    1989-03-01

    We have cloned and sequenced two cDNA molecules that code for parts of two forms of human hexabrachion. The smaller clone has a sequence that corresponds to the previously published sequence of a cDNA clone coding for a part of chicken hexabrachion [Jones, F. S., Burgoon, M. P., Hoffman, S., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1988) Proc. Natl. Acad. Sci. USA 85, 2186-2190]. It has eight consecutive domains similar to the type III homology units from fibronectin, several epidermal growth factor repeats, and a domain similar to the beta and gamma chains of fibrinogen. The larger clone has 5' and 3' ends that are identical to the smaller clone but also has an alternatively spliced 1.9-kilobase internal segment. The unique segment contains remarkable repeats of potential glycosylation sites and an additional seven type III homology units. PMID:2466295

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

    SciTech Connect

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

    1994-09-01

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

  6. Alternative splicing in the synaptic protein interaction site of rat Ca(v)2.2 (alpha (1B)) calcium channels: changes induced by chronic inflammatory pain.

    PubMed

    Asadi, Sareh; Javan, Mohammad; Ahmadiani, Abolhassan; Sanati, Mohammad Hossein

    2009-09-01

    Voltage-gated N-type Ca(2+) channels play a central role in regulation of neurotransmitter release. The II-III linker of N-type calcium channel alpha(1B) subunit (exons 17-21 in rats) contains synaptic protein interaction (synprint) site, which plays a pivotal role in efficient neurotransmission. Using RT-PCR analysis of rat hippocampus and lumbar spinal cord transcripts, the expressions of Ca(v)2.2 exon 18a and a novel Ca(v)2.2 splice variant, which were detected in this study, have been investigated, and they showed tissue-specific pattern. The new variant contains a large deletion in II-III linker that produces a stop codon, which is predicted to produce a two-domain-truncated channel. To study the effect of inflammatory pain on the expression pattern of these variants, animals were treated with intraplantar formalin, and the amount of splice variants in lumbar spinal cord was measured. The results showed that chronic inflammatory pain increases the Ca(v)2.2 mRNA levels lacking exon 18a and decreases the amount of full-length variants, which do not have any deletion. Determining different Ca(v)2.2 splice variants in rat nervous system and the impact of inflammatory pain on the splicing pattern suggest a possible regulatory role for calcium channel alternative splicing. PMID:19125229

  7. 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.

  8. Evidence for widespread association of mammalian splicing and conserved long-range RNA structures.

    PubMed

    Pervouchine, Dmitri D; Khrameeva, Ekaterina E; Pichugina, Marina Yu; Nikolaienko, Oleksii V; Gelfand, Mikhail S; Rubtsov, Petr M; Mironov, Andrei A

    2012-01-01

    Pre-mRNA structure impacts many cellular processes, including splicing in genes associated with disease. The contemporary paradigm of RNA structure prediction is biased toward secondary structures that occur within short ranges of pre-mRNA, although long-range base-pairings are known to be at least as important. Recently, we developed an efficient method for detecting conserved RNA structures on the genome-wide scale, one that does not require multiple sequence alignments and works equally well for the detection of local and long-range base-pairings. Using an enhanced method that detects base-pairings at all possible combinations of splice sites within each gene, we now report RNA structures that could be involved in the regulation of splicing in mammals. Statistically, we demonstrate strong association between the occurrence of conserved RNA structures and alternative splicing, where local RNA structures are generally more frequent at alternative donor splice sites, while long-range structures are more associated with weak alternative acceptor splice sites. As an example, we validated the RNA structure in the human SF1 gene using minigenes in the HEK293 cell line. Point mutations that disrupted the base-pairing of two complementary boxes between exons 9 and 10 of this gene altered the splicing pattern, while the compensatory mutations that reestablished the base-pairing reverted splicing to that of the wild-type. There is statistical evidence for a Dscam-like class of mammalian genes, in which mutually exclusive RNA structures control mutually exclusive alternative splicing. In sum, we propose that long-range base-pairings carry an important, yet unconsidered part of the splicing code, and that, even by modest estimates, there must be thousands of such potentially regulatory structures conserved throughout the evolutionary history of mammals. PMID:22128342

  9. Differential GC content between exons and introns establishes distinct strategies of splice-site recognition.

    PubMed

    Amit, Maayan; Donyo, Maya; Hollander, Dror; Goren, Amir; Kim, Eddo; Gelfman, Sahar; Lev-Maor, Galit; Burstein, David; Schwartz, Schraga; Postolsky, Benny; Pupko, Tal; Ast, Gil

    2012-05-31

    During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus "marking" them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition. PMID:22832277

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

    PubMed Central

    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.

    2009-01-01

    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(Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5? 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. PMID:19304800

  11. 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.

  12. Splicing predictions reliably classify different types of alternative splicing.

    PubMed

    Busch, Anke; Hertel, Klemens J

    2015-05-01

    Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5' or 3' splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

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

    PubMed

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

    2013-06-01

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

  14. [Myosonographic and electromyographic (EMC) evaluation of donor-site morbidity after microsurgical TRAM flap].

    PubMed

    Plogmeier, K; Schneider, W; Jost, S; Feistner, H; von Rohden, L; Fessel, L

    2003-01-01

    Due to improved microsurgical techniques the role of autologous breast reconstruction is expanding. One reason is the decreased donor-site morbidity compared to other techniques. Perforator based flaps (DIEP flaps) seemingly decrease the damage of the abdominal wall due to flap harvesting. However, complication rate in perforator flaps is still considered to be higher than in TRAM flaps. Additionally, operating time is increased due to the meticulous microsurgical dissection of perforators. To evaluate whether donor-site morbidity is caused by harvesting a part of the rectus muscle (as TRAM flaps), 15 patients after unilateral muscle sparing TRAM flap underwent EMG and myosonographic examination of the rectus muscle. EMG is the only technique to assess the muscle functionally, as the electrical activity of the muscle is recorded, and motor unit recruitment can be shown. EMG examination clearly revealed that harvesting of a muscle sparing TRAM flap did not impair the rectus muscle compared to the intact contralateral muscle. However, motor units were enlarged. Myosonography (in contrast to MRI) detected particular damage of isolated muscle fibres indicating nerve-related muscle changes. Taking all findings together, harvesting of a part of the rectus muscle shows a distinct damage to the muscle. The muscle however still acts functionally. This indicates that problems after harvesting a TRAM flap (bulging, hernias etc.) are not due to the partial removal of muscle, but due to the general dissection of the abdominal wall, as seen in other abdominal operations. PMID:12772062

  15. The influence of systemic growth hormone administration on the healing time of skin graft donor sites in a pig model.

    PubMed

    Ghofrani, A; Hller, D; Schuhmann, K; Saldern, S; Messmer, B J

    1999-08-01

    A more rapid healing of skin graft donor sites has often been observed during ultimoratio therapies with growth hormone in adults who have suffered extremely severe burns. The purpose of this animal experimental study was to examine the influence of systemic growth hormone administration on the healing time of skin graft donor sites under standardized conditions in pigs. The animals were 14 (7 experimental and 7 control) male, sexually mature, German domestic pigs, in which 30 skin graft donor sites 8 cm x 4 cm and 0.6 mm deep were created. Fifteen each of the skin graft donor sites were bandaged with the same material [hydrocolloid bandage (Varihaesive E) and PVP-iodine gauze (Braunovidon Gaze)]. The test period was 15 days for each pig, whereby recombinant growth hormone (0.5 IU/kg body weight per day) was applied subcutaneously in the experimental group. The bandages were changed under brief narcosis every 2 days, during which one skin-punch biopsy was taken per skin graft donor site, and blood samples were drawn for determination of the serum IGF-1 values. Photographic documentation was also recorded. The biopsies were examined histologically (hematoxylin and eosin stain) and immunohistochemically (collagen IV and VII, and laminin), whereby histologically the start of keratinization was assessed as a healing criterion. The serum IGF-1 values in the growth hormone group were statistically significantly higher than in the control group. Immunohistochemically, a complete basal membrane was observed in both the experimental and the control group after the 7th or 8th day. A clearly elevated serum IGF-1 level correlated in the growth hormone group with the skin graft donor sites healing. It could thus be demonstrated both clinically and histologically that systemic application of growth hormone results in a statistically significantly more rapid healing of the skin graft donor sites by 2 days earlier than in the control group. PMID:10654690

  16. [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

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

    PubMed Central

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

    1993-01-01

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

  18. 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

  19. Therapeutic Potential of Splice-Switching Oligonucleotides

    PubMed Central

    Bauman, John; Jearawiriyapaisarn, Natee

    2009-01-01

    Alternative splicing enables a single pre-messenger RNA transcript to yield multiple protein isoforms, making it a major contributor to the diversity of the proteome. While this process is essential for normal development, aberrations in alternative splicing are the cause of a multitude of human diseases. Methods for manipulating alternative splicing would thus be of therapeutic value. Chemically modified antisense oligonucleotides that alter alternative splicing by directing splice site selection have been developed to achieve this end. These splice-switching oligonucleotides (SSOs) have been applied to correct aberrant splicing, induce expression of a therapeutic splice variant, or induce expression of a novel therapeutic splice variant in a number of disease-relevant genes. Recently, in vivo efficacy of SSOs has been reported using animal disease models, as well as in results from the first clinical trial. PMID:19125639

  20. 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

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

    PubMed

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

    2015-07-01

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

  2. 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

  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. 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

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

    PubMed

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

    2015-01-01

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

  6. Internal Ribosome Entry Site Structural Motifs Conserved among Mammalian Fibroblast Growth Factor 1 Alternatively Spliced mRNAs

    PubMed Central

    Martineau, Yvan; Le Bec, Christine; Monbrun, Laurent; Allo, Valrie; Chiu, Ing-Ming; Danos, Olivier; Moine, Herv; Prats, Herv; Prats, Anne-Catherine

    2004-01-01

    Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5? untranslated regions (5? UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5? UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5? UTR databases. PMID:15314170

  7. Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs.

    PubMed

    Martineau, Yvan; Le Bec, Christine; Monbrun, Laurent; Allo, Valrie; Chiu, Ing-Ming; Danos, Olivier; Moine, Herv; Prats, Herv; Prats, Anne-Catherine

    2004-09-01

    Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases. PMID:15314170

  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. Adjusting the attB site in donor plasmid improves the efficiency of ?C31 integrase system.

    PubMed

    Xie, Fei; Ma, Qingwen; Jiang, Shizhong; Ren, Zhaorui; Wang, Juan; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2012-07-01

    ?C31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ?C31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ?C31 integrase system. PMID:22489575

  10. 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

  11. 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

  12. 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

  13. 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.

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

    PubMed

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

    2015-01-01

    Central quadriceps tendon (CQT) graft has been successfully used as a viable autograft option in cruciate ligament reconstruction of the knee. The prime emphasis in the majority of the literature is given to surgical details of quadriceps graft harvesting and outcome of cruciate ligament reconstruction. There is less discussion about donor site morbidity in CQT graft, and it is less frequent as compared to that in bone patellar tendon bone graft. We report an extremely unusual case of late quadriceps tendon rupture at the donor site following anterior cruciate ligament reconstruction using CQT graft. PMID:26475883

  15. 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/.

  16. 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

  17. Adenosine deaminase deficiency with mosaicism for a "second-site suppressor" of a splicing mutation: decline in revertant T lymphocytes during enzyme replacement therapy.

    PubMed

    Arredondo-Vega, Francisco X; Santisteban, Ines; Richard, Eva; Bali, Pawan; Koleilat, Majed; Loubser, Michael; Al-Ghonaium, Abdulaziz; Al-Helali, Mariam; Hershfield, Michael S

    2002-02-01

    Four patients from 3 Saudi Arabian families had delayed onset of immune deficiency due to homozygosity for a novel intronic mutation, g.31701T>A, in the last splice acceptor site of the adenosine deaminase (ADA) gene. Aberrant splicing mutated the last 4 ADA amino acids and added a 43-residue "tail" that rendered the protein unstable. Mutant complementary DNA (cDNA) expressed in Escherichia coli yielded 1% of the ADA activity obtained with wild-type cDNA. The oldest patient, 16 years old at diagnosis, had greater residual immune function and less elevated erythrocyte deoxyadenosine nucleotides than his 4-year-old affected sister. His T cells and Epstein-Barr virus (EBV) B cell line had 75% of normal ADA activity and ADA protein of normal size. DNA from these cells and his whole blood possessed 2 mutant ADA alleles. Both carried g.31701T>A, but one had acquired a deletion of the 11 adjacent base pair, g.31702-12, which suppressed aberrant splicing and excised an unusual purine-rich tract from the wild-type intron 11/exon 12 junction. During ADA replacement therapy, ADA activity in T cells and abundance of the "second-site" revertant allele decreased markedly. This finding raises an important issue relevant to stem cell gene therapy. PMID:11807006

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

    PubMed Central

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

    2015-01-01

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

  19. 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. RNA splicing and genes

    SciTech Connect

    Sharp, P.A.

    1988-11-25

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

  1. Recursive splicing in long vertebrate genes

    PubMed Central

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

    2015-01-01

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

  2. Recursive splicing in long vertebrate genes.

    PubMed

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

    2015-05-21

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

  3. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

    PubMed Central

    Schneider, Cornelius; Agafonov, Dmitry E.; Schmitzov, Jana; Hartmuth, Klaus; Fabrizio, Patrizia; Lhrmann, Reinhard

    2015-01-01

    Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast Bact spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the Bact crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing. PMID:26393790

  4. Control of the vascular endothelial growth factor internal ribosome entry site (IRES) activity and translation initiation by alternatively spliced coding sequences.

    PubMed

    Bornes, Stphanie; Boulard, Mathieu; Hieblot, Corinne; Zanibellato, Catherine; Iacovoni, Jason S; Prats, Herv; Touriol, Christian

    2004-04-30

    The vascular endothelial growth factor-A (VEGF) gene locus contains eight exons that span 14 kb. Alternative splicing generates multiple, different mRNAs that in turn translate into at least five protein isoforms. While the canonical AUG start codon is located at position 1039 in exon 1, there also exists an upstream, in-frame CUG initiation codon that drives expression of L-VEGF, containing an additional 180 amino acids. Two separate internal ribosome entry sites (IRES) regulate the activity of each initiation codon. Thus the 5'-UTR of VEGF, which comprises the majority of exon 1, consists of IRES B, the CUG, IRES A, and the AUG, from 5' to 3'. Previously, it has been shown that IRES B regulates initiation at the CUG and IRES A regulates AUG usage. In this study, we have found evidence that the exon content of the VEGF mRNA, determined through alternative splicing, controls IRES A activity. While the CUG is most efficient at initiating translation, transcripts that lack both exons 6 and 7 and therefore contain an exon 5/8 junction lack AUG-initiated translation. The process of splicing is not responsible for this start codon selection since transfection of genomic and cDNA VEGF sequences give the same expression pattern. We hypothesize that long range tertiary interactions in the VEGF mRNA regulate IRES activity and thus control start codon selection. This is the first report describing the influence of alternatively spliced coding sequences on codon selection by modulating IRES activity. PMID:14764596

  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. Species-Dependent Splice Recognition of a Cryptic Exon Resulting from a Recurrent Intronic CEP290 Mutation that Causes Congenital Blindness

    PubMed Central

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

    2015-01-01

    A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo. PMID:25761237

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

    PubMed

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

    2015-01-01

    A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo. PMID:25761237

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

    PubMed Central

    2012-01-01

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

  10. Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1.

    PubMed Central

    Arrigo, S J; Weitsman, S; Zack, J A; Chen, I S

    1990-01-01

    Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins. Images PMID:2384924

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

    PubMed Central

    Smalle, Jan A

    2013-01-01

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

  12. 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

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

    PubMed Central

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

    2013-01-01

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

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2014-02-01

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

  18. [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

  19. 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

  20. Versatility of retroauricular mastoid donor site: a convenient valuable warehouse of various free graft tissues in cosmetic and reconstructive surgery.

    PubMed

    Cho, Jeong Mok; Jeong, Jae Hoon; Woo, Kevin Volt; Lee, Yoon Ho

    2013-01-01

    Soft-tissue deficiency is a critical issue in facial cosmetic and reconstructive surgery. Harvesting autografts from other anatomical sites has been a common practice in overcoming soft-tissue insufficiency for many years. However, donor-site complications and visible scars are of important concerns. Therefore, we would like to introduce an alternative donor site of free-tissue grafts and its inherent advantages: the retroauricular mastoid area located along the mastoid hair line. From August 1991 to June 2011, we performed facial reconstructive surgeries for cosmetic correction of disfigurements from both congenital and complications of previous cosmetic procedures on a total of 213 patients. These patients had undergone either 1 or more facial cosmetic surgeries in the past. In this study, our primary goal focused on revising facial asymmetries or defects from previous surgical scars, tissue contraction, undercorrection, or underdevelopment. For autograft harvesting, we incised an elliptical shape along the retroauricular hairline. We then harvested sufficient amount of skin, dermal fat, fascia, or a piece of the mastoid bone if needed. After harvesting, we closed the incisional area and covered it with a compressive dressing. In evaluation of our results, we compared the preoperative photographs with postoperative and constructed a survey on patient satisfaction. Overall, the patients in this study were greatly satisfied with their surgical results. No major complications were reported. As a result of our long-term study, we believe that the retroauricular mastoid area has been shown to be an indispensable donor site for a variety of autograft tissues in terms of safety, convenience, and versatility of its unique structural composition consisting of skin, dermal fat, fascia, and bone. PMID:24036825

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

    PubMed Central

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

    1999-01-01

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

  2. 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

  3. Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11-48K and U11/U12-65K genes.

    PubMed

    Niemel, Elina H; Verbeeren, Jens; Singha, Prosanta; Nurmi, Visa; Frilander, Mikko J

    2015-11-01

    Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5' splice sites (5'ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50nt downstream from the target 3' splice site (3'ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3'ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5'ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5'ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions. PMID:26479860

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

    PubMed

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

    2016-03-01

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

  5. 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

  6. 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

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

    PubMed

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

    2013-01-01

    Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1. PMID:23342054

  8. 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 parasites 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 parasites transcriptome. PMID:21245033

  9. Chromatin's thread to alternative splicing regulation.

    PubMed

    Iannone, Camilla; Valcrcel, Juan

    2013-12-01

    Intron removal (pre-mRNA splicing) is a necessary step for expression of most genes in higher eukaryotes. Alternative splice site selection is a prevalent mechanism that diversifies genome outputs and offers ample opportunities for gene regulation in these organisms. Pre-mRNA splicing occurs co-transcriptionally and is influenced by features in chromatin structure, including nucleosome density and epigenetic modifications. We review here the molecular mechanisms by which the reciprocal interplay between chromatin and RNA processing can contribute to alternative splicing regulation. PMID:23912688

  10. 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

  11. A quantitative analysis of donor site morbidity after vascularized fibula transfer.

    PubMed

    Youdas, J W; Wood, M B; Cahalan, T D; Chao, E Y

    1988-01-01

    Eleven patients with free vascularized fibula graft transplants to the upper extremity were studied for donor-side morbidity effects. Based on their surgical follow-up times, the patients were divided into either group 1 (5 +/- 2 months) or group 2 (21 +/- 8 months) to examine the effects of surgical trauma more closely. Gait evaluation was performed using two walkways containing different ground conditions. Knee and ankle-foot motion was measured using a three-dimensional electrogoniometer. Basic temporodistance factors and ground reactions were obtained from foot switches, instrumented floor mats, and a force plate. A modified Cybex II isokinetic dynamometer was used to quantitate ankle-foot strengths. Moderate knee and ankle-foot motion changes were seen while walking on the level, sideslope, and ramp in patients evaluated less than 10 months after their surgical procedure. Minimal joint motion effects were discovered in patients with follow-up times longer than 10 months. Muscle strengths were significantly impaired in both groups, especially foot inversion and eversion. An inverse relationship existed between the length of resected fibula and ankle evertor muscle strength. Donor side morbidity does not appear to be caused by surgical soft-tissue trauma exclusively. These patients need to be studied for several more years to determine the long-term functional effects of free vascularized fibular graft transfer. PMID:3404318

  12. Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

    PubMed Central

    Schwartz, Schraga; Silva, Joo; Burstein, David; Pupko, Tal; Eyras, Eduardo; Ast, Gil

    2008-01-01

    Introns are among the hallmarks of eukaryotic genes. Splicing of introns is directed by three main splicing signals: the 5? splice site (5?ss), the branch site (BS), and the polypyrimdine tract/3?splice site (PPT-3?ss). To study the evolution of these splicing signals, we have conducted a systematic comparative analysis of these signals in over 1.2 million introns from 22 eukaryotes. Our analyses suggest that all these signals have dramatically evolved: The PPT is weak among most fungi, intermediate in plants and protozoans, and strongest in metazoans. Within metazoans it shows a gradual strengthening from Caenorhabditis elegans to human. The 5?ss and the BS were found to be degenerate among most organisms, but highly conserved among some fungi. A maximum parsimony-based algorithm for reconstructing ancestral position-specific scoring matrices suggested that the ancestral 5?ss and BS were degenerate, as in metazoans. To shed light on the evolutionary variation in splicing signals, we have analyzed the evolutionary changes in the factors that bind these signals. Our analysis reveals coevolution of splicing signals and their corresponding splicing factors: The strength of the PPT is correlated to changes in key residues in its corresponding splicing factor U2AF2; limited correlation was found between changes in the 5?ss and U1 snRNA that binds it; but not between the BS and U2 snRNA. Thus, although the basic ability of eukaryotes to splice introns has remained conserved throughout evolution, the splicing signals and their corresponding splicing factors have considerably evolved, uniquely shaping the splicing mechanisms of different organisms. PMID:18032728

  13. Porcine dermal collagen as a wound dressing for skin donor sites and deep partial skin thickness burns.

    PubMed

    Gao, Z R; Hao, Z Q; Li, Y; Im, M J; Spence, R J

    1992-12-01

    Collagen was extracted by pepsin digestion from porcine skin, and collagen membrane was prepared by salt precipitation. The porcine collagen membrane was evaluated as a burn wound dressing in deep partial skin thickness burn wounds in rats. Burn wounds, 4 x 4 cm, were inflicted by exposure of skin to 75 degrees C for 15 s followed by de-epithelialization. Wound healing was assessed by planimetry of epithelialization on day 10 after injury. Open wounds exhibited 24 per cent of wound area re-epithelialized. Collagen membrane dressing significantly improved the healing to 69 per cent of wound area (P < 0.0001). In a completely separate experiment, the porcine collagen membrane was applied as a wound dressing to the donor sites of burn patients, and its effect on wound healing was compared with that of a petroleum jelly gauze dressing. The donor sites covered with petroleum jelly gauze had re-epithelialized by an average of 14.5 days (ranging from 13 to 16 days) after wounding. The wounds dressed with collagen membrane demonstrated a significant increase in the healing rate. Complete re-epithelialization was observed by 10.3 days (ranging from 10 to 12 days) after wounding (P < 0.0001). PMID:1489500

  14. 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

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

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

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

  16. 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

  17. 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.

  18. A novel splice site mutation in collagen type VII gene in a Chinese family with dominant dystrophic epidermolysis bullosa pruriginosa.

    PubMed

    Jiang, Wei; Bu, Dingfang; Yang, Yong; Zhu, Xuejun

    2002-01-01

    Dystrophic epidermolysis bullosa pruriginosa, a subtype of epidermolysis bullosa dystrophica and a heterogeneous inherited disease, is characterized by pruritus, excoriated nodular prurigo-like lesions, skin fragility, altered anchoring fibrils and loss of dermal-epidermal adhesion. Mutation in type VII collagen gene (COL7A1) is thought to be implicated in the underlying change for dystrophic epidermolysis bullosa pruriginosa. We report here a large family of dominant dystrophic epidermolysis bullosa pruriginosa. Mutation analysis using polymerase chain reaction and direct sequencing demonstrated a novel nucleotide substitution of 6899A-->G in exon 87 in one COL7A1 allele of the proband and 18 affected family members. This substitution was not found in 100 normal alleles. Polymerase chain reaction and sequencing of the cDNA, reverse transcribed from the proband's peripheral lymphocyte RNA, suggest that this mutation causes aberrant COL7A1 mRNA splicing of exon 87 skipping. Clinical features and pedigree analysis suggest that 6899A-->G substitution is a mutation with full penetrance and variable expressivity. PMID:12353709

  19. 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.

  20. Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome.

    PubMed

    Bothwell, Susan P; Farber, Leslie W; Hoagland, Adam; Nussbaum, Robert L

    2010-10-01

    The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P(2)) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P(2) 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome. PMID:20872266

  1. 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

  2. T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.

    PubMed Central

    Qian, L; Theodor, L; Carter, M; Vu, M N; Sasaki, A W; Wilkinson, M F

    1993-01-01

    The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing. Images PMID:8441406

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Direct Repression of Splicing by transformer-2

    PubMed Central

    Chandler, Dawn S.; Qi, Junlin; Mattox, William

    2003-01-01

    The Drosophila melanogaster sex determination factor Tra2 positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs but negatively affects the splicing of the M1 intron in tra2 pre-mRNA. Retention of the M1 intron is known to be part of a negative-feedback mechanism wherein the Tra2 protein limits its own synthesis, but the mechanism responsible for accumulation of M1-containing RNA is unknown. Here we show that the recombinant Tra2 protein specifically represses M1 splicing in Drosophila nuclear extracts. We find that the Tra2 protein binds directly to several sites in and near the M1 intron and that, when Tra2 binding is competed with other RNAs, the splicing of M1 is restored. Mapping the RNA sequences functionally required for M1 repression identified both a 34-nucleotide (nt) A/C-rich sequence immediately upstream of the M1 5? splice site and a region within the intron itself. The AC-rich sequence is largely composed of a repeated 4-nt sequence that also forms a subrepeat within the repeated 13-nt splicing enhancer elements of fru and dsx RNAs. Although required for repression, the element also enhances M1 splicing in the absence of Tra2. We propose that Tra2 represses M1 splicing by interacting with multiple sequences in the pre-mRNA and interfering with enhancer function. PMID:12861004

  9. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-? -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 ? -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and ? -globin sequences, we show that the NS1 5' splice site was effectively utilized by the ? -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 ? -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from ? -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.

  10. 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

  11. Identification of two splicing mutations in the collagen type VII gene (COL7A1) of a patient affected by the localisata variant of recessive dystrophic epidermolysis bullosa.

    PubMed Central

    Gardella, R.; Belletti, L.; Zoppi, N.; Marini, D.; Barlati, S.; Colombi, M.

    1996-01-01

    Collagen type VII gene (COL7A1) has been demonstrated to be altered in several variants of dystrophic epidermolysis bullosa (DEB), with either recessive or dominant mode of inheritance. We have identified two mutations in a patient affected by a localisata variant of recessive DEB (L-RDEB), which is characterized by the less severe phenotype of the syndrome. These mutations are the first splicing mutations so far described for COL7A1 in DEB. One mutation is a paternally inherited A-->G transition at position -2 of the donor splicing site of intron 3, which results in three aberrant mRNAs, depending on the skipping of exon 3, the usage of a cryptic donor site inside exon 3, or the maintenance of intron 3. The second mutation is a maternally inherited G-->A transition at position -1 of the donor splicing site of intron 95, which induces the activation of a cryptic donor site 7 nt upstream the normal site and gives rise to a deleted mRNA, in addition to the normal one. All aberrant mRNAs show a shift of the reading frame, thus generating premature termination codons of translation. Allele-specific analysis of the transcripts has shown that the maternal mutation does not completely abolish the correct splicing of COLVII pre-mRNA, thus allowing, in the patient, the synthesis of a certain level of a functional protein. This result is compatible with the mild clinical L-RDEB phenotype observed in our patient. Images Figure 1 Figure 2A & B Figure 2C Figure 3A & B Figure 3C Figure 4 Figure 5 PMID:8755915

  12. Discovery of Candidate Disease Genes in ENUInduced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2014-01-01

    The 22q11.2 deletion syndrome (22q11DS) is thought to be a contiguous gene syndrome caused by haploinsufficiency for a variable number of genes with overlapping function during the development of the craniofacial, pharyngeal and cardiac structures. The complexity of genetic and developmental anomalies resulting in 22q11DS has made attributing causation to specific genes difficult. The CRKL gene resides within the common 3-Mb region, most frequently affected in 22q11DS, and has been shown to play an essential role in the development of tissues affected in 22q11DS. Here, we report the characterisation of a mouse strain we named snoopy, harbouring a novel Crkl splice-site mutation that results in a loss of Crkl expression. The snoopy strain exhibits a variable phenotype that includes micrognathia, pharyngeal occlusion, aglossia and holoprosencephaly, and altered retinoic acid and endothelin signalling. Together, these features are reminiscent of malformations occurring in auriculocondylar syndrome and agnathia-otocephaly complex, 2 conditions not previously associated with the CRKL function. Comparison of the features of a cohort of patients harbouring small 22q11.2 deletions centred over the CRKL gene, but sparing TBX1, highlights the role of CRKL in contributing to the craniofacial features of 22q11DS. These analyses demonstrate the central role of Crkl in regulating signalling events in the developing oropharyngeal complex and its potential to contribute to dysmorphology. PMID:25565927

  14. 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

  15. MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis.

    PubMed

    Koh, Cheryl M; Bezzi, Marco; Low, Diana H P; Ang, Wei Xia; Teo, Shun Xie; Gay, Florence P H; Al-Haddawi, Muthafar; Tan, Soo Yong; Osato, Motomi; Sab, Arianna; Amati, Bruno; Wee, Keng Boon; Guccione, Ernesto

    2015-07-01

    Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here we show that during lymphomagenesis in E-myc transgenic mice, MYC directly upregulates the transcription of the core small nuclear ribonucleoprotein particle assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for the core biogenesis of small nuclear ribonucleoprotein particles, effective pre-messenger-RNA splicing, cell survival and proliferation. Our results demonstrate that MYC maintains the splicing fidelity of exons with a weak 5' donor site. Additionally, we identify pre-messenger-RNAs that are particularly sensitive to the perturbation of the MYC-PRMT5 axis, resulting in either intron retention (for example, Dvl1) or exon skipping (for example, Atr, Ep400). Using antisense oligonucleotides, we demonstrate the contribution of these splicing defects to the anti-proliferative/apoptotic phenotype observed in PRMT5-depleted E-myc B cells. We conclude that, in addition to its well-documented oncogenic functions in transcription and translation, MYC also safeguards proper pre-messenger-RNA splicing as an essential step in lymphomagenesis. PMID:25970242

  16. 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.

  17. 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

  18. The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro

    SciTech Connect

    Furdon, P.J.; Kole, R.

    1988-02-01

    The authors have shown previously that truncation of the human ..beta..-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. They now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit ..beta..-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a ''poison'' sequence that can inhibit the splicing of an upstream intron.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5' splice site.

    PubMed

    Boesler, Carsten; Rigo, Norbert; Agafonov, Dmitry E; Kastner, Berthold; Urlaub, Henning; Will, Cindy L; Lhrmann, Reinhard

    2015-11-01

    Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation. PMID:26385511

  5. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  8. 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

  9. Prolyl 4-hydroxylase genes are subjected to alternative splicing in roots of maize seedlings under waterlogging

    PubMed Central

    Zou, Xiling; Jiang, Yuanyuan; Zheng, Yonglian; Zhang, Meidong; Zhang, Zuxin

    2011-01-01

    Background In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize. Methods A comprehensive genome-wide analysis of P4H genes of maize (zmP4H genes) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Key Results Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites was strongly correlated with selection of donor and receptor sites, and ambiguous junction sites due to small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harboured a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. Reverse transcriptionPCR (RTPCR) indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4H genes were under specific control by post-transcriptional regulation under waterlogging stress in the line HZ32. Conclusions Our results provide a framework for future dissection of the function of the emerging zmP4H family and suggest that AS might have an important role in the regulation of the expression profile of this gene family under waterlogging stress. PMID:21969257

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

    PubMed

    Uralo?lu, Muhammet; Livao?lu, Murat; Agdo?an, zgr; Mungan, Sevdegl; Alhan, Etem; Karaal, Naci

    2014-02-01

    The objective of this study was to investigate the healing effect of five different products on split-thickness skin graft (STSG) donor sites and full-thickness cutaneous wounds (FTCWs) using an occlusive dressing model. Six groups were included: 1 control and 5 experimental groups, with a total of 24 rats, using an occlusive dressing model. STSG donor sites and FTCWs were established in two separate areas, to the right and left on the animals' backs. Wound sites were dressed with one of the following materials: fine mesh gauze, microporous polysaccharide hemosphere (MPH), clinoptilolite, alginate, hydrogel or biosynthetic wound dressing (Biobran() ). These materials were compared in terms of healing rate, healing quality and histopathological findings. Occlusive dressings were applied to each wound on days 0, 3, 5, 7, 10 and 14. Area measurements were taken using images of each dressing. The alginate and clinoptilolite groups gave the best healing rate results for both STSG donor sites (P = 0003) and FTCWs (P = 0003). MPH came third in each group. The alginate group produced better results in terms of healing quality criteria, followed by hydrogel, MPH, clinoptilolite and Biobran() , in that order. Statistically significant results were obtained in all groups compared to the control group (P < 00007). Rapid and good healing quality for both the STSG donor sites and FTCWs were obtained with alginate. Healing with clinoptilolite and MPH was rapid, but poor quality, while slower but good healing quality was obtained with hydrogel. Slower and worse quality healing was obtained with Biobran() . PMID:22943661

  13. 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...

  14. Quantitative evaluation of all hexamers as exonic splicing elements.

    PubMed

    Ke, Shengdong; Shang, Shulian; Kalachikov, Sergey M; Morozova, Irina; Yu, Lin; Russo, James J; Ju, Jingyue; Chasin, Lawrence A

    2011-08-01

    We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 3' splice sites tend to be single stranded, and effective 5' splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms. PMID:21659425

  15. Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

    PubMed Central

    Dietrich, R C; Shukla, G C; Fuller, J D; Padgett, R A

    2001-01-01

    Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo. PMID:11680842

  16. Progressive valgus deformity of the donor-site ankle after extraperiosteal harvesting the fibular shaft in children. Treatment with osteotomy and synostosis at one session.

    PubMed

    Van der Veen, Fientje J C; Strackee, Simon D; Besselaar, Philip P

    2015-10-01

    After extraperiosteal harvesting of the fibular shaft in children, progressive valgus deformity of the donor-site ankle may pose a serious problem. We present three illustrative case-histories: three children became functionally impaired and required surgery. Pathogenesis, natural history and surgical options are discussed. A supramalleolar osteotomy combined with a distal fibular-tibial synostosis is a good option to correct valgus and eliminate instability in one surgical session. PMID:26719617

  17. 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

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

    SciTech Connect

    Vermaas, W.F.J.; Rutherford, A.W.; Hansson, O. )

    1988-11-01

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

  19. 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.

  20. 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.

  1. 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

  2. Single-molecule imaging of transcriptionally coupled and uncoupled splicing.

    PubMed

    Vargas, Diana Y; Shah, Khyati; Batish, Mona; Levandoski, Michael; Sinha, Sourav; Marras, Salvatore A E; Schedl, Paul; Tyagi, Sanjay

    2011-11-23

    Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally. PAPERCLIP: PMID:22118462

  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. Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection.

    PubMed

    Alsina, L; Colobran, R; de Sevilla, M F; Catal, A; Vias, L; Ricart, S; Plaza, A M; Lois, S; Juan, M; Pujol-Borrell, R; Martinez-Gallo, M

    2014-08-01

    Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753+1G>T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448-13G>A) at the 3' acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype. PMID:24825797

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

    PubMed

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

    2014-10-01

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

  6. Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini.

    PubMed Central

    Benjamin, H W; Kleckner, N

    1992-01-01

    Tn10 transposition is accomplished without extensive replication of the transposon sequences. Replicative cointegrate formation is precluded by efficient separation of transposon sequences from flanking donor DNA at an early stage in the transposition reaction. We report here that excision of Tn10 from its donor site occurs by a pair of flush double-strand breaks. Breaks occur at each end of the element precisely between the terminal base pair of the element and the first base pair of flanking DNA. This observation provides definitive evidence that cleavage of both strands of the element occurs under the direct control of Tn10 transposase protein. It is highly likely that transposase itself is directly responsible for these cleavages. The implications of this possibility are discussed. Images PMID:1316613

  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. Chromatin and alternative splicing.

    PubMed

    All, M; Schor, I E; Muoz, M J; de la Mata, M; Agirre, E; Valcrcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing. PMID:21289049

  9. 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

  10. 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 CAPER?. 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

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

    PubMed Central

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

    2016-01-01

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

  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. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exonintron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing mutations underlying human disease. PMID:22160217

  14. 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

  15. 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

  16. Use of Polymethyl Methacrylate-Based Cement for Cosmetic Correction of Donor-Site Defect following Transposition of Temporalis Myofascial Flap and Evaluation of Results after Adjuvant Radiotherapy.

    PubMed

    Mandlik, Dushyant; Gupta, Karan; Patel, Daxesh; Patel, Purvi; Toprani, Rajendra; Patel, Kaustubh

    2015-11-01

    Background?Temporalis myofascial flap is a versatile flap for reconstruction of the oral cavity defects, but results in an esthetically compromised deformity at the donor site. We used polymethyl methacrylate (PMMA) cement to correct the volume loss defect caused by temporalis myofascial flap and evaluated its results before and after adjuvant radiotherapy. Methods?We discuss our experience of using PMMA cement to augment donor-site deformity in 25 patients (17 males, 8 females) between years 2005 and 2009. The primary defect was a result of the ablative surgery for squamous cell carcinoma of the upper alveolar and the buccoalveolar sulcus. A modified curved hemicoronal incision was used as an access for better cosmetic outcome. The volume of cement required was decided during the surgery. Results?All patients are in regular follow-up, alive and free of complications at implant site, except one patient who developed wound dehiscence. The condition of the implant was evaluated by postoperative computed tomographic scan, repeated after adjuvant radiotherapy in cases required. There were no radiation-induced changes in the contour and volume of the implants. Cosmetic result of the implant was reported satisfactory by the patients postoperatively. Conclusion?Restoration of the temporal area defect after the temporalis myofascial flap harvest with the use of PMMA cement is an easy and safe method, with excellent esthetic results. The implant is stable and resistant to any changes in contour and loss of volume even after adjuvant radiotherapy, with no added morbidity to the patients. PMID:26280521

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

    PubMed Central

    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-? (IFN-?) 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-? 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

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

    PubMed

    g, Norbert; Flipphi, Michel; Karaffa, Levente; Scazzocchio, Claudio; Fekete, Erzsbet

    2015-12-01

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

  19. Pre-mRNA splicing is a determinant of nucleosome organization.

    PubMed

    Keren-Shaul, Hadas; Lev-Maor, Galit; Ast, Gil

    2013-01-01

    Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5' splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5' splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization. PMID:23326444

  20. Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1.

    PubMed Central

    Amendt, B A; Hesslein, D; Chang, L J; Stoltzfus, C M

    1994-01-01

    The human immunodeficiency virus type 1 (HIV-1) RNA follows a complex splicing pathway in which a single primary transcript either remains unspliced or is alternatively spliced to more than 30 different singly and multiply spliced mRNAs. We have used an in vitro splicing assay to identify cis elements within the viral genome that regulate HIV-1 RNA splicing. A novel splicing regulatory element (SRE) within the first tat coding exon has been detected. This element specifically inhibits splicing at the upstream 3' splice site flanking this tat exon. The element only functions when in the sense orientation and is position dependent when inserted downstream of a heterologous 3' splice site. In vivo, an HIV-1 SRE mutant demonstrated a decrease in unspliced viral RNA, increased levels of single- and double-spliced tat mRNA, and reduced levels of env and rev mRNAs. In addition to the negative cis-acting SRE, the flanking 5' splice site downstream of the first tat coding exon acts positively to increase splicing at the upstream 3' splice sites. These results are consistent with hypotheses of bridging interactions between cellular factors that bind to the 5' splice site and those that bind at the upstream 3' splice site. Images PMID:8196635

  1. 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.

  2. 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

  3. The alternative role of DNA methylation in splicing regulation.

    PubMed

    Lev Maor, Galit; Yearim, Ahuvi; Ast, Gil

    2015-05-01

    Although DNA methylation was originally thought to only affect transcription, emerging evidence shows that it also regulates alternative splicing. Exons, and especially splice sites, have higher levels of DNA methylation than flanking introns, and the splicing of about 22% of alternative exons is regulated by DNA methylation. Two different mechanisms convey DNA methylation information into the regulation of alternative splicing. The first involves modulation of the elongation rate of RNA polymerase II (Pol II) by CCCTC-binding factor (CTCF) and methyl-CpG binding protein 2 (MeCP2); the second involves the formation of a protein bridge by heterochromatin protein 1 (HP1) that recruits splicing factors onto transcribed alternative exons. These two mechanisms, however, regulate only a fraction of such events, implying that more underlying mechanisms remain to be found. PMID:25837375

  4. Grouted sleeve tubular splices

    SciTech Connect

    Grundy, P.

    1995-12-31

    Using the classical theory of bending of cylindrical shells the key parameters in the shear transfer between inner and outer members of plain pipe splices are identified. Assuming no slip the analysis establishes that shear transfer occurs at each end of the splice with a characteristic wavelength. The generation of radial stress in the grout, which enhances shear transfer, is discussed. First slip at higher loads progresses from the ends, leading to a residual stress state which enhances resistance to further slip. The enhancement of capacity through chemical prestress is discussed.

  5. 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

  6. 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.

  7. 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

  8. Mechanisms and Regulation of Alternative Pre-mRNA Splicing.

    PubMed

    Lee, Yeon; Rio, Donald C

    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

  9. Alternative splicing generates secretory isoforms of human CD1.

    PubMed Central

    Woolfson, A; Milstein, C

    1994-01-01

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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Pre-mRNA splicing in the absence of an SR protein RS domain

    PubMed Central

    Zhu, Jun; Krainer, Adrian R.

    2000-01-01

    SR proteins are essential pre-mRNA splicing factors that act at the earliest stages of splice-site recognition and spliceosome assembly, as well as later in the splicing pathway. SR proteins consist of one or two RNA-recognition motifs and a characteristic arginine/serine-rich C-terminal RS domain. The RS domain, which is extensively phosphorylated, mediates the subcellular localization of individual SR proteins and also functions as a splicing activation module, apparently by engaging in proteinprotein interactions. The RS domain of SF2/ASF is dispensable for the concentration-dependent effects of this SR protein on alternative splice-site selection. However, this RS domain is highly conserved phylogenetically, and was shown to be required for constitutive splicing in vitro and for cell viability. Here, we demonstrate that the RS domain of SF2/ASF is, in fact, dispensable for splicing of several substrates, including constitutive and enhancer-dependent pre-mRNAs. The requirement for this RS domain is substrate specific, and correlates with the strength of the splicing signals. When the 3? splice site is weak, both the SF2/ASF RS domain and U2AF35 are required for splicing. These results show the existence of an RS domain-independent function of SR proteins in constitutive and enhancer-dependent splicing, and suggest mechanisms for their role in enhancer function besides U2AF recruitment. PMID:11124808

  15. 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

  16. 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

  17. 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

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

    PubMed Central

    Allemand, Eric; Gattoni, Renata; Bourbon, Henri-Marc; Stevenin, James; Cceres, Javier F.; Soret, Johann; Tazi, Jamal

    2001-01-01

    The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5? alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo. PMID:11158320

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Long-term effect on foot and ankle donor site following vascularized fibular graft resection in children.

    PubMed

    Sulaiman, Abdul Razak; Wan, Zulmi; Awang, Shukrimi; Che Ahmad, Aminudin; Halim, Ahmad Sukari; Ahmad Mohd Zain, Rajaei

    2015-09-01

    This study was carried out to evaluate the long-term effect on the donor side of the foot and ankle following vascularized fibular graft resection in children. Eight patients underwent resection of the fibula for the purpose of a vascularized fibular graft by a surgical team who practiced leaving at least 6?cm residual distal fibula. The age of these children at the time of surgery was between 3 and 12 years. They were reviewed between 3 and 12 years after surgery. Two patients who underwent resection of the middle shaft of the fibula at 3 and 5 years of age developed abnormal growth of the distal tibia, leading to ankle valgus. They were treated with growth modulation of the distal tibial physis and supramalleolar osteotomy with tibiofibular synostosis. Another patient who underwent the entire proximal fibula resection at the age of 6 years had developed hindfoot valgus because of weakness of the tibialis posterior muscle. He required talonavicular fusion and flexor hallucis to tibialis posterior muscle transfer. Patients operated at the age of older than 8 years neither had ankle nor hindfoot deformity. We concluded that resection of the middle shaft of the fibula for the purpose of a vascularized fibula graft, leaving a 6?cm distal fibular stump in children younger than 6 years old, may give rise to abnormal growth of the distal tibial physis, leading to valgus ankle. The entire proximal fibular resection for the similar purpose in a 6-year-old child may give rise to weakness of tibialis posterior and hindfoot valgus. PMID:26049965

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

    PubMed

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

    2015-11-01

    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

  10. 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

  11. Design principles for bifunctional targeted oligonucleotide enhancers of splicing.

    PubMed

    Owen, Nicholas; Zhou, Haiyan; Malygin, Alexey A; Sangha, Jason; Smith, Lindsay D; Muntoni, Francesco; Eperon, Ian C

    2011-09-01

    Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a 'tail' of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns. PMID:21602265

  12. Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis

    PubMed Central

    Stadler, Michael B; Shomron, Noam; Yeo, Gene W; Schneider, Aniket; Xiao, Xinshu; Burge, Christopher B

    2006-01-01

    Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences. PMID:17121466

  13. 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

  14. 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

  15. 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

  16. 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

  17. Splicing patterns of the Chinese hamster MT-II gene

    SciTech Connect

    Grady, D.L.; Hildebrand, C.E.; Jackson, P.J.; Ratliff, R.L.; Walters, R.A.; Moyzis, R.K.

    1986-05-01

    Heavy metal induction of the synthesis of metallothioneins (MTs) provides an ideal model system for basic mechanistic studies of gene expression. Previous work from their laboratory has shown that a cell line designated 20 OT1, resistant to 200 ..mu..M CdCl/sub 2/, has coordinately amplified the MT-I and MT-II genes 10-20 fold. A region encompassing 14 kilobases (Kb) of overlapping DNA clones isolated from this cell line includes the structural genes for Chinese hamster MT-I and MT-II proteins. The MT-I gene is located 6 Kb downstream from the MT-II gene and is in the same transcriptional orientation. Analysis of the complete nucleotide sequence of the MT-II gene uncovered an alternative splice site in the first intron of this gene. To determine whether splicing occurs in vivo at this alternative splice site, oligonucleotide probes were synthesized. These probes consisted of 30 nucleotides bracketing either the normal or alternative splice sites. Northern blot analysis indicated that the alternative site is rarely used in the normal processing of the primary MT-II transcript. They suggest that efficient splicing does not occur at this alternative site due to inaccessibility of the proximal lariat site. Intron specific probes have been used to determine if there is a specific order to intron removal for this gene transcript. These studies indicate that the second intron is preferentially removed before the first intron.

  18. 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.

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

    PubMed

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

    2012-01-27

    Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.81.1nM) complex with ESS3 via site-specific interactions with the loop. PMID:22154809

  20. Acute intermittent porphyria: characterization of two novel mutations in the porphobilinogen deaminase gene, one amino acid deletion (453-455delAGC) and one splicing aceptor site mutation (IVS8-1G>T).

    PubMed

    De Siervi, A; Mendez, M; Parera, V E; Varela, L; Batlle, A M; Rossetti, M V

    1999-10-01

    A partial deficiency of Porphobilinogen deaminase (PBG-D) is responsible for acute intermittent porphyria (AIP). AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000. Here, two new mutations and three previously reported were found in the PBG-D gene in 12 Argentinean AIP patients corresponding to 5 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced. The new mutations are 453-455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8-1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used. Both mutations produce amino acid deletion without frameshift effect. To further characterize the 453-455delAGC mutation, the pKK-PBGD construct for the mutant allele was expressed in E. coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele. Finally, three missense mutations, previously reported, were identified in three unrelated families. PMID:10502788

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

    PubMed Central

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

    2013-01-01

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

  2. The rare TXNRD1_v3 ("v3") splice variant of human thioredoxin reductase 1 protein is targeted to membrane rafts by N-acylation and induces filopodia independently of its redox active site integrity.

    PubMed

    Cebula, Marcus; Moolla, Naazneen; Capovilla, Alexio; Arnr, Elias S J

    2013-04-01

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

  3. 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

  4. 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.

  5. 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

  6. Palatal bone dimensions on cone beam computed tomography. Implications for the palate as autogenous donor site: an observational study.

    PubMed

    El Nahass, H; Naiem, S N

    2016-01-01

    This study analyzed cone beam computed tomography (CBCT) records. Radiographic image analysis included: (1) the width and height of the palatal vault, and diameter and location of the incisive foramen; (2) the potential block graft dimensions and their correlation to arch dimensions; (3) the potential graft size and its variation by sex. CBCT scans of 76 patients were included, 42 from female patients and 34 from male patients (mean age 42.3 years). The mean palatal width was 35.23.4mm, while the mean palatal height was 15.22.9mm. The mean diameter of the incisive foramen was 3.11.3.mm. The mean potential osteotomy diameter was 7.81.5mm. The mean osteotomy length varied according to site: central incisor region, 5.92.0mm; lateral incisor region, 5.22.1mm; canine region, 4.71.9mm; premolar region, 4.11.7mm. A positive correlation was observed between the osteotomy diameter and the palatal width: a greater osteotomy length was obtained from the more anterior teeth position. Males presented significantly greater osteotomy diameter and length compared to females. The palate represents a potential site for the harvest of autogenous bone block grafts for the reconstruction of ridge defects. PMID:26443273

  7. Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

    PubMed Central

    Roman, J; Rubin, M N; Woodson, S A

    1999-01-01

    Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site. PMID:9917062

  8. SAGE2Splice: Unmapped SAGE Tags Reveal Novel Splice Junctions

    PubMed Central

    Kuo, Byron Yu-Lin; Chen, Ying; Bohacec, Slavita; Johansson, jvind; Wasserman, Wyeth W; Simpson, Elizabeth M

    2006-01-01

    Serial analysis of gene expression (SAGE) not only is a method for profiling the global expression of genes, but also offers the opportunity for the discovery of novel transcripts. SAGE tags are mapped to known transcripts to determine the gene of origin. Tags that map neither to a known transcript nor to the genome were hypothesized to span a splice junction, for which the exon combination or exon(s) are unknown. To test this hypothesis, we have developed an algorithm, SAGE2Splice, to efficiently map SAGE tags to potential splice junctions in a genome. The algorithm consists of three search levels. A scoring scheme was designed based on position weight matrices to assess the quality of candidates. Using optimized parameters for SAGE2Splice analysis and two sets of SAGE data, candidate junctions were discovered for 5%6% of unmapped tags. Candidates were classified into three categories, reflecting the previous annotations of the putative splice junctions. Analysis of predicted tags extracted from EST sequences demonstrated that candidate junctions having the splice junction located closer to the center of the tags are more reliable. Nine of these 12 candidates were validated by RT-PCR and sequencing, and among these, four revealed previously uncharacterized exons. Thus, SAGE2Splice provides a new functionality for the identification of novel transcripts and exons. SAGE2Splice is available online at http://www.cisreg.ca. PMID:16683015

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    SciTech Connect

    Morcos, Paul A. . E-mail: pmorcos@gene-tools.com

    2007-06-29

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a {beta}-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

  14. 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

  15. Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing

    PubMed Central

    Selman, Mohammed; Dankar, Samar K; Forbes, Nicole E; Jia, Jian-Jun; Brown, Earl G

    2012-01-01

    Little is known about the processes that enable influenza A viruses to jump into new host species. Here we show that the non-structural protein1 nucleotide substitution, A374G, encoding the D125G(GAT?GGT) mutation, which evolved during the adaptation of a human virus within a mouse host, activates a novel donor splice site in the non-structural gene, hence producing a novel influenza A viral protein, NS3. Using synonymous 125G mutations that do not activate the novel donor splice site, NS3 was shown to provide replicative gain-of-function. The protein sequence of NS3 is similar to NS1 protein but with an internal deletion of a motif comprised of three antiparallel ?-strands spanning codons 126 to 168 in NS1. The NS1-125G(GGT) codon was also found in 33 natural influenza A viruses that were strongly associated with switching from avian to mammalian hosts, including human, swine and canine populations. In addition to the experimental human to mouse switch, the NS1-125G(GGT) codon was selected on avian to human transmission of the 1997 H5N1 and 1999 H9N2 lineages, as well as the avian to swine jump of 1979 H1N1 Eurasian swine influenza viruses, linking the NS1 125G(GGT) codon with host adaptation and switching among multiple species. PMID:26038410

  16. 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

  17. Accurate Splicing of HDAC6 Pre-mRNA Requires SON

    PubMed Central

    Battini, Vishnu Priya; Bubulya, Athanasios; Bubulya, Paula A.

    2015-01-01

    Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Our lab previously reported that the SR-like protein SON maintains organization of pre-mRNA splicing factors in nuclear speckles as well as splicing of many human transcripts including mRNAs coding for the chromatin-modifying enzymes HDAC6, ADA and SETD8. However, the mechanism by which SON maintains accurate splicing is unknown. To build tools for understanding SON-dependent pre-mRNA splicing, we constructed a minigene reporter plasmid driving expression of the genomic sequence spanning exons 26 through 29 of HDAC6. Following SON depletion, we observed altered splicing of HDAC6 reporter transcripts that showed exclusion of exons 27 and 28, reflecting the splicing patterns of endogenous HDAC6 mRNA. Importantly, loss of HDAC6 biological function was also observed, as indicated by truncated HDAC6 protein and corresponding absence of aggresome assembly activities of HDAC6 binding-of-ubiquitin zinc finger (BUZ) domain. We therefore propose that SON-mediated splicing regulation of HDAC6 is essential for supporting protein degradation pathways that prevent human disease. PMID:25782155

  18. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing.

    PubMed

    Nguyen, Anh H; Lee, Jong Uk; Sim, Sang Jun

    2016-02-18

    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 ?-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 ?M 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. PMID:26847719

  19. 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.

  20. Technique for Minimizing Donor-site Morbidity after Pedicled TRAM-Flap Breast Reconstruction: Outcomes by a Single Surgeons 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, 436 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

  1. 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

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

    PubMed

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

    2015-10-01

    The aim of this study was to analyse changes in the volume of the chin after harvest of a bone graft for secondary reconstruction of an alveolar cleft. Cone-beam computed tomographic (CT) scans of 27 patients taken preoperatively, and immediately and one year postoperatively, were analysed, and 3-dimensional hard-tissue reconstructions made. The hard-tissue segmentation of the scan taken one year postoperatively was subtracted from the segmentation of the preoperative scan to calculate the alteration in the volume of bone at the donor site (chin). A centrally-orientated persistent concavity at the buccal side of the chin was found (mean (range) 160 (0-500) mm(3)). At the lingual side of the chin, a central concavity remained (mean (range) volume 20 (0-80) mm(3)). Remarkably, at the periphery of this concavity there was overgrowth of new bone (mean (range) volume 350 (0-1600) mm(3)). Re-attachment of the muscles of the tongue resulted in a significantly larger central lingual defect one year postoperatively (p=0.01). We also measured minor alterations in volume of the chin at one year. Whether these alterations influence facial appearance and long term bony quality is to be the subject of further research. PMID:26051866

  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. 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

  5. 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

  6. 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

  7. 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.; ren, 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 intronexon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of ?-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4279 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-4279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. PMID:25737553

  8. NOUGHT MAY ENDURE BUT MUTABILITY*: SPLICEOSOME DYNAMICS AND THE REGULATION OF SPLICING

    PubMed Central

    Smith, Duncan J.; Query, Charles C.; Konarska, Maria M.

    2008-01-01

    SUMMARY The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction. PMID:18570869

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

    PubMed Central

    Xu, Dong-Qing; Mattox, William

    2006-01-01

    Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within proteincoding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in disease, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict if a given mutation will have effects on splicing based on sequence alone. Here we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary nonpolyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659 indicating that their primary effect is on splicing. Surprisingly the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion. PMID:16357104

  10. 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

  11. Splicing and alternative splicing in rice and humans.

    PubMed

    E, Zhiguo; Wang, Lei; Zhou, Jianhua

    2013-09-01

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

  12. 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.

  13. Donor Tag Game

    MedlinePLUS

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

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

    PubMed

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

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease. PMID:26031236

  15. 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

  16. 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.

  17. 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

  18. 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

  19. SpliceVista, a Tool for Splice Variant Identification and Visualization in Shotgun Proteomics Data*

    PubMed Central

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

    2014-01-01

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

  20. 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

  1. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

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

  2. 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

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

    PubMed

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

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions. PMID:25589247

  4. 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

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

    PubMed Central

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

    2007-01-01

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

  6. Hyperfine and nuclear quadrupole tensors of nitrogen donors in the Q(A) site of bacterial reaction centers: correlation of the histidine N(?) tensors with hydrogen bond strength.

    PubMed

    Taguchi, Alexander T; O'Malley, Patrick J; Wraight, Colin A; Dikanov, Sergei A

    2014-08-01

    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 (14)N and in (15)N 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 (15)N Davies ENDOR, X- and Q-band (14,15)N HYSCORE, and X-band (14)N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a((14)N) = 2.3 MHz, T = 0.3 MHz for His-M219 N? and a((14)N) = 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((14)N). The nuclear quadrupole coupling constants were estimated as e(2)Qq/4h = 0.38 MHz, ? = 0.97 and e(2)Qq/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 Townes-Dailey 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((14)N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a((14)N) and ? will provide an important structural characterization tool in future studies of semiquinone-binding proteins. PMID:25026433

  7. 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

  8. Exon and intron definition in pre-mRNA splicing.

    PubMed

    De Conti, Laura; Baralle, Marco; Buratti, Emanuele

    2013-01-01

    One of the fundamental issues in RNA splicing research is represented by understanding how the spliceosome can successfully define exons and introns in a huge variety of pre-mRNA molecules with nucleotide-precision. Since its first description, researchers in this field have identified and characterized many fundamental elements and players capable of affecting the splicing process, both in a negative and positive manner. Indeed, it can be argued that today we know a great deal about the forces that make an exon, an exon and an intron, an intron. As will be discussed in this review, these decisions are a result of a complex combinatorial control resulting from many different factors/influences. Most importantly, these influences act across several levels of complexity starting from the relatively simple interaction between two consensus 5' and 3' splice sites to much more complex factors: such as the interplay between silencer or enhancer sequences, transcriptional processivity, genomic milieu, nucleosome positioning, and histone modifications at the chromatin level. Depending on local contexts, all these factors will act either antagonistically or synergistically to decide the exon/intron fate of any given RNA sequence. At present, however, what we still lack is a precise understanding of how all these processes add up to help the spliceosome reach a decision. Therefore, it is expected that future challenges in splicing research will be the careful characterization of all these influences to improve our ability to predict splicing choices in different organisms or in specific contexts. PMID:23044818

  9. Identifying Splicing Regulatory Elements with de Bruijn Graphs

    PubMed Central

    Badr, Eman

    2014-01-01

    Abstract Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation. PMID:25393830

  10. 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

  11. 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

  12. Neuronal calcium channels: Splicing for optimal performance

    PubMed Central

    Gray, Annette C; Raingo, Jesica; Lipscombe, Diane

    2007-01-01

    Calcium ion channels coordinate an astounding number of cellular functions. Surprisingly, only 10 Cav?1 subunit genes encode the structural cores of all voltage-gated calcium channels. What mechanisms exist to modify the structure of calcium channels and optimize their coupling to the rich spectrum of cellular functions? Growing evidence points to the contribution of post-translational alternative processing of calcium channel RNA as the main mechanism for expanding the functional potential of this important gene family. Alternative splicing of RNA is essential during neuronal development where fine adjustments in protein signaling promote and inhibit cell-cell interactions and underlie axonal guidance. However, attributing a specific functional role to an individual splice isoform or splice site has been difficult. In this regard, studies of ion channels are advantageous because their function can be monitored with precision, allowing even subtle changes in channel activity to be detected. Such studies are especially insightful when coupled with information about isoform expression patterns and cellular localization. In this paper we focus on two sites of alternative splicing in the N-type calcium channel Cav2.2 gene. We first describe cassette exon 18a that encodes a 21 amino acid segment in the II-III intracellular loop region of Cav2.2. Here we show that e18a is up-regulated in the nervous system during development. We discuss these new data in light of our previous reports showing that e18a protects the N-type channel from cumulative inactivation. Second, we discuss our published data on exons e37a and e37b, which encode 32 amino acids in the intracellular C-terminus of Cav2.2. These exons are expressed in a mutually exclusive manner. Exon e37a-containing Cav2.2 mRNAs and their resultant channels express at higher density in dorsal root ganglia and, as we showed recently, e37a increases N-type channel sensitivity to G protein-mediated inhibition, as compared to generic e37b-containing N-type channels. PMID:17512586

  13. 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

  14. 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

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

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

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

  16. 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.

  17. 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

  18. Exon First Nucleotide Mutations in Splicing: Evaluation of In Silico Prediction Tools

    PubMed Central

    Grodeck, Lucie; Lockerov, Pavla; Rav?ukov, Barbora; Buratti, Emanuele; Baralle, Francisco E.; Duek, Ladislav; Freiberger, Tom

    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

  19. An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing

    PubMed Central

    2010-01-01

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

  20. 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

  1. Alternative splicing: Functional diversity among voltage-gated calcium channels and behavioral consequences?

    PubMed Central

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

    2012-01-01

    Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal CaV channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinsons disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of CaV channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of CaV channel structures and functions. The precise composition of CaV channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of CaV splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of CaV pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels. PMID:23022282

  2. Functional Characterization of the spf/ash Splicing Variation in OTC Deficiency of Mice and Man

    PubMed Central

    Viecelli, Hiu Man; Rfenacht, Veronique; Prez, Beln; Ugarte, Magdalena; Hberle, Johannes; Thny, 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

  3. Two splicing factors carrying serine-arginine motifs, TSR1 and TSR1IP, regulate splicing, mRNA stability, and rRNA processing in Trypanosoma brucei.

    PubMed

    Gupta, Sachin Kumar; Chikne, Vaibhav; Eliaz, Dror; Tkacz, Itai Dov; Naboishchikov, Ilana; Carmi, Shai; Waldman Ben-Asher, Hiba; Michaeli, Shulamit

    2014-01-01

    In trypanosomes, mRNAs are processed by trans-splicing; in this process, a common exon, the spliced leader, is added to all mRNAs from a small RNA donor, the spliced leader RNA (SL RNA). However, little is known regarding how this process is regulated. In this study we investigated the function of two serine-arginine-rich proteins, TSR1 and TSR1IP, implicated in trans-splicing in Trypanosoma brucei. Depletion of these factors by RNAi suggested their role in both cis- and trans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNAs was changed, suggesting that these proteins have a role in regulating only a subset of T. brucei mRNAs. Mass-spectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNA stability, translation, and rRNA processing. We further demonstrate changes in the stability of mRNA as a result of depletion of the two TSR proteins. In addition, rRNA defects were observed under the depletion of U2AF35, TSR1, and TSR1IP, but not SF1, suggesting involvement of SR proteins in rRNA processing. PMID:24922194

  4. 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

  5. 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.

  6. 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

  7. SRSF1-Regulated Alternative Splicing in Breast Cancer.

    PubMed

    Anczukw, Olga; Akerman, Martin; Clry, Antoine; Wu, Jie; Shen, Chen; Shirole, Nitin H; Raimer, Amanda; Sun, Shuying; Jensen, Mads A; Hua, Yimin; Allain, Frdric 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

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

    PubMed

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

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig.1). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70% of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies. PMID:242223

  9. 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.

  10. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicols; Giono, Luciana E; Cambindo Botto, Adrin E; Muoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing. PMID:26296319

  11. ASGS: an alternative splicing graph web service

    PubMed Central

    Bollina, Durgaprasad; Lee, Bernett T. K.; Tan, Tin Wee; Ranganathan, Shoba

    2006-01-01

    Alternative transcript diversity manifests itself a prime cause of complexity in higher eukaryotes. The Alternative Splicing Graph Server (ASGS) is a web service facilitating the systematic study of alternatively spliced genes of higher eukaryotes by generating splicing graphs for the compact visual representation of transcript diversity from a single gene. Taking a set of transcripts in General Feature Format as input, ASGS identifies distinct reference and variable exons, generates a transcript splicing graph, an exon summary, splicing events classification and a single line graph to facilitate experimental analysis. This freely available web service can be accessed at . PMID:16845045

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. Our Future Donors

    ERIC Educational Resources Information Center

    Miller, Richard E.

    2004-01-01

    The rhetorical advantages and dangers involved in casting the students as "future donors" are explained. The way in which the institutions have to change for casting its students as future donors is described.

  17. 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

  18. 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

  19. Splicing remodels the let-7 primary microRNA to facilitate Drosha processing in Caenorhabditis elegans.

    PubMed

    Mondol, Vanessa; Ahn, Byoung Chan; Pasquinelli, Amy E

    2015-08-01

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that use partial base-pairing to recognize and regulate the expression of messenger RNAs (mRNAs). Mature miRNAs arise from longer primary transcripts (pri-miRNAs) that are processed to a shorter hairpin precursor miRNA (pre-miRNA) by the Microprocessor complex. In Caenorhabditis elegans the primary let-7 (pri-let-7) transcript undergoes trans-splicing, where pri-let-7 is cleaved at a 3' splice site and the splice-leader-1 (SL1) sequence is appended at the 5' end. Here we investigate the role of this splicing event in the biogenesis of let-7 miRNA. We hypothesized that splicing changes the secondary structure of the pri-let-7 transcript, creating a more favorable substrate for recognition by the Microprocessor. Supporting this idea, we detected conspicuous structural differences between unspliced and SL1-spliced pri-let-7 transcripts using in vitro ribonuclease (RNase) assays. Through the generation of transgenic worm strains, we found that the RNA secondary structure produced by splicing, as opposed to the act of splicing itself, optimizes processing of pri-let-7 by the Microprocessor in vivo. We also observed that the endogenous spliced, but not the unspliced, pri-let-7 transcripts bind to the Microprocessor and accumulate upon its depletion. We conclude that splicing is a key step in generating pri-let-7 transcripts with a structure that enables downstream processing events to produce appropriate levels of mature let-7. PMID:26081559

  20. 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

  1. 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

  2. Pre-mRNA splicing is a determinant of histone H3K36 methylation.

    PubMed

    Kim, Soojin; Kim, Hyunmin; Fong, Nova; Erickson, Benjamin; Bentley, David L

    2011-08-16

    A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3' splice site in intron 2 or in both introns 1 and 2 of an integrated ?-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5' ends and toward 3' ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5' ends in favor of 3' ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification. PMID:21807997

  3. 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

  4. 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

  5. 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.

  6. Donor corneal tissue evaluation.

    PubMed

    Saini, J S; Reddy, M K; Sharma, S; Wagh, S

    1996-03-01

    Proper evaluation of donor cornea is critical to the success of corneal transplantation. Attention must be paid to the cause of death and ocular condition as several general and ocular diseases constitute contraindications for donor corneal usage. Death to enucleation time should be noted. Gross examination and slit lamp biomicroscopy are mandatory for the evaluation of the donor eye while specular microscopy adds another useful dimension to information regarding donor cornea. This article provides a comprehensive review of all the aspects of donor corneal evaluation as practised today worldwide. PMID:8828299

  7. 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

  8. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    PubMed

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) ?? and ?? splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. PMID:25822725

  9. 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.

  10. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  11. A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers.