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Sample records for 5prime splice donor

  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. Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5[prime] end

    SciTech Connect

    Corson, G.M.; Chalberg, S.C.; Charbonneau, N.L.; Sakai, L.Y. ); Dietz, H.C. )

    1993-08-01

    Fibrillin is an important structural protein of the extracellular matrix. It is a large cysteine-rich glycoprotein with extensive intrachain disulfide bonds, likely contributed by multiple EGF-like repeats. The authors have previously published 6.9 kb of FBN1 cDNA sequence. FBN1 cDNA clones that extend the sequence 3089 bp in the 5[prime] direction are described in this report. The deduced primary structure suggests that fibrillin in composed of multiple domains. The most predominant features the presence of 43 calcium binding EGF-like repeats. They demonstrate here that fibrillin molecules bind calcium. In addition, three alternatively spliced exons at the 5[prime] end are described. Analysis of 5.8 kb of surrounding genomic sequence revealed a 1.8-kb CpG island spanning the alternatively spliced exons and the next downstream exon. Since FBN1 is the gene responsible for Marfan syndrome, the information presented here will be useful in identifying new mutations and in understanding the function of fibrillin in the pathogenesis of the disease. 42 refs., 7 figs.

  3. 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.

  4. Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals

    PubMed Central

    2010-01-01

    Background Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals. Results Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts. Conclusions Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts. PMID:21062477

  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.

  6. 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.

  7. 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.

  8. 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.

  9. Antisense suppression of donor splice site mutations in the dystrophin gene transcript

    PubMed Central

    Fletcher, Sue; Meloni, Penny L; Johnsen, Russell D; Wong, Brenda L; Muntoni, Francesco; Wilton, Stephen D

    2013-01-01

    We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis. PMID:24498612

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

    PubMed Central

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

    2013-01-01

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

  11. 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.

  12. 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.

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

    PubMed

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

    2015-12-15

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

  14. 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.

  15. Hepatitis B virus DNA splicing in Lebanese blood donors and genotype A to E strains: implications for hepatitis B virus DNA quantification and infectivity.

    PubMed

    El Chaar, Mira; El Jisr, Tamima; Allain, Jean-Pierre

    2012-10-01

    Hepatitis B virus (HBV) is one of the major viruses transmissible by blood that causes chronic infection in immunocompromised individuals. The study of 61 HBV carrier blood donors from Lebanon revealed multiple patterns of spliced HBV DNA. HBV DNA splicing was examined and quantified in samples of five genotypes and in seroconversion panels. The Lebanese sample median viral load was 1.5 ×10(2) IU/ml. All strains were genotype D, serotype ayw; 35 clustered as subgenotype D1 and 7 clustered as subgenotype D2. Three splice variants (SP1, SP1A, and Pol/S) were observed in 12 high-viral-load samples. Twenty samples of each genotype, A to E, were tested for the presence of HBV spliced DNA and SP1-specific splice variant. An unspliced HBV genome was dominant, but 100% of strains with a viral load of ≥10(5) copies/ml contained various proportions of spliced DNA. SP1 was detected in 56/100 (56%) samples in levels that correlated with the overall viral load. HBV DNA quantification with S (unspliced) and X (total DNA) regions provided different levels of viral load, with the difference corresponding to spliced DNA. During the highly infectious window period, the SP1 variant became detectable shortly after the hepatitis B surface antigen (HBsAg), suggesting a correlation between the initiation of splicing and the production of detectable levels of HBsAg. The quantification of HBV DNA with primers located outside and inside the spliced region might provide different estimations of viral load and differentiate between infectious and defective viral genomes. The role of splicing neoproteins in HBV replication and interaction with the host remains to be determined.

  16. Splicing of the large intron present in the nonstructural gene of minute virus of mice is governed by TIA-1/TIAR binding downstream of the nonconsensus donor.

    PubMed

    Choi, Eun-Young; Pintel, David

    2009-06-01

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

  17. The cyclization of arabinosyladenine-5-prime-phosphorimidazolide

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1991-01-01

    When arabinosyladenine-5-prime-phosphorimidazolide is allowed to decompose in aqueous solution at room temperature and pH 7.2, depending on the buffer, 5-24 percent is converted to the 2-prime,5-prime-cyclic phosphate (V). Although the extent of cyclization is much greater than for adenosine-5-prime-phosphorimidazolide, cyclization is less efficient than hydrolysis and so would not substantially decrease the efficiency of condensation reactions in aqueous solution. The significance of this result for prebiotic chemistry is discussed.

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

    SciTech Connect

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

    2004-12-20

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

  19. Isolation of an insulin-like growth factor II cDNA with a unique 5 prime untranslated region from human placenta

    SciTech Connect

    Shen, Shujane; Daimon, Makoto; Wang, Chunyeh; Ilan, J. ); Jansen, M. )

    1988-03-01

    Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5{prime} untranslated region (5{prime}-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A){sup +} RNA was probed with either the 5{prime}-UTR of the isolated human placental IGF-II cDNA or the 5{prime}-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5{prime}-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5{prime}-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5{prime}-UTR consisting of a base sequence dissimilar to that of either IGF-II 5{prime}-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5{prime}-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    1992-02-01

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

  2. Whole-genome sequencing of two probands with hereditary spastic paraplegia reveals novel splice-donor region variant and known pathogenic variant in SPG11

    PubMed Central

    Chan, Anne Yin-Yan; Au, Wing Chi; Shen, Yun; Chan, Ting Fung

    2016-01-01

    Hereditary spastic paraplegias (HSPs) are a group of heterogeneous neurodegenerative disorders, which are often presented with overlapping phenotypes such as progressive paraparesis and spasticity. To assist the diagnosis of HSP subtypes, next-generation sequencing is often used to provide supporting evidence. In this study, we report the case of two probands from the same family with HSP symptoms, including bilateral lower limb weakness, unsteady gait, cognitive decline, dysarthria, and slurring of speech since the age of 14. Subsequent whole-genome sequencing revealed that the patients are compound heterozygous for variants in the SPG11 gene, including the paternally inherited c.6856C>T (p.Arg2286*) variant and the novel maternally inherited c.2316+5G>A splice-donor region variant. Variants in SPG11 are the common cause of autosomal recessive spastic paraplegia type 11. According to the ClinVar database, there are already 101 reported pathogenic variants in SPG11 that are associated with HSPs. To our knowledge, this is the first report of SPG11 variants in our local population. The novel splice variant identified in this study enriches the catalog of SPG11 variants, potentially leading to better genetic diagnosis of HSPs. PMID:27900367

  3. 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

  4. Recurrent disruption of the Imu splice donor site in t(14;18) positive lymphomas: a potential molecular basis for aberrant downstream class switch recombination.

    PubMed

    Ruminy, Philippe; Jardin, Fabrice; Penther, Dominique; Picquenot, Jean-Michel; Parmentier, Françoise; Buchonnet, Gérard; Bertrand, Philippe; Tilly, Hervé; Bastard, Christian

    2007-08-01

    t(14;18) positive lymphomas are mature germinal center B-cell neoplasms. In agreement with this cellular origin, most have somatically mutated immunoglobulin variable genes and the IGH@ locus has almost always been reorganized by class switch recombination (CSR). However, contrasting with normal B-cells, a majority of cases still express an IgM while the constant genes are normally rearranged only on the non-productive allele. Concurrently, aberrant intra-allelic junctions involving downstream switch regions, with a lack of engagement of the switch mu (Smu), often accumulate on the functional alleles, suggesting some recurrent CSR perturbation during the onset of the disease. To clarify these surprising observations, we addressed the accessibility of the Smu to the CSR machinery in a large series of patients by characterizing the mutations that are expected to accumulate at this place upon CSR activation. Our data indicate that the Smu is mutated in a large majority of cases, often on both alleles, indicating that these cells usually reach a differentiation stage where CSR is activated and where this region remains accessible. Interestingly, we also identified a significant cluster of mutations at the splicing donor site of the first exon of the Smu germline transcripts, on the functional allele. This location suggests a possible relation with CSR perturbations in lymphoma and the clustering points to a probable mechanism of selection. In conclusion, our data suggest that an acquired mutation at the splicing donor site of the Smu transcripts may participate in the selection of lymphoma cells and play a significant role during the onset of the disease.

  5. 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.

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

    PubMed Central

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

    1994-01-01

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

  7. Novel point mutation in the splice donor site of exon-intron junction 6 of the androgen receptor gene in a patient with partial androgen insensitivity syndrome.

    PubMed

    Sammarco, I; Grimaldi, P; Rossi, P; Cappa, M; Moretti, C; Frajese, G; Geremia, R

    2000-09-01

    Androgen receptor (AR) gene mutations have been shown to cause androgen insensitivity syndrome with altered sexual differentiation in XY individuals, ranging from a partial insensitivity with male phenotype and azoospermia to a complete insensitivity with female phenotype and the absence of pubic and axillary sexual hair after puberty. In this study we present an 11-yr-old XY girl, with clinical manifestations peculiar for impaired androgen biological action, including female phenotype, blind-ending vagina, small degree of posterior labial fusion, and absence of uterus, fallopian tubes, and ovaries. At the time of the diagnosis the patient had a FSH/LH ratio according to the puberal stage, undetectable 17beta-estradiol, and high levels of testosterone (80.1 ng/mL). After bilateral gonadectomy, performed at the age of 11 yr, histological examination showed small embryonic seminiferous tubules containing prevalently Sertoli cells and occasional spermatogonia together with abundant fibrous tissue. Molecular study of the patient showed a guanine to thymine transversion in position +5 of the donor splice site in the junction between exon 6 and intron 6 of the AR gene. The result of RT-PCR amplification of the AR messenger ribonucleic acid from cultured genital skin fibroblasts of the patient suggests that splicing is defective, and intron 6 is retained in most of the receptor messenger ribonucleic acid molecules. We show by immunoblotting that most of the expressed protein lacks part of the C-terminal hormone-binding domain, and a small amount of normal receptor is observed. This is probably responsible for the reduced binding capacity in genital skin fibroblasts of the patient. The molecular basis of the alteration in this case is a novel, uncommon mutation, leading to a phenotype indicative of a partial androgen insensitivity syndrome, Quigley's grade 5.

  8. Oligomerizations of deoxyadenosine bis-phosphates and of their 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers - Effects of a pyrophosphate-linked, poly(T) analog

    NASA Technical Reports Server (NTRS)

    Visscher, J.; Bakker, C. G.; Schwartz, Alan W.

    1990-01-01

    The effect of a 3-prime-5-prime pyrophosphate-linked oligomer of pTp on oligomerizations of pdAp and of its 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers was investigated, using HPLC to separate the reaction mixtures; peak detection was by absorbance monitoring at 254 nm. It was expected that the dimers would form stable complexes with the template, with the degree of stability depending upon the internal linkage of each dimer. It was found that, although the isomers differ substantially in their oligomerization behavior in the absence of template, the analog-template catalyzes the oligomerization to about the same extent in all three cases.

  9. 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.

  10. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  11. 5[prime] to 3[prime] nucleic acid synthesis using 3[prime]-photoremovable protecting group

    DOEpatents

    Pirrung, M.C.; Shuey, S.W.; Bradley, J.C.

    1999-06-01

    The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5[prime] to 3[prime] nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5[prime] end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

  12. Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

    PubMed

    Peijnenburg, A; Van den Berg, R; Van Eggermond, M J; Sanal, O; Vossen, J M; Lennon, A M; Alcaïde-Loridan, C; Van den Elsen, P J

    2000-01-01

    MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

  13. Effect of cold exposure on thyroxine 5 prime -deiodinase activity in iron-deficient rats

    SciTech Connect

    Brigham, D.E.; Beard, J.L. )

    1991-03-11

    When exposed to cold, severely anemic iron-deficient (ID) rats become hypothermic, fail to adequately increase metabolic rate, and have lower interscapular brown adipose tissue (IBAT) thyroxine 5{prime}-deiodinase (5{prime}-DI) activity compared to control (CN) rats. Less severely anemic rats and CN rats were exposed to 4C for 6h. Rectal temperatures were measured hourly, and VO{sub 2} was measured both prior to and throughout the cold exposure period. IBAT 5{prime}-DI activity was measured in cold-exposed ID and CN rats and compared to ID and CN rats that were not cold exposed. During cold exposure, both ID and CN rats increased metabolic rate similarly. IBAT 5{prime}-DI activity also increased similarly in both groups after cold exposure. These results show that moderately anemic DI rats acutely increase metabolic rate and IBAT 5{prime}-DI activity in the cold. This suggests brown fat production of thyroid hormone is not limiting thermoregulatory performance.

  14. NMR studies of two spliced leader RNAs using isotope labeling

    SciTech Connect

    Lapham, J.; Crothers, D.M.

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

  15. Modulation of 2{prime}-5{prime} oligoadenylate synthetase by environmental stress in the marine sponge Geodia cydonium

    SciTech Connect

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G.; Kuusksalu, A.; Kelve, M.

    1997-07-01

    Recently the authors established the presence of relatively high amounts of 2{prime}-5{prime} oligoadenylates (2{prime}-5{prime} A) and 2{prime}-5{prime} oligoadenylate synthetase (2{prime}-5{prime} A synthetase) in the marine sponge Geodia cydonium. Here they determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2{prime}-5{prime} A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2{prime}-5{prime} A content and the activity of 2{prime}-5{prime} A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26 C), cold shock (6 C), pH shock (pH 6), and hypertonic shock and subsequent incubation for 18 h under ambient conditions (16 C). No response was observed after exposure of sponges to an alkaline (pH 10) or hypotonic environment. Similar changes have been found for the expression of heat shock protein HSP70 in G. cydonium. These results show that 2{prime}-5{prime} A in sponges may be useful as a novel biomarker for environmental monitoring.

  16. Genomic organization and 5{prime}-flanking DNA sequence of the murine stomatin gene (Epb72)

    SciTech Connect

    Gallagher, P.G.; Turetsky, T.; Mentzer, W.C. |

    1996-06-15

    Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5{prime}-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over {approximately}25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5{prime}-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins. 12 refs., 2 figs., 1 tab.

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

    PubMed Central

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

    1995-01-01

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

  18. 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.

  19. 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.

  20. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

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

    PubMed Central

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

    2015-01-01

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

  2. An interspecific plant hybrid shows novel changes in parental splice forms of genes for splicing factors.

    PubMed

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

    2010-04-01

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

  3. Expression of splice variants of mts1 gene in normal and neoplastic human tissues

    SciTech Connect

    Ambartsumyan, N.S. |; Grigorian, M.S.; Lukanidin, E.M.

    1995-09-01

    Data on cloning of cDNA corresponding to human mts1 gene transcripts are presented. By comparing nucleotide sequences of the genomic DNA clone and cDNA of mts1, it was shown that human osteosarcoma OHS cells contain two alternative splice variants of mts1 transcripts. Alternative splicing occurs in the 5{prime}-untranslated region of the mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1(var), demonstrate similar stability in the cells, and each contains one open reading frame for the MTS1 protein. However, the two types of transcripts are translated with different effectiveness. The level of transcription of mts1 splice variants in different normal and neoplastic tissues and cell lines varies significantly. The role of alternative splicing as the mechanism responsible for posttranscriptional regulation of mts1 gene expression is discussed. 31 refs., 5 figs.

  4. 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.

  5. Understanding splicing regulation through RNA splicing maps.

    PubMed

    Witten, Joshua T; Ule, Jernej

    2011-03-01

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

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

    PubMed

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

    2009-11-01

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

  7. RNA polymerase II pauses at the 5 prime end of the transcriptionally induced Drosophila hsp70 gene

    SciTech Connect

    O'Brien, T.; Lis, J.T. )

    1991-10-01

    An RNA polymerase II molecule is associated with the 5{prime} end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides. Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here the authors report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5{prime} end of hsp70 was transcribed approximately five times during the 25-min heat shock that they used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

  8. Soluble minerals in chemical evolution. I - Adsorption of 5-prime-AMP on CaSO4 - A model system

    NASA Technical Reports Server (NTRS)

    Orenberg, J. B.; Chan, S.; Calderon, J.; Lahav, N.

    1985-01-01

    The adsorption of 5-prime-AMP onto solid CaSO4-2H2O was studied in a saturated suspension as a function of pH and electrolyte concentration. The adsorption is pH-dependent and is directly correlated with the charge on the 5-prime-AMP molecule which is determined by the state of protonation of the N-1 nitrogen of the purine ring and the phosphate oxygens. It is proposed that the binding that occurs between the nucleotide and the salt is electrostatic in nature. The adsorption decreases with increasing ionic strength of the solution which means that in a fluctuating environment of wetting and drying cycles, a biomolecule similar to 5-prime-AMP could be expected to desorb during the drying phase. The results indicate that CaSO4-2H2O can serve as a concentrating surface for biomolecules. The significance of this is discussed with regard to the possible role of soluble minerals and their surfaces in a geochemical model consistent with the evolution of the earth and the origin of life.

  9. Alternative splicing and genomic structure of the Wilms tumor gene WT1

    SciTech Connect

    Haber, D.A. Massachusetts General Hospital Cancer Center, Charlestown ); Sohn, R.L.; Buckler, A.J.; Pelletier, J.; Call, K.M.; Housman, D.E. )

    1991-11-01

    The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5{prime} splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggest that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.

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

    SciTech Connect

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

    1993-03-01

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

  11. The peculiarities of large intron splicing in animals.

    PubMed

    Shepard, Samuel; McCreary, Mark; Fedorov, Alexei

    2009-11-16

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

  12. Analysis of the 5{prime} junctions of R2 insertions with the 28S gene: Implications for non-LTR retrotransposition

    SciTech Connect

    George, J.A.; Burke, W.D.; Eickbush, T.H.

    1996-03-01

    R2 elements are non-long terminal repeat retrotransposable elements that insert into 28S rRNA genes of most insect species. The single open reading frame of R2 encodes a protein with both endonuclease activity, which cleaves the target site, and reverse transcriptase activity, which uses this cleavage to prime reverse transcription. This target-primed reverse transcription mechanism is also used by group II introns. Little is known of the mechanism by which the 5{prime} end of R2 is integrated after reverse transcription. We have determined the 5{prime} junction sequence of 94 R2 elements from 14 different species of Drosophila. Only 37% of the full-length elements contained precise 5{prime} junctions; the remainder contained deletions of the 28S gene and/or insertions of additional sequences. Because the 5{prime} junctions of truncated copies were similar to full-length elements, no sequences at the 5{prime} end of R2 appear to be required for element integration. A model in which the R2 reverse transcriptase is capable of switching templates from the R2 RNA transcript to the upstream 28S gene can best explain the observed 5{prime} junction sequences. This template jumping is analogous to the template switching of retroviral reverse transcriptases during formation of the double-stranded integration products. 44 refs., 5 figs.

  13. Characterization of a splicing mutation in group A xeroderma pigmentosum

    SciTech Connect

    Satokata, Ichiro; Tanaka, Kiyoji; Miura, Naoyuki; Miyamoto, Iwai; Okada, Yoshio ); Satoh, Yoshiaki Tokyo Medical and Dental Univ. ); Kondo, Seiji )

    1990-12-01

    The molecular basis of group A xeroderma pigmentosum (WP) was investigated by comparison of the nucleotide sequences of multiple clones of the XP group A complementing gene (XPAC) from a patient with group A XP with that of a normal gene. The clones showed a G {r arrow} C substitution at the 3{prime} splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotide to AC. Nucleotide sequencing of cDNAs amplified by the polymerase chain reaction revealed that this single base substitution abolishes the canonical 3{prime} splice site, thus creating two abnormally spliced mRNA forms. The larger form is identical with normal mRNA except for a dinucleotide deletion at the 5{prime} end of exon 4. This deletion results in a frameshift with premature translation termination in exon 4. The smaller form has a deletion of the entire exon 3 and the dinucleotide at the 5{prime} end of exon 4. The result of a transfection study provided additional evidence that this single base substitution is the disease-causing mutation. This single base substitution creates a new cleavage site for the restriction nuclease AlwNI. Analysis of AlwNI restriction fragment length polymorphism showed a high frequency of this mutation in Japanese patients with group A XP: 16 of 21 unrelated Japanese patients were homozygous and 4 were heterozygous for this mutation. However, 11 Caucasians and 2 Blacks with group A XP did not have this mutant allele. The polymorphic AlwNI restriction fragments are concluded to be useful for diagnosis of group A XP in Japanese subjects, including prenatal cases and carriers.

  14. A five prime splice-region G yields C mutation in exon 1 of the human. beta. -globin gene inhibits pre-mRNA splicing: A mechanism for. beta. sup + -thalassemia

    SciTech Connect

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M. ); Gattoni, R.; Stevenin, J. ); Chibani, J. )

    1989-02-01

    The authors have characterized a Mediterranean {beta}-thalassemia allele containing a sequence change at codon 30 that alters both {beta}-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G {yields} C transversion at position {minus}1 of intron 1 reduces severely the utilization of the normal 5{prime} splice site since the level of the Arg {yields} Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position {minus}1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5{prime} splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

  15. Splicing plastic optical fibers

    NASA Astrophysics Data System (ADS)

    Carson, Susan D.; Salazar, Roberto A.

    1991-12-01

    Polymethylmethacrylate (PMMA) plastic optical fiber (500 micrometers diameter, fluoropolymer cladding) has been spliced using a fused silica sleeve and a variety of solvent/PMMA solutions as adhesives. Mechanical splicing using index matching fluid has also been investigated. To ensure good bonding and minimize scattering, fiber ends are polished prior to application of adhesive. Using an LED ((lambda) max approximately 640 nm), losses are routinely less than 1.0 dB/splice, and some adhesive formulations have exhibited losses as low as 0.2 dB/splice. Five-meter fibers with as many as ten splices/fiber have been monitored over a period of several months. No fiber has exhibited an increase in optical loss with time.

  16. Statistical analysis of mammalian pre-mRNA splicing sites.

    PubMed Central

    Gelfand, M S

    1989-01-01

    222 donor and 222 acceptor (including 206 pairs) non-homologous splicing sites were studied. Well known features of these were confirmed and some novel observations were made. It is (1) cCAGGGag signal in (-60)-(-58) region of acceptor sites; (2) strong complementarity between regions (-69)-(-55) and (-36)-(-22) of some of the acceptor sites, and (3) small but statistically significant correlation between discrimination energies of corresponding donor and acceptor sites. PMID:2528123

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

    PubMed

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

    2015-09-01

    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.

  18. Distribution of a length polymorphism 5{prime} to exon 1 of the antithrombin III (ATIII) gene in the Chinese

    SciTech Connect

    Low, P.S.; Liu, Y.; Saha, N.

    1994-09-01

    A length polymorphism at the 5{prime} untranslated region of the ATIII gene has been described as having been detected by polymerase chain reaction (PCR) with a frequency of 0.75 for the short allele (S) in the Caucasian population. This length polymorphism of the ATIII gene has been studied in 251 Chinese healthy subjects. Genomic DNA was amplified by PCR with primers of published sequences. Fragments of the amplified DNA were separated by agarose gel electrophoresis (3% NuSieve and 1% Seakem GTG) and photographed on a UV transilluminator. The frequency of the short allele (S) was found to be significantly lower (0.37) than that in the Caucasians (0.75). The distribution of genotypes of this polymorphism of the ATIII gene was at Hardy-Weinberg equilibrium. The large difference of allelic frequencies in the Mongoloid and Caucasian populations makes it a useful marker for population studies.

  19. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  20. pH profile of the adsorption of nucleotides onto montmorillonite. II - Adsorption and desorption of 5-prime-AMP in iron-calcium montmorillonite systems

    NASA Technical Reports Server (NTRS)

    Banin, A.; Lawless, J. G.; Mazzurco, J.; Church, F. M.; Margulies, L.; Orenberg, J. B.

    1985-01-01

    The interaction of 5-prime-AMP with montmorillonite saturated with various ratios of two metals found ubiquitously on the surface of earth, that is, iron and calcium, is investigated. Adsorption and desorption of the nucleotide were studied in the pH range of 2-12 at three levels of addition: 0.080, 0.268 and 0.803 mmole 5-prime-AMP per gram of clay. Two desorption stages were employed - H2O wash and NaOH extraction (pH = 12.0). 5-prime-AMP was preferentially adsorbed on the Fe-containing clays relative to the Ca clay. The nucleotide was fully recovered by the two desorption stages, mostly by the NaOH extraction. The evidence at hand indicates that 5-prime-AMP reaction with clay is affected by electrostatic interactions involving both attraction and repulsion forces. Some specific adsorption, possibly the result of covalent bonding and complex formation with the adsorbed ion, cannot be ruled out for iron but does not appear to operate for calcium. Changes in pH cause varying degrees of attaction and repulsion of 5-prime-AMP and may have been operating on the primitive earth, leading to sequences of adsorption and release of this biomolecule.

  1. Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

    PubMed Central

    Turton, Keren B.; Esnault, Stephane; Delain, Larissa P.; Mosher, Deane F.

    2016-01-01

    Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites. PMID:27768061

  2. RNA helicases in splicing.

    PubMed

    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.

  3. 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.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2013-05-01

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

  6. Alternative splicing interference by xenobiotics.

    PubMed

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

  7. 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.

  8. SpliceDisease database: linking RNA splicing and disease.

    PubMed

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

    2012-01-01

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

  9. Aberrant splicing and transcription termination caused by P element insertion into the intron of a Drosophila gene

    SciTech Connect

    Horowitz, H.; Berg, C.A.

    1995-01-01

    Insertional mutagenesis screens using the P[lacZ, rosy{sup +}] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5{prime} untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce an mRNA composed of 5{prime} sequences from the mutated gene (in this case, pipsqueak) and 3{prime} sequences from within the P[lacZ, rosy{sup +}] element. These truncated RNAs could yield proteins with dominant mutant effects. 43 refs., 4 figs.

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

    PubMed

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

    2010-02-17

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

  11. Conformational dynamics of single pre-mRNA molecules during in vitro splicing.

    PubMed

    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

    2010-04-01

    The spliceosome is a complex small nuclear RNA (snRNA)-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 uses 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 observed 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.

  12. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

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

    PubMed

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

    2010-09-01

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

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

    PubMed

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

    2009-10-16

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

  15. Transcription and splicing: when the twain meet.

    PubMed

    Brody, Yehuda; Shav-Tal, Yaron

    2011-01-01

    Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

  16. Donor Tag Game

    MedlinePlus

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

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

    PubMed

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

    2011-07-01

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

  18. RNA splicing: disease and therapy.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2011-05-01

    The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.

  19. 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.

  20. The neurogenetics of alternative splicing

    PubMed Central

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

    2016-01-01

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

  1. Alternative RNA splicing and cancer.

    PubMed

    Liu, Sali; Cheng, Chonghui

    2013-01-01

    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.

  2. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

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

  3. 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.

  4. SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis.

    PubMed

    Feng, Jinlin; Li, Jingjing; Gao, Zhaoxu; Lu, Yaru; Yu, Junya; Zheng, Qian; Yan, Shuning; Zhang, Wenjiao; He, Hang; Ma, Ligeng; Zhu, Zhengge

    2015-07-01

    Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

  5. 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.

  6. Cellular stress and RNA splicing.

    PubMed

    Biamonti, Giuseppe; Caceres, Javier F

    2009-03-01

    In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

  7. Splicing of aged fibers

    NASA Astrophysics Data System (ADS)

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

    1993-11-01

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

  8. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  9. 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.

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

    PubMed Central

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

    2011-01-01

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

  11. Synthesis and characterization of ( sup 3 H)-5 prime azido-N-1-naphthylphthlamic acid, a photolabile N-1-naphthylphthalamic acid analog

    SciTech Connect

    Voet, J.G.; Dodge, B.; Harris, K.; Jacobs, M.; Larkin, L.; Bader, S.; Schnitzler, G.; Sutherland, J. )

    1990-05-01

    The NPA (N-1-naphthylphthalamic acid) receptor is an important protein involved in the regulation of transport of indole-3-acetic acid (IAA). In our attempt to isolate and characterize this protein we have previously synthesized and characterized a photolabile analog of NPA, 5{prime}-azido-NPA (Az-NPA) and shown it to be a competitor of NPA for binding sites on the NPA receptor as well as an inhibitor of auxin transport. We have now synthesized and characterized ({sup 3}H)-Az-NPA. The precursor, 2,3,4,5-Br-5{prime}-amino-NPA was dehydrohalogenated with tritium gas by Research Products International. The amino group was converted to an azido group and the product purified by HPLC. ({sup 3}H)-Az-NPA was found to be photolabile and to co-chromatograph with our synthetic unlabeled Az-NPA. Furthermore, the tritiated material was found to bind to zucchini hypocotyl cell membranes in a manner competitive with NPA as well as unlabeled Az-NPA. Photolysis of zucchini phase-partitioned plasma membranes in the presence of ({sup 3}H)-Az-NPA resulted in covalent association of tritium with the membranes. Much of this covalent association could be prevented by prior treatment of the membranes with excess NPA.

  12. Biological effects of exogenous adenosine 5 prime -triphosphate on cultured mammalian cells: Evidence for a receptor mechanism and its regulation by desensitization

    SciTech Connect

    Gonzalez, F.A.

    1989-01-01

    Exogenous adenosine 5{prime}-triphosphate (ATP) mobilized intracellular calcium in human carcinoma A43l cells and in Swiss 3T3 and 3T6 mouse fibroblasts by increasing inositol trisphosphate similar to well down growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bradykinin (BK), serum). Calcium mobilization was examined by video imaging of fura-2 fluorescence is single cells, following the radioactive isotope {sup 45}Ca, and monitoring the decrease in fluorescence of cells loaded with chlortetracycline. Uridine 5{prime}-triphosphate, but not other nucleotides, mimicked ATP. Single-cell analysis revealed synchronous responses in 10 sec to ATP, BK or serum, while PDGF (3T3) and EGF (A431) produced slower signals with significant cell-to-cell variation. PDGF desensitized 3T3 cells to ATP and BK added 100 sec later but ATP or BK did not desensitized to PDGF. Homologous desensitization was seen with all agonists. Heterologous desensitization was also observed in A431 cells where ATP desensitized to serum, but serum did not to ATP. ATP-stimulated calcium entry was detected after 10 sec in A431 cells, but not in Swiss 3T6 cells. Entry started before significant efflux had occurred and did not fit the capacitance model of Putney. A 2-3 hr ATP pretreatment produced a homologous desensitization state that required 20 hr to disappear, probably due to down-regulation of the putative ATP receptors.

  13. Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human.

    PubMed

    Clark, Francis; Thanaraj, T A

    2002-02-15

    By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/~thanaraj/altExtron/.

  14. Homologous SV40 RNA trans-splicing: a new mechanism for diversification of viral sequences and phenotypes.

    PubMed

    Eul, Joachim; Patzel, Volker

    2013-11-01

    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.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. 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.

  18. Complexities of 5'splice site definition: implications in clinical analyses.

    PubMed

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

    2012-06-01

    In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.

  19. RNA splicing and splicing regulator changes in prostate cancer pathology.

    PubMed

    Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

    2017-04-05

    Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

  20. Targeting RNA Splicing for Disease Therapy

    PubMed Central

    Havens, Mallory A.; Duelli, Dominik M.

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics. PMID:23512601

  1. Targeting RNA splicing for disease therapy.

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2005-10-01

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

  3. Linkage disequilibrium between polymorphisms at the 5{prime} untranslated region and intron 5 (Dde I) of the antithrombin III (ATIII) gene in the Chinese

    SciTech Connect

    Tay, J.S.H.; Liu, Y.; Low, P.S.

    1994-09-01

    A length polymorphism at the 5{prime} untranslated region of exon 1 and an RFLP (Dde I) in intron 5 (nt 160) of the ATIII gene were amplified by polymerase chain reaction with primers of published sequences. DNA fragments were size-fractionated by agarose gel electrophoresis (3% NuSieve and 1% Seakem GTG) and photographed over a UV transilluminator. A strong linkage disequilibrium was observed between these two polymorphisms of the ATIII gene in the Chinese ({chi}{sup 2} = 63.7; {triangle} 0.42, P < 0.001). The estimated frequencies of the three haplotypes were found to be 0.37 for SD+, 0.40 for LD+ and 0.23 for LD-.

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

    PubMed Central

    Purcell, D F; Martin, M A

    1993-01-01

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

  5. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

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

    PubMed Central

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

    2016-01-01

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

  7. Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

    PubMed Central

    Jin, Y; Dietz, H C; Montgomery, R A; Bell, W R; McIntosh, I; Coller, B; Bray, P F

    1996-01-01

    Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17. PMID:8878424

  8. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  9. Volunteer donor apheresis.

    PubMed

    Waxman, Dan A

    2002-02-01

    Volunteer donor apheresis has evolved from early plasmapheresis procedures that collected single components into technically advanced multicomponent procedures that can produce combinations of red blood cells, platelets, and plasma units. Blood collection and utilization is increasing annually in the United States. The number of apheresis procedures is also increasing such that single donor platelet transfusions now exceed platelet concentrates from random donors. Donor qualifications for apheresis vary from those of whole blood. Depending on the procedure, the donor weight, donation interval, and platelet count must be taken into consideration. Adverse effects of apheresis are well known and fortunately occur in only a very small percentage of donors. The recruitment of volunteer donors is one of the most challenging aspects of a successful apheresis program. As multicomponent apheresis becomes more commonplace, it is important for collection centers to analyze the best methods to recruit and collect donors.

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

    Sargent, Kevin M.; Clopton, Debra T.; Lu, Ningxia; Pohlmeier, William E.

    2015-01-01

    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

  12. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, 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.

  13. Evolutionary conservation of alternative splicing in chicken

    PubMed Central

    Katyal, S.; Gao, Z.; Liu, R.-Z.; Godbout, R.

    2013-01-01

    Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

  14. Alcoholism and alternative splicing of candidate genes.

    PubMed

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

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

    PubMed

    Sêton Bocco, Steven; Csűrös, Miklós

    2016-08-25

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

  16. Splice Sites Seldom Slide: Intron Evolution in Oomycetes

    PubMed Central

    Sêton Bocco, Steven; Csűrös, Miklós

    2016-01-01

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

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

    SciTech Connect

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

    1988-04-01

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

  18. Conserved RNA secondary structures promote alternative splicing.

    PubMed

    Shepard, Peter J; Hertel, Klemens J

    2008-08-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site strength, splicing regulators, the exon/intron architecture, and the process of pre-mRNA synthesis itself. RNA secondary structures have also been proposed to influence alternative splicing as stable RNA secondary structures that mask splice sites are expected to interfere with splice-site recognition. Using structural and functional conservation, we identified RNA structure elements within the human genome that associate with alternative splice-site selection. Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease.

  19. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

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

  20. The chicken FMR1 gene is highly conserved with a CCT 5{prime} - untranslated repeat and encodes an RNA-binding protein

    SciTech Connect

    Price, D.K.; Zhang, F.; Ashley, C.T. Jr.; Warren, S.T.

    1996-01-01

    The transcriptional silencing of the human gene, fragile X metal retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5{prime}-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of metal retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3{prime}-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs. 49 refs., 7 figs.

  1. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    PubMed Central

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks. PMID:24101931

  2. 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

  3. 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.

  4. A synonymous CHRNE mutation responsible for an aberrant splicing leading to congenital myasthenic syndrome.

    PubMed

    Richard, Pascale; Gaudon, Karen; Fournier, Emmanuel; Jackson, Christopher; Bauché, Stéphanie; Haddad, Hafedh; Koenig, Jeanine; Echenne, Bernard; Hantaï, Daniel; Eymard, Bruno

    2007-05-01

    Congenital myasthenic syndromes (CMSs) are rare hereditary disorders transmitted in a recessive or dominant pattern, and are caused by mutations in the genes encoding proteins of the neuromuscular junction. They are classified in three groups depending on the origin of the molecular defect. Postsynaptic defects are the most frequent and have been reported to be partly due to abnormalities of the acetylcholine receptor, and particularly to mutations in CHRNE, the gene encoding the acetylcholine receptor epsilon-subunit. In a Portuguese patient with a mild form of recessive CMS, CHRNE sequencing identified an unknown homozygous transition. This variation affects the third nucleotide of the glycine 285 condon, and leads to a synonymous variant. Analysis of transcripts demonstrated that this single change creates a new splice donor site located 4 nucleotides upstream of the normal site, leading to a deletion and generating a frameshift in exon 9 followed by a premature termination codon. This paper relates the identification of a synonymous mutation in CHRNE that creates a new splice donor site leading to an aberrant splicing of pre-mRNAs and so to their instability. This is the first synonymous mutation in CHRNE known to generate a cryptic splice site, and mRNA quantification strongly suggests that it is the disease-causing mutation.

  5. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.

  6. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

    PubMed Central

    Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

  7. 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.

  8. O-Glycosyl Donors

    NASA Astrophysics Data System (ADS)

    López, J. Cristóbal

    O-Glycosyl donors, despite being one of the last successful donors to appear, have developed themselves into a burgeoning class of glycosyl donors. They can be classified in two main types: O-alkyl and O-aryl (or hetaryl) glycosyl donors. They share, however, many characteristics, they can be (1) synthesized from aldoses, either by modified Fisher glycosidation (O-alkyl) or by nucleophilic aromatic substitution (O-aryl or O-hetaryl), (2) stable to diverse chemical manipulations, (3) directly used for saccharide coupling, and (4) chemoselectively activated. Among these, n-pentenyl glycosides stand apart. They were the first O-alkyl glycosyl donors to be described and have paved the way to many conceptual developments in oligosaccharide synthesis. The development of the chemoselectivity-based "armed-disarmed" approach for saccharide coupling, including its stereoelectronic or torsional variants, now extended to other kinds of glycosyl donors, was first recognized in n-pentenyl glycosides. The chemical manipulation of the anomeric substituent in the glycosyl donor to induce reactivity differences between related species (sidetracking) was also introduced in n-pentenyl glycosides. An evolution of this concept, the "latent-active" strategy for glycosyl couplings, first described in thioglycosyl donors (vide infra), has been elegantly applied to O-glycosyl donors. Thus, allyl and vinyl glycosides, 2-(benzyloxycarbonyl)benzyl (BCB) glycosides and 2'-carboxybenzyl (CB) glycosides are useful "latent-active" glycosyl pairs. Finally, unprotected 3-methoxy-2-pyridyl (MOP) glycosides have been used in glycosylation processes with moderate success.

  9. Stereochemical control over Mn(II)-Thio versus Mn(II)-Oxy coordination in adenosine 5 prime -O-(1-thiodiphosphate) complexes at the active site of creatine kinase

    SciTech Connect

    Smithers, G.W.; Sammons, R.D.; Goodhart, P.J.; LoBrutto, R.; Reed, G.H. )

    1989-02-21

    The stereochemical configurations of the Mn(II) complexes with the resolved epimers of adenosine 5{prime}-O-(1-thiodiphosphate) (ADP{alpha}S), bound at the active site of creatine kinase, have been determined in order to assess the relative strengths of enzymic stereoselectivity versus Lewis acid/base preferences in metal-ligand binding. Electron paramagnetic resonance (EPR) data have been obtained for Mn(II) in anion-stabilized, dead-end (transition-state analogue) complexes, in ternary enzyme-Mn{sup II}ADP{alpha}S complexes, and in the central complexes of the equilibrium mixture. The modes of coordination of Mn(II) at P{sub alpha} in the nitrate-stabilized, dead-end complexes with each epimer of ADP{alpha}S were ascertained by EPR measurements with (R{sub p})-({alpha}-{sup 17}O)ADP{alpha}S and (S{sub p})-({alpha}-{sup 17}O)ADP{alpha}S. A reduction in the magnitude of the {sup 55}Mn hyperfine coupling constant in the spectrum for the complex containing (S{sub p})-ADP{alpha}S is indicative of Mn(II)-thio coordination at P{sub alpha}. The results indicate that a strict discrimination for a unique configuration of the metal-nucleotide substrate is expressed upon binding of all of the substrates to form the active complex (or an analogue thereof). This enzymic stereoselectivity provides sufficient binding energy to overcome an intrinsic preference for the hard Lewis acid Mn(II) to coordinate to the hard Lewis base oxygen.

  10. In vivo and in vitro dermal penetration of 2,4,5,2 prime ,4 prime , 5 prime -hexachlorobiphenyl in young and adult rats

    SciTech Connect

    Shah, P.V.; Sumler, M.R. ); Fisher, H.L.; Hall, L.L. )

    1989-10-01

    Penetration of 2,4,5,2{prime},4{prime},5{prime}-({sup 14}C)hexachlorobiphenyl (HCB) through skin of young (33 days) and adult (82 days) female Fischer 344 rats was determined in vivo and by two in vitro methods. In vivo dermal penetration at 120 hr was 45% in young and 43% in adults. At 72 hr in vivo dermal penetration was 35% in young and 26% in adults compared to 1.5% for young and 1.0% for adult as measured with a continuous flow in vitro system and 2.9% for young and 1.9% for adults as measured with a static in vitro system. Most of the dermally absorbed HCB remained in the body as only 4.9 and 2.6% of that absorbed was excreted by young and adult rats, respectively, at the end of 120 hr. Significant differences in dermal penetration and kinetics of HCB between young and adult female rats were observed. The elimination of ECB-derived material was approximately six times higher in feces than in urine. A physiological pharmacokinetic model was fitted to the organ and tissue radioactivity distribution data. Parameters in the model determined from dermal dosing of female Fischer 344 rats were in reasonable agreement with those reported in the literature for adult male Sprague-Dawley rats (iv dose). The rate constant for dermal penetration was 0.83 {times} 10{sup {minus}4} min{sup {minus}1} for adults and 0.96 {times} 10{sup {minus}4} min{sup {minus}1} for young. The delay or lag time parameter for dermal penetration was 4.4 hr in adults and 1.1 hr in young.

  11. Epistatic selection of a sequence 5{prime} of the gene responsible for cystic fibrosis may account for the high frequency of this disease in the Caucasian population

    SciTech Connect

    Macek, M. Jr. |; Nash, E.; Cutting, G.R.

    1994-09-01

    Cystic fibrosis (CF) is one of the more common lethal autosomal recessive disorders in Caucasian populations. Numerous hypotheses including genetic drift, founder effect, sex ratio, segregation distortions and various forms of heterozygote advantage have been proposed to explain the relatively high frequency of CF alleles. The observation of high linkage disequilibrium between markers at the 5{prime} end of CFTR and mutations that cause CF raised the possibility of epistatic selection. CF-linked marker allele frequencies were determined in 417 elderly individuals from a stable Czech population that survived high levels of infant and childhood mortality in the pre-antibiotic era. These data were compared with allele frequencies of 646 contemporary newborns and 345 young adults drawn from the same population who had significantly lower mortality rates in the antibiotic era. Allele frequencies of markers CS7/Hhal and KM19/Pstl from the D7S23 locus are significantly different (p<0.05) between elderly female and male subjects in this population. Furthermore, there is a significant difference in the allele frequencies of marker CS7/Hhal when newborn females and elderly women are compared (p<0.05). Taken together, these data suggest that the allele status at the CS7 region influenced female survival in the period of high infant and childhood mortality in the pre-antibiotic era. Under this selective pressure, CFTR mutations that occurred on the {open_quotes}favorable{close_quotes} background would marginally increase in frequency in each successive generation and more ancient mutations residing on this background would become the most frequent in the general population.

  12. Tyrosinase-positive oculocutaneous albinism in Southern African blacks: P gene-associated haplotypes suggest a major mutation in the 5{prime} region of the gene

    SciTech Connect

    Ramsay, M.; Stevens, G.; Beukering, J. van

    1994-09-01

    Tyrosinase-positive oculocutaneous albinism (ty-pos OCA) occurs with a prevalence of 1 in 3900 among Southern African (SA) blacks. The major contributors to morbidity and mortality are skin cancer and decreased visual acuity. Two distinct phenotypes occur, namely individuals with ephelides (darkly pigmented patches) and those without. There is complete concordance with regard to ephelus status among siblings. The disorder is linked to markers on chromosome 15q11.2-q12, and no obligatory cross-overs were observed with polymophic markers at the human homolog, P, of the mouse pink eyed dilute gene, p. Contrary to what has been shown for Caucasoid ty-pos OCA, this condition shows locus homogeneity among SA blacks. The P gene is an excellent candidate for ty-pos OCA and mutations in this gene will confirm its role in causing the common form of albinism in SA. Numerous P gene mutations have been described in other populations. In an attempt to detect mutations, the P gene cDNA was used to search for structural rearrangements or polymorphisms. Six polymorphisms (plR10/Scal, 912/Xbal, 912/HincII, 912/TaqI, 1412/TaqI [two systems] and 1412/HindIII) were detected with subclones of the P cDNA and haplotypes were determined in each family. None were clearly associated with an albinism-related rearrangement. However, strong linkage disequilibrium was observed with alleles at loci toward the 5{prime} region of the gene ({triangle}=0.65, 0.57 and 0.80 for the three polymorphisms detected with the 912 subclone), suggesting a major ty-pos OCA mutation in this region. Haplotype analysis provides evidence for a major mutation associated with the same haplotype in individuals with ephelides (8/12 OCA chromosomes) and those without ephelides (24:30). The presence of other ty-pos OCA associated haplotypes indicates several other less common mutations.

  13. Bioaccumulation of dietary 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl and induction of hepatic arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss)

    SciTech Connect

    Costa, E.G. da; Curtis, L.R.

    1995-10-01

    Juvenile rainbow trout (Oncorhynchus mykiss) were fed either 5 or 20 {micro}g, 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (245-HxCB)/g diet (wet wt.) for 4,8, or 12 weeks. Hepatic xenobiotic-metabolizing enzyme activities and dietary 245-HxCB accumulation in liver, muscle, and remaining carcass were determined. Liver-to-body weight ratios were not altered by either of the two 245-HxCB concentrations. Relative growth rate increased with time but was not altered by 245-HxCB concentration. Bioaccumulation of 245-HxCB was dose and time dependent in all tissues without reaching apparent stayed state. Hepatic arylhydrocarbon hydroxylase (AHH) activities increased with 245-HxCB dose and with time. Ethoxyresorufin-O-deethylase (EROD) activities also increased in fish fed 20 {micro}g 245-HxCB/g diet. No 245-HxCB-induced changes in uridine diphosphoglucuronosyl transferase (UDP-DT) or NADPH-dytochrome-c reductase (NCCR) activities were determined. High-resolution GC-MS analysis of the 245-HxCB standard revealed trace (0.4--0.5%) contamination by two mono-ortho pentachlorobiphenyls (PnCBs): 2,3,3{prime},4,4{prime}-PnCB and 2,3,4,4{prime},5-PnCB. Total liver accumulation of these contaminants was inversely related with corresponding EROD and AHH activities and estimated to contribute minimally to their induction. Results from this study suggested that long-term dietary 245-HxCB exposure induced cytochrome P4501A activities in rainbow trout liver.

  14. RNA Splicing in a New Rhabdovirus from Culex Mosquitoes▿†

    PubMed Central

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

    2011-01-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae. PMID:21507977

  15. 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.

  16. Cryptic splice sites and split genes.

    PubMed

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

    2011-08-01

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

  17. Cryptic splice sites and split genes

    PubMed Central

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

    2011-01-01

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

  18. Pharmacology of Modulators of Alternative Splicing

    PubMed Central

    Morris, Jonathan C.; Oltean, Sebastian; Donaldson, Lucy F.

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. PMID:28034912

  19. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  20. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

  1. Recursive splicing in long vertebrate genes.

    PubMed

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

    2015-05-21

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

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

    PubMed

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

    2015-05-01

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

  3. Donor Telomere Length SAA

    Cancer.gov

    A new NCI study has found that, among patients with severe aplastic anemia who received a hematopoietic cell transplant from an unrelated donor, those whose donor white blood cells had longer telomeres had higher survival rates five-years after transplant

  4. Rich Donors, Poor Countries

    ERIC Educational Resources Information Center

    Thomas, M. A.

    2012-01-01

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

  5. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

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

    PubMed

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

    2014-03-01

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

  7. An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

    PubMed Central

    Chabot, B; Blanchette, M; Lapierre, I; La Branche, H

    1997-01-01

    The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites. PMID:9121425

  8. [Motivations of oocytes donors].

    PubMed

    Cauvin, P

    2009-01-01

    Oocyte donation is a complex situation that requires the applicant couple to deal with the presence of the donor in the history of the child conception. Accepting the eggs is not the same thing than accepting the donor. Her place in the child's life depends on how his parents will accept her phantasmal reality beyond her real person. Paying attention to the story told by the donors on their motivations may help parents internalize this conception to three. We show from two clinical observations, that the generosity of donors is connected to personal issues that do not relate to unborn child or its parents. If there are two mothers in oocyte donation, they are not really in competition because there are also two children: the child conceived through donation is that of the project of the couple, the child to which the donor thinks, is and will remain in phantasmal domain, i.e. linked to the personal history of the donor. We also show that the psychological interview fully responds to the donor expectations when it seeks to highlight her motives.

  9. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.

    PubMed

    Wu, J Y; Maniatis, T

    1993-12-17

    Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

  10. Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.

    PubMed

    Cristea, Sandra; Gregory, Philip D; Urnov, Fyodor D; Cost, Gregory J

    2011-02-08

    Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.

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

    PubMed Central

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

    2012-01-01

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

  12. The incredible complexity of RNA splicing.

    PubMed

    Robert, Christelle; Watson, Mick

    2016-12-30

    Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets.

  13. Multiple links between transcription and splicing.

    PubMed

    Kornblihtt, Alberto R; de la Mata, Manuel; Fededa, Juan Pablo; Munoz, Manuel J; Nogues, Guadalupe

    2004-10-01

    Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

  14. The Characterizations of Different Splicing Systems

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

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

    2015-01-01

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

  16. Tumor microenvironment-associated modifications of alternative splicing.

    PubMed

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

    2014-02-01

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

  17. Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

    PubMed Central

    Gantla, S; Bakker, C T; Deocharan, B; Thummala, N R; Zweiner, J; Sinaasappel, M; Roy Chowdhury, J; Bosma, P J; Roy Chowdhury, N

    1998-01-01

    Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression. PMID:9497253

  18. Laparoscopic donor nephrectomy.

    PubMed

    Deger, S; Giessing, M; Roigas, J; Wille, A H; Lein, M; Schönberger, B; Loening, S A

    2005-01-01

    Laparoscopic live donor nephrectomy (LDN) has removed disincentives of potential donors and may bear the potential to increase kidney donation. Multiple modifications have been made to abbreviate the learning curve while at the same time guarantee the highest possible level of medical quality for donor and recipient. We reviewed the literature for the evolution of the different LDN techniques and their impact on donor, graft and operating surgeon, including the subtleties of different surgical accesses, vessel handling and organ extraction. We performed a literature search (PubMed, DIMDI, medline) to evaluate the development of the LDN techniques from 1995 to 2003. Today more than 200 centres worldwide perform LDN. Hand-assistance has led to a spread of LDN. Studies comparing open and hand-assisted LDN show a reduction of operating and warm ischaemia times for the hand-assisted LDN. Different surgical access sites (trans- or retroperitoneal), different vessel dissection approaches, donor organ delivery techniques, delivery sites and variations of hand-assistance techniques reflect the evolution of LDN. Proper techniques and their combination for the consecutive surgical steps minimize both warm ischaemia time and operating time while offering the donor a safe minimally invasive laparoscopic procedure. LDN has breathed new life into the moribund field of living kidney donation. Within a few years LDN could become the standard approach in living kidney donation. Surgeons working in this field must be trained thoroughly and well acquainted with the subtleties of the different LDN techniques and their respective advantages and disadvantages.

  19. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    PubMed

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program.

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

    PubMed Central

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

    1993-01-01

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

  1. MAASE: An alternative splicing database designed for supporting splicing microarray applications

    PubMed Central

    ZHENG, CHRISTINA L.; KWON, YOUNG-SOO; LI, HAI-RI; ZHANG, KUI; COUTINHO-MANSFIELD, GABRIELA; YANG, CANZHU; NAIR, T. MURLIDHARAN; GRIBSKOV, MICHAEL; FU, XIANG-DONG

    2005-01-01

    Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication. PMID:16251387

  2. SAW: A Method to Identify Splicing Events from RNA-Seq Data Based on Splicing Fingerprints

    PubMed Central

    Ning, Kang; Fermin, Damian

    2010-01-01

    Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons. PMID:20706591

  3. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  4. Identification of a splice-site mutation in the human growth hormone-variant gene.

    PubMed Central

    MacLeod, J N; Liebhaber, S A; MacGillivray, M H; Cooke, N E

    1991-01-01

    The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analysis of the elongated hGH-V mRNA demonstrated retention of the first 12 bases of intron 2, resulting from both a base substitution at the intron 2 splice-donor dinucleotide (GT----AT) and activation of a cryptic splice-donor site 12 bases downstream. Survey of a total of 60 additional chromosomes failed to reveal additional incidence of this mutation. The mutation, which we have designated hGH-Vintron 2, pos 1 (G----A), represents both an initial example of a nondeletional mutation within the hGH-V gene and corresponding structural alteration in the encoded hGH-V hormone. Images Figure 1 Figure 2 Figure 4 PMID:2035535

  5. 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; Sabò, Arianna; Amati, Bruno; Wee, Keng Boon; Guccione, Ernesto

    2015-07-02

    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 Eµ-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 Eµ-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.

  6. HLA-A*68020103 shows an eight nucleotides deletion within intron 2 but has normal mRNA splicing and serological recognition.

    PubMed

    Balas, A; Sánchez-García, F; Bustamante, L; García-Sánchez, F; Vicario, J L

    2007-09-01

    A novel A*68020103 allele was completely characterized by sequencing in a Spanish bone marrow donor. A*68020103 has an eight nucleotides deletion at the 5'-end of intron 2, when compared with other A*6802 alleles. This alteration does not affect either its mRNA splicing process or serological detection.

  7. Alternative splicing in cancer: implications for biology and therapy.

    PubMed

    Chen, J; Weiss, W A

    2015-01-02

    Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

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

    PubMed

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

    1993-08-01

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

  9. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  10. Lessons from non-canonical splicing

    PubMed Central

    Ule, Jernej

    2016-01-01

    Recent improvements in experimental and computational techniques used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons, and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimises their potential to disrupt gene expression. While non-canonical splicing can lead to aberrant transcripts that cause many diseases, we also explain how it can be exploited for new therapeutic strategies. PMID:27240813

  11. RNA-splicing endonuclease structure and function.

    PubMed

    Calvin, K; Li, H

    2008-04-01

    The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes.

  12. HOLLYWOOD: a comparative relational database of alternative splicing.

    PubMed

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  13. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2001-10-01

    2001. Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of Caenorhabditis elegans . 11... Caenorhabditis 36 Lorson, C.L. et al. (1999) A single nucleotide in the Biol. 77, 277-291 elegans . Nature 402, 835-838 SMN gene regulates splicing and is...terminate (Birse et al., 1998; Proudfoot, 2000), mutation of the poly(A) signal resulted in the accumulation to high levels in the nuclear fraction of

  14. Targeting RNA-splicing for SMA treatment.

    PubMed

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

    The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease.

  15. CDH1 Missense Variant c.1679C>G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site

    PubMed Central

    Bacares, Ruben; Salo-Mullen, Erin; Somar, Joshua; Lehrich, Deborah A.; Fasaye, Grace-Ann; Coit, Daniel G.; Tang, Laura H.; Stadler, Zsofia K.; Zhang, Liying

    2016-01-01

    Disease-causing germline mutations in CDH1 cause Hereditary Diffuse Gastric Cancer (HDGC). For patients who meet the HDGC screening criteria, the identification and classification of the sequence variants found in CDH1 are critical for risk management of patients. In this report, we describe a germline CDH1 c.1679C>G (p.T560R) variant identified in a 50 year old man who was diagnosed with gastric cancer with a strong family history of gastric cancer (one living brother was diagnosed with gastric cancer at 63 and another brother died of gastric cancer at 45). cDNA analysis, involving fragment analysis and cloning, indicated that the p.T560R mutation created a novel 5’ splice donor site, which led to a novel transcript with a 32 nucleotide deletion in exon 11. This abnormal transcript putatively produces a truncated CDH1 protein (E-cadherin) of 575 amino acids instead of 882. We also demonstrated that the variant completely abolishes normal splicing as the mutant allele does not generate any normal transcript. Furthermore, the CDH1 c.1679C>G (p.T560R) variant segregated with gastric cancer in all three family members affected with gastric cancer in this family. These results support the conclusion that CDH1 c.1679C>G (p.T560R) variant is a pathogenic mutation and contributes to HDGC through disruption of normal splicing. PMID:27880784

  16. CDH1 Missense Variant c.1679C>G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5' Splice Site.

    PubMed

    Yelskaya, Zarina; Bacares, Ruben; Salo-Mullen, Erin; Somar, Joshua; Lehrich, Deborah A; Fasaye, Grace-Ann; Coit, Daniel G; Tang, Laura H; Stadler, Zsofia K; Zhang, Liying

    2016-01-01

    Disease-causing germline mutations in CDH1 cause Hereditary Diffuse Gastric Cancer (HDGC). For patients who meet the HDGC screening criteria, the identification and classification of the sequence variants found in CDH1 are critical for risk management of patients. In this report, we describe a germline CDH1 c.1679C>G (p.T560R) variant identified in a 50 year old man who was diagnosed with gastric cancer with a strong family history of gastric cancer (one living brother was diagnosed with gastric cancer at 63 and another brother died of gastric cancer at 45). cDNA analysis, involving fragment analysis and cloning, indicated that the p.T560R mutation created a novel 5' splice donor site, which led to a novel transcript with a 32 nucleotide deletion in exon 11. This abnormal transcript putatively produces a truncated CDH1 protein (E-cadherin) of 575 amino acids instead of 882. We also demonstrated that the variant completely abolishes normal splicing as the mutant allele does not generate any normal transcript. Furthermore, the CDH1 c.1679C>G (p.T560R) variant segregated with gastric cancer in all three family members affected with gastric cancer in this family. These results support the conclusion that CDH1 c.1679C>G (p.T560R) variant is a pathogenic mutation and contributes to HDGC through disruption of normal splicing.

  17. Living Donor Liver Transplantation

    MedlinePlus

    ... instructions before and after surgery. • Have a compatible blood type. • Have an emotional tie with the recipient. • Not ... test is to find out if the donor's blood type matches the recipient’s blood type. Next, the transplant ...

  18. Dialing for Donors

    ERIC Educational Resources Information Center

    Schaffhauser, Dian

    2012-01-01

    When times get tough, grown children often turn to their parents for help--for some extra cash, even somewhere to stay. For colleges and universities, that role is filled by alumni donors. In 2011, with education budgets slashed across the country, giving accounted for 6.5 percent of college expenditures, according to the Council for Aid to…

  19. Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals

    PubMed Central

    Piton, Amélie; Jouan, Loubna; Rochefort, Daniel; Dobrzeniecka, Sylvia; Lachapelle, Karine; Dion, Patrick A; Gauthier, Julie; Rouleau, Guy A

    2013-01-01

    A large-scale sequencing screen of X-linked synaptic genes in individuals with autism spectrum disorder (ASD) or schizophrenia (SCZ), two common neurodevelopmental disorders, identified many variants most of which have no easily predictable effect on gene function. In this report, we evaluated the impact of these rare missense and silent variants on gene splicing. For this purpose, we used complementary in silico analyses, in vitro minigene-based assays and RNA prepared from lymphoblastoid cells derived from patients with these mutations. Our goal was to identify the variants which might either create or disrupt an acceptor splice site, a donor splice site or an exonic splicing enhancer, thus leading to aberrant splicing that could be involved in the pathogenesis of ASD or SCZ. We identified truncating mutations in distinct X-linked gamma-aminobutyric acid A (GABAA) receptor subunit-encoding genes, GABRQ and GABRA3, in two different families. Furthermore, missense and silent variants in nuclear RNA export factor 5 and histone deacetylase 6 were shown to partially disrupt the protein. While genes from the GABAergic pathway have previously been thought to be involved in the pathophysiology of ASD, this is the first report of ASD patients with truncating mutations in GABA receptors genes. PMID:23169495

  20. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

  1. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  2. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

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

  3. A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection.

    PubMed

    Hoffmann, Steve; Otto, Christian; Doose, Gero; Tanzer, Andrea; Langenberger, David; Christ, Sabina; Kunz, Manfred; Holdt, Lesca M; Teupser, Daniel; Hackermüller, Jörg; Stadler, Peter F

    2014-02-10

    Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (http://www.bioinf.uni-leipzig.de/Software/segemehl/).

  4. 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

    2015-01-09

    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.

  5. Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

    PubMed

    Vetrini, Francesco; Tammaro, Roberta; Bondanza, Sergio; Surace, Enrico M; Auricchio, Alberto; De Luca, Michele; Ballabio, Andrea; Marigo, Valeria

    2006-05-01

    An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

  6. Variation in alternative splicing across human tissues

    PubMed Central

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

    2004-01-01

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

  7. Vitamin D and alternative splicing of RNA.

    PubMed

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  8. Being a Living Donor: Risks

    MedlinePlus

    ... Living Donation / Being a Living Donor / Risks Facts History Organs Frequently Asked Questions Discussing Living Donation Types Related Non-Related Non-Directed Paired Donation Blood Type Incompatible Positive Crossmatch Being a Living Donor ...

  9. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    PubMed

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  10. The behavior of bonded doubler splices for composite sandwich panels

    NASA Technical Reports Server (NTRS)

    Zeller, T. A.; Weisahaar, T. A.

    1980-01-01

    The results of an investigation into the behavior of adhesively bonded doubler splices of two composite material sandwich panels are presented. The splices are studied from three approaches: analytical; numerical (finite elements); and experimental. Several parameters that characterize the splice are developed to determine their influence upon joint strength. These parameters are: doubler overlap length; core stiffness; laminate bending stiffness; the size of the gap between the spliced sandwich panels; and room and elevated temperatures. Similarities and contrasts between these splices and the physically similar single and double lap joints are discussed. The results of this investigation suggest several possible approaches to improving the strength of the sandwich splices.

  11. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    SciTech Connect

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. )

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  12. Systematic screening for mutations in the 5{prime}-regulatory region of the human dopamine D{sub 1} receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder

    SciTech Connect

    Cichon, S.; Noethen, M.M.; Stoeber, G.

    1996-07-26

    A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5{prime}-flanking region of the dopamine D{sub 1} receptor (DRD1) gene. This region has previously been shown to contain a functional promoter. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5{prime}-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder. 31 refs., 3 tabs.

  13. National Marrow Donor Program

    DTIC Science & Technology

    2009-08-14

    Recipient Pair HLA typing project to characterize class I and class II alleles of donor/recipient paired samples from NMDP’s Repository was initiated...24 • Initiated investigation of the first class II non-ARS mismatch (DRB1*140101/1454) where both alleles have been seen in the same genotype... MHC Major Histocompatibility Complex B-LCLs B-Lymphocytic Cell Lines MICA MHC Class I-Like Molecule, Chain A BARDA Biomedical Advanced Research and

  14. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  15. Alternative splicing modulates stem cell differentiation.

    PubMed

    Fu, Ru-Huei; Liu, Shih-Ping; Ou, Chen-Wei; Yu, Hsiu-Hui; Li, Kuo-Wei; Tsai, Chang-Hai; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2009-01-01

    Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell-cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.

  16. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  17. Complications of donor apheresis.

    PubMed

    Winters, Jeffrey L

    2006-07-01

    A decreasing blood donor pool in the presence of increasing blood transfusion demands has resulted in the need to maximally utilize each blood donor. This has led to a trend in the increasing use of automated blood collections. While apheresis donation shares many reactions and injuries with whole blood donation, because of the differences, unique complications also exist. Overall, evidence in the literature suggests that the frequency of reactions to apheresis donation is less than that seen in whole blood donation, though the risk of reactions requiring hospitalization is substantially greater. The most common apheresis-specific reaction is hypocalcemia due to citrate anticoagulation, which, while usually mild, has the potential for severely injuring the donor. Other reactions to apheresis donation are uncommon (e.g., hypotension) or rare (e.g., air embolism). More worrisome, and in need of additional study, are the long-term effects of apheresis donation. Recent evidence suggests that repeated apheresis platelet donations may adversely effect thrombopoiesis as well as bone mineralization. Granulocyte donation has also been implicated in unexpected long-term consequences.

  18. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  19. The 20S Proteasome Splicing Activity Discovered by SpliceMet

    PubMed Central

    Textoris-Taube, Kathrin; Janek, Katharina; Keller, Christin; Henklein, Petra; Kloetzel, Peter Michael; Zaikin, Alexey

    2010-01-01

    The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected. PMID:20613855

  20. Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

    PubMed

    de Longevialle, Andéol Falcon; Small, Ian D; Lurin, Claire

    2010-07-01

    Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

  1. Analysis of differential splicing suggests different modes of short-term splicing regulation

    PubMed Central

    Topa, Hande; Honkela, Antti

    2016-01-01

    Motivation: Alternative splicing is an important mechanism in which the regions of pre-mRNAs are differentially joined in order to form different transcript isoforms. Alternative splicing is involved in the regulation of normal physiological functions but also linked to the development of diseases such as cancer. We analyse differential expression and splicing using RNA-sequencing time series in three different settings: overall gene expression levels, absolute transcript expression levels and relative transcript expression levels. Results: Using estrogen receptor α signaling response as a model system, our Gaussian process-based test identifies genes with differential splicing and/or differentially expressed transcripts. We discover genes with consistent changes in alternative splicing independent of changes in absolute expression and genes where some transcripts change whereas others stay constant in absolute level. The results suggest classes of genes with different modes of alternative splicing regulation during the experiment. Availability and Implementation: R and Matlab codes implementing the method are available at https://github.com/PROBIC/diffsplicing. An interactive browser for viewing all model fits is available at http://users.ics.aalto.fi/hande/splicingGP/ Contact: hande.topa@helsinki.fi or antti.honkela@helsinki.fi Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307611

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

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2014-07-01

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

  4. Targeting MDM4 Splicing in Cancers

    PubMed Central

    Bardot, Boris; Toledo, Franck

    2017-01-01

    MDM4, an essential negative regulator of the P53 tumor suppressor, is frequently overexpressed in cancer cells that harbor a wild-type P53. By a mechanism based on alternative splicing, the MDM4 gene generates two mutually exclusive isoforms: MDM4-FL, which encodes the full-length MDM4 protein, and a shorter splice variant called MDM4-S. Previous results suggested that the MDM4-S isoform could be an important driver of tumor development. In this short review, we discuss a recent set of data indicating that MDM4-S is more likely a passenger isoform during tumorigenesis and that targeting MDM4 splicing to prevent MDM4-FL protein expression appears as a promising strategy to reactivate p53 in cancer cells. The benefits and risks associated with this strategy are also discussed. PMID:28230750

  5. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  6. Systematic identification and analysis of exonic splicing silencers.

    PubMed

    Wang, Zefeng; Rolish, Michael E; Yeo, Gene; Tung, Vivian; Mawson, Matthew; Burge, Christopher B

    2004-12-17

    Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

  7. Experimental study of lap splice bolted connection

    NASA Astrophysics Data System (ADS)

    Zhao, Dehui; Tian, Lishan; Jiang, Wenqiang; An, Liqiang; Zhang, Ziyang

    2017-01-01

    The bolted connection is prone to slip under external load in the lattice transmission tower, which will affect the internal forces and deformation of tower. In order to better simulate the effect of bolt connection slippage on transmission tower, the load deformation relationship should be established. In this paper, the single lap splice bolt connection under tension load is tested and the load displacement curve is obtained. Furthermore, the existing model of single lap splice bolted connection is modified, which will plays an important role in the influence of the bolt slippage of the transmission lines towers more accurately and reasonably.

  8. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  9. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  10. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Electrical connections or splices; suitability... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically...

  11. 30 CFR 75.514 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Electrical connections or splices; suitability... COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Electrical Equipment-General § 75.514 Electrical connections or splices; suitability. All electrical connections or splices...

  12. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Electrical connections or splices; suitability... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically...

  13. Schizophyllum commune has an extensive and functional alternative splicing repertoire

    PubMed Central

    Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

    2016-01-01

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically. PMID:27659065

  14. Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level

    PubMed Central

    Liu, Wei; Han, Bing; Zhu, Wenjiao; Cheng, Tong; Fan, Mengxia; Wu, Jiajun; Yang, Ying; Zhu, Hui; Si, Jiqiang; Lyu, Qifeng; Chai, Weiran; Zhao, Shuangxia; Song, Huaidong; Kuang, Yanping; Qiao, Jie

    2017-01-01

    Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5′ splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads. PMID:28367994

  15. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

    PubMed

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé; Emiliani, Stéphane; Gallois-Montbrun, Sarah

    2016-12-20

    Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

  16. Splicing-directed therapy in a new mouse model of human accelerated aging.

    PubMed

    Osorio, Fernando G; Navarro, Claire L; Cadiñanos, Juan; López-Mejía, Isabel C; Quirós, Pedro M; Bartoli, Catherine; Rivera, José; Tazi, Jamal; Guzmán, Gabriela; Varela, Ignacio; Depetris, Danielle; de Carlos, Félix; Cobo, Juan; Andrés, Vicente; De Sandre-Giovannoli, Annachiara; Freije, José M P; Lévy, Nicolas; López-Otín, Carlos

    2011-10-26

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino-based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide-based therapies for treating human diseases of accelerated aging.

  17. [Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

    PubMed

    Pepe, Carolina M; Saraco, Nora I; Baquedano, María Sonia; Guercio, Gabriela; Vaiani, Elisa; Berensztein, Esperanza; Rivarola, Marco A; Belgorosky, Alicia

    2007-01-01

    P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

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

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

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

  19. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2000-10-01

    erythematosus and sarcoidosis . Arthritis Rheum. 41: 1505-15 10. Eldridge A.G., Y. Li, P.A. Sharp, and B.J. Blencowe. 1999. The SRml60/300 splicing coactivator...J. Hum. Genet. 59:279-286. erythematosus and sarcoidosis . Arthritis Rheum. 41:1505-1510. Matsumoto, K., K.M. Wassarman, and A.P. Wolffe. 1998. Nuclear

  20. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  1. Emerging roles of BRCA1 alternative splicing.

    PubMed

    Orban, T I; Olah, E

    2003-08-01

    Germline mutations of the BRCA1 gene predispose individuals mainly to the development of breast and/or ovarian cancer. However, the exact function of the gene is still unclear, although the encoded proteins are involved in various cellular processes, including transcriptional regulation and DNA repair pathways. Several BRCA1 splice variants are found in different tissues, but in spite of intense investigations, their regulation and possible functions are poorly understood at the moment. This review summarises current knowledge on the roles of these splice variants and the mechanisms responsible for their formation. Because alternative splicing is now widely accepted as an important source of genetic diversity, elucidating the functions of the BRCA1 splice variants would help in the understanding of the exact role(s) of this tumour suppressor. This should help to resolve the current paradox that, despite its seemingly vital cellular functions, mutations of this gene are associated with tissue specific tumour formation predominantly in the breast and the ovary.

  2. Alternative splicing: a pivotal step between eukaryotic transcription and translation.

    PubMed

    Kornblihtt, Alberto R; Schor, Ignacio E; Alló, Mariano; Dujardin, Gwendal; Petrillo, Ezequiel; Muñoz, Manuel J

    2013-03-01

    Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

  3. Second order limit language in variants of splicing system

    NASA Astrophysics Data System (ADS)

    Ahmad, Muhammad Azrin; Sarmin, Nor Haniza; Heng, Fong Wan; Yusof, Yuhani

    2014-07-01

    The cutting and pasting processes that occur in DNA molecules have led to the formulation of splicing system. Since then, there are few models used to model the splicing system. The splicing language, which is the product of splicing system, can be categorized into two, namely the adult and limit language. In this research, limit language is extended to the second order limit language. Few problems are approached which lead to the formation of second order limit language which is then analyzed using various types of splicing system.

  4. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

    PubMed

    Listerman, Imke; Sapra, Aparna K; Neugebauer, Karla M

    2006-09-01

    Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

  5. [Psychosomatic selection of living liver donors].

    PubMed

    Erim, Y; Senf, W

    2001-01-01

    In the Essen University Clinic for Psychotherapy and Psychosomatics, between January and December 2000, 54 potential liver donors and 12 kidney donors were examined. All the kidney donors were found to be suitable; 7 potential liver donors were rejected on psychosomatic grounds. Reasons for the rejection were addiction (1 donor), suspected financial dependency of the donor on the recipient (1 donor) and, in the case of one donor not related to the recipient, the apparent lack of a special emotional attachment. During the actual evaluation interview, 4 potential donors reversed their original decision. Such a psychosomatic evaluation is a great help for donors in clarifying their motives and their decision.

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

    PubMed Central

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

    2016-01-01

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

  7. 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.

  8. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan T; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz.

  9. [Clinical evaluation of living donor].

    PubMed

    Scolari, Maria Piera; Comai, G; La Manna, G; Liviano D'Arcangelo, G; Monti, M; Feliciangeli, G; Stefoni, S

    2009-01-01

    When possible, living donor transplantation represents the best therapeutic strategy for patients suffering from chronic renal failure. Studying the donor allows a complete and thorough clinical, laboratory and instrumental assessment that guarantees good organ function whilst protecting the health of the donor. The main parameters considered within this framework are age, renal function, nephrological complications, comorbidities (diabetes, hypertension, obesity, etc.), malignancies, and infection. Moreover, particular attention is paid to the sociopsychological aspects of the donation, particularly related to the donor, the recipient, and the entire family situation.

  10. Functional association between promoter structure and transcript alternative splicing.

    PubMed

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

    1997-10-14

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

  11. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery.

    PubMed

    Garanto, Alejandro; Chung, Daniel C; Duijkers, Lonneke; Corral-Serrano, Julio C; Messchaert, Muriël; Xiao, Ru; Bennett, Jean; Vandenberghe, Luk H; Collin, Rob W J

    2016-06-15

    Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.

  12. Donor cell leukemia.

    PubMed

    Ruiz-Arguelles, Alejandro

    2012-04-01

    Minimal residual disease refers to the tumour cells that are still present in a given patient after completion of a therapeutic scheme. The demonstration and quantification of residual neoplastic cells has a crucial impact in clinical decision making, for it might prompt continuation of treatment, while the absence of such cells might serve as evidence to withdraw therapy. Therefore, both sensitivity and specificity of the methods used to unravel residual neoplastic cells must be highly reliable and robust. Flow cytometry has been widely used for this purpose, and its clinical performance depends mainly on the criteria of interpretation, rather than in the technique by itself; molecular biology techniques have proved to be highly sensitive and specific but unfortunately they cannot be used in all patients or in all types of leukemia. Finally, the development of donor cell leukemia in transplanted patients, might mimic residual disease and add more confusion to an already controversial issue. These topics are discussed in this paper.

  13. Multiple splicing defects in an intronic false exon.

    PubMed

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  14. Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3′ splice-site selection. PMID:9858550

  15. 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.

  16. Urinary biomarkers after donor nephrectomy.

    PubMed

    Hoogendijk-van den Akker, Judith M; Warlé, Michiel C; van Zuilen, Arjan D; Kloke, Heinrich J; Wever, Kim E; d'Ancona, Frank C H; Ӧzdemir, Denise M D; Wetzels, Jack F M; Hoitsma, Andries J

    2015-05-01

    As the beginning of living-donor kidney transplantation, physicians have expressed concern about the possibility that unilateral nephrectomy can be harmful to a healthy individual. To investigate whether the elevated intra-abdominal pressure (IAP) during laparoscopic donor nephrectomy causes early damage to the remaining kidney, we evaluated urine biomarkers after laparoscopic donor nephrectomy. We measured albumin and alpha-1-microglobulin (α-1-MGB) in urine samples collected during and after open and laparoscopic donor nephrectomy and laparoscopic cholecystectomy and colectomy. Additionally, kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were measured in urine samples collected during and after laparoscopic donor nephrectomy and colectomy. The same biomarkers were studied in patients randomly assigned to standard or low IAP during laparoscopic donor nephrectomy. We observed a peak in urinary albumin excretion during all procedures. Urine α-1-MGB rose in the postoperative period with a peak on the third postoperative day after donor nephrectomy. Urine α-1-MGB did not increase after laparoscopic cholecystectomy and colectomy. After laparoscopic nephrectomy, we observed slight increases in urine KIM-1 during surgery and in urine NGAL at day 2-3 after the procedure. After laparoscopic colectomy, both KIM-1 and NGAL were increased in the postoperative period. There were no differences between the high- and low-pressure procedure. Elevated urinary α-1-MGB suggests kidney damage after donor nephrectomy, occurring irrespective of IAP during the laparoscopic procedure.

  17. Reevaluating the dead donor rule.

    PubMed

    Collins, Mike

    2010-04-01

    The dead donor rule justifies current practice in organ procurement for transplantation and states that organ donors must be dead prior to donation. The majority of organ donors are diagnosed as having suffered brain death and hence are declared dead by neurological criteria. However, a significant amount of unrest in both the philosophical and the medical literature has surfaced since this practice began forty years ago. I argue that, first, declaring death by neurological criteria is both unreliable and unjustified but further, the ethical principles which themselves justify the dead donor rule are better served by abandoning that rule and instead allowing individuals who have suffered severe and irreversible brain damage to become organ donors, even though they are not yet dead and even though the removal of their organs would be the proximal cause of death.

  18. Evolution of Nova-Dependent Splicing Regulation in the Brain

    PubMed Central

    Živin, Marko; Darnell, Robert B

    2007-01-01

    A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

  19. Heritability of alternative splicing in the human genome

    PubMed Central

    Kwan, Tony; Benovoy, David; Dias, Christel; Gurd, Scott; Serre, David; Zuzan, Harry; Clark, Tyson A.; Schweitzer, Anthony; Staples, Michelle K.; Wang, Hui; Blume, John E.; Hudson, Thomas J.; Sladek, Rob; Majewski, Jacek

    2007-01-01

    Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5′ splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled. PMID:17671095

  20. The influence of Argonaute proteins on alternative RNA splicing.

    PubMed

    Batsché, Eric; Ameyar-Zazoua, Maya

    2015-01-01

    Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO.

  1. 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.

  2. [EDAS, databases of alternatively spliced human genes].

    PubMed

    Nurtdinov, R N; Neverov, A D; Mal'ko, D B; Kosmodem'ianskiĭ, I A; Ermakova, E O; Ramenskiĭ, V E; Mironov, A A; Gel'fand, M S

    2006-01-01

    EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.

  3. Solution behavior and complete sup 1 H and sup 13 C NMR assignments of the coenzyme B sub 12 derivative (5 prime -deoxyadenosyl)cobinamide using modern 2D NMR experiments, including 600-MHz sup 1 H NMR data

    SciTech Connect

    Pagano, T.G.; Yohannes, P.G.; Marzilli, L.G. ); Hay, B.P.; Scott, J.R.; Finke, R.G. )

    1989-02-15

    Two-dimensional (2D) NMR methods have been used to assign completely the {sup 1}H and {sup 13}C NMR spectra of the (5{prime}-deoxyadenosyl)cobinamide cation (AdoCbi{sup +}) in D{sub 2}O. Most of the {sup 1}H spectral assignments were made by using 2D homonuclear shift correlation spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), absorption-mode (phase sensitive) 2D nuclear Overhauser effect (NOE) spectroscopy, and spin-locked NOE spectroscopy (also called ROESY, for rotating-frame Overhauser enhancement spectroscopy). Most of the protonated carbon resonances were assigned by using {sup 1}H-detected heteronuclear multiple-quantum coherence (HMQC) spectroscopy. The nonprotonated carbon resonances, as well as the remaining unassigned {sup 1}H and {sup 13}C NMR signals, were assigned from long-range {sup 1}H-{sup 13}C connectivities determined from {sup 1}H-detected multiple-bond heteronuclear multiple-quantum coherence spectroscopy (HMBC). Comparison of the {sup 13}C chemical shifts and {sup 1}H NOEs of AdoCbi{sup +} with those of coenzyme B{sup 12} ((5{prime}-deoxyadenosyl)cobalamin) and its benzimidazole-protonated, base-off form indicates that the electronic properties and structure of AdoCbi{sup +} are similar to that of coenzyme B{sup 12} in the protonated, base-off form. The {sup 13}C chemical shifts of most of the carbons of AdoCbi{sup +} do not vary significantly from those of base-off, benzimidazole-protonated coenzyme B{sup 12}, indicating that the electronic environment of the corrin ring is also similar in both compounds. However, significant differences in the chemical shifts of some of the corresponding carbons of the b, d, e, and f corrin side chains in AdoCbi{sup +} and in base-off, benzimidazole-protonated coenzyme B{sub 12} indicate that the positions of these side chains may be different in AdoCbi{sup +} compared to base-off coenzyme B{sup 12}.

  4. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  5. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

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

    PubMed

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

    2004-09-15

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

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

    PubMed

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

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

  8. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

    PubMed Central

    Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

    2007-01-01

    Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

  9. Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing.

    PubMed

    Defesche, J C; Schuurman, E J M; Klaaijsen, L N; Khoo, K L; Wiegman, A; Stalenhoef, A F H

    2008-06-01

    In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3'-splice donor site in exon 4 and R385R was associated with a new 5'-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay.

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

    PubMed

    Mazloomian, Alborz; Meyer, Irmtraud M

    2015-01-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

    Královicová, Jana; Vorechovsky, Igor

    2007-01-01

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

  13. Living kidney donors and ESRD.

    PubMed

    Ross, Lainie Friedman

    2015-07-01

    There are more than 325 living kidney donors who have developed end-stage renal disease and have been listed on the Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) deceased donor kidney wait list. The OPTN/UNOS database records where these kidney donors are listed and, if they donated after April 1994, where that donation occurred. These 2 locations are often not the same. In this commentary, I examine whether a national living donor registry should be created and whether transplantation centers should be notified when one of their living kidney donors develops end-stage renal disease. I consider and refute 5 potential objections to center notification. I explain that transplantation centers should look back at these cases and input data into a registry to attempt to identify patterns that could improve donor evaluation protocols. Creating a registry and mining the information it contains is, in my view, our moral and professional responsibility to future patients and the transplantation endeavor. As individuals and as a community, we need to acknowledge the many unknown risks of living kidney donation and take responsibility for identifying these risks. We then must share information about these risks, educate prospective donors about them, and attempt to minimize them.

  14. 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.

  15. A multi-agent system simulating human splice site recognition.

    PubMed

    Vignal, L; Lisacek, F; Quinqueton, J; d'Aubenton-Carafa, Y; Thermes, C

    1999-06-15

    The present paper describes a method detecting splice sites automatically on the basis of sequence data and models of site/signal recognition supported by experimental evidences. The method is designed to simulate splicing and while doing so, track prediction failures, missing information and possibly test correcting hypotheses. Correlations between nucleotides in the splice site regions and the various elements of the acceptor region are evaluated and combined to assess compensating interactions between elements of the splicing machinery. A scanning model of the acceptor region and a model of interaction between the splicing complexes (exon definition model) are also incorporated in the detection process. Subsets of sites presenting deficiencies of several splice site elements could be identified. Further examination of these sites helps to determine lacking elements and refine models.

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

    PubMed Central

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

    2015-01-01

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

  17. Targeting Splicing in the Treatment of Human Disease

    PubMed Central

    Suñé-Pou, Marc; Prieto-Sánchez, Silvia; Boyero-Corral, Sofía; Moreno-Castro, Cristina; El Yousfi, Younes; Suñé-Negre, Josep Mª; Hernández-Munain, Cristina; Suñé, Carlos

    2017-01-01

    The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event. PMID:28245575

  18. Test and Analysis of Spliced DI-BSCCO HTS Tapes

    NASA Astrophysics Data System (ADS)

    Fetisov, S. S.; Sotnikov, D. V.; Radchenko, I. P.; Vysotsky, V. S.; Osabe, G.; Kinoshita, K.; Fujikami, J.; Kobayashi, S.; Yamazaki, K.

    For some applications, short unit lengths of HTS wires should be spliced if longer lengths are necessary and short unit lengths of HTS wires should be utilize by applying the splice technology to reduce the total wire cost in the application. The splice technology has been developed for DI-BSCCO Type HT-CA tapes by Sumitomo Electric and spliced tapes were tested in Russian Cable Institute. The test program included: measurements of splice's resistance, critical current anisotropy, thermo cycling tolerance, mechanical properties, overload tests and magnetization measurements. In the paper the results of tests are presented and discussed. The test results demonstrated that splices can be used for cable production if twisting and bending limitations are taken into account.

  19. HEXEvent: a database of Human EXon splicing Events.

    PubMed

    Busch, Anke; Hertel, Klemens J

    2013-01-01

    HEXEvent (http://hexevent.mmg.uci.edu) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

  20. Visualizing group II intron catalysis through the stages of splicing

    PubMed Central

    Marcia, Marco; Pyle, Anna Marie

    2012-01-01

    SUMMARY Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report fourteen crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing, in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, adopting a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome. PMID:23101623

  1. [Kidney grafts from elderly donors].

    PubMed

    Hiesse, Christian; Pessione, Fabienne; Cohen, Sophie

    2003-06-07

    FROM AN EPIDEMIOLOGICAL POINT OF VIEW: The epidemiology of renal transplantation had greatly changed over the past 10 years. The increasing number of patients with renal failure and candidates for transplantation increases the demand for grafts, whereas the sampling rate of organs remains stable. The mean age of the donors is rising, hence underlining the question of the use of organs of so-called "borderline" quality. THE WEAK POINTS OF ELDERLY GRAFTS: Aging of the kidneys affects the structure of the parenchyma and renal function, which decreases, notably in hypertensive persons. The elderly graft exhibits a critical mass of nephrons that is insufficient to fulfil the functional requirements of a poorly equipped recipient. The recipient is more sensitive to the added agressions: prolonged ischemia and immunological and medicinal agressions. THE RESULTS OF RENAL GRAFT FROM ELDERLY DONORS: They are quantitatively and qualitatively inferior to those of renal transplants from "ideal" donors. The donor's age is a significant factor influencing negatively influences the survival of the transplanted kidney, but dependent on past vascular history. Good results regarding the maintenance of dialysis are obtained by selecting the donors and by avoiding added risk factors. THE ASSESSMENT OF A GRAFT FROM AN ELDERLY DONOR: This, basically, relies on clinical criteria: donor's history, cause of death and accurate measurement of the renal function. A biopsy of the graft, at the time of sampling, provides useful information. TRANSPLANTATION STRATEGY OF A GRAFT FROM AN ELDERLY DONOR: Donor-recipient matching by age is a common approach. Grafting of both kidneys in the same recipient is a method presently under assessment. The episode of ischemia must be reduced and the immunosuppressive therapy adapted.

  2. Identification of a novel synonymous mutation in the human β -Ureidopropionase Gene UPB1 affecting pre-mRNA splicing.

    PubMed

    Meijer, J; Nakajima, Y; Zhang, C; Meinsma, R; Ito, T; Van Kuilenburg, A B P

    2013-01-01

    β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO2. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C >T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G >A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C >T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92_104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.

  3. Identification of common genetic variation that modulates alternative splicing.

    PubMed

    Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

    2007-06-01

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  4. Superconducting cable-in-conduit low resistance splice

    DOEpatents

    Artman, Thomas A.

    2003-06-24

    A low resistance splice connects two cable-in-conduit superconductors to each other. Dividing collars for arranging sub-cable units from each conduit are provided, along with clamping collars for mating each sub-cable wire assembly to form mated assemblies. The mated assemblies ideally can be accomplished by way of splicing collar. The mated assemblies are cooled by way of a flow of coolant, preferably helium. A method for implementing such a splicing is also described.

  5. How to Motivate Whole Blood Donors to Become Plasma Donors

    PubMed Central

    2014-01-01

    This study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. A sample of 924 donors was randomized to one of three conditions: control; information only by nurse; and information plus self-positive image message by nurse (SPI). Participants in the control condition only received a leaflet describing the plasma donation procedure. In the two experimental conditions the leaflet was explained face-to-face by a nurse. The dependent variables were the proportion of new plasma donors and the number of donations at six months. Overall, 141 (15.3%) new plasma donors were recruited at six months. There were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (P < .001); the two experimental conditions did not differ. Also, compared to the control condition, those in the experimental conditions (all Ps < .001) gave plasma more often (information only by nurse:  d = .26; SPI: d = .32); the SPI intervention significantly outperformed (P < .05) the information only by nurse condition. The results suggest that references to feelings of SPI such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation. PMID:25530909

  6. How to motivate whole blood donors to become plasma donors.

    PubMed

    Godin, Gaston; Germain, Marc

    2014-01-01

    This study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. A sample of 924 donors was randomized to one of three conditions: control; information only by nurse; and information plus self-positive image message by nurse (SPI). Participants in the control condition only received a leaflet describing the plasma donation procedure. In the two experimental conditions the leaflet was explained face-to-face by a nurse. The dependent variables were the proportion of new plasma donors and the number of donations at six months. Overall, 141 (15.3%) new plasma donors were recruited at six months. There were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (P < .001); the two experimental conditions did not differ. Also, compared to the control condition, those in the experimental conditions (all Ps < .001) gave plasma more often (information only by nurse:  d = .26; SPI: d = .32); the SPI intervention significantly outperformed (P < .05) the information only by nurse condition. The results suggest that references to feelings of SPI such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation.

  7. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    SciTech Connect

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. )

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  8. Mis-Spliced Lr34 Transcript Events in Winter Wheat

    PubMed Central

    Fang, Tilin; Carver, Brett F.; Hunger, Robert M.; Yan, Liuling

    2017-01-01

    Lr34 in wheat is a non-race-specific gene that confers resistance against multiple fungal pathogens. The resistant allele Lr34 and the susceptible allele Lr34s can be distinguished by three polymorphisms that cause alternation of deduced amino acid sequences of Lr34 at the protein level. In seedlings of a cultivar carrying the resistant Lr34r allele, only a portion (35%) of its transcripts was correctly spliced and the majority (65%) of its transcripts were incorrectly spliced due to multiple mis-splicing events. Lr34 mis-splicing events were also observed at adult plant age when this gene exerts its function. All of the mis-spliced Lr34r cDNA transcripts observed in this study resulted in a premature stop codon due to a shift of the open reading frame; hence, the mis-spliced Lr34r cDNAs were deduced to encode incomplete proteins. Even if a cultivar has a functional Lr34 gene, its transcripts might not completely splice in a correct pattern. These findings suggested that the partial resistance conferred by a quantitative gene might be due to mis-splicing events in its transcripts; hence, the resistance of the gene could be increased by eliminating or mutating regulators that cause mis-splicing events in wheat. PMID:28135317

  9. Evolutionary Insights into RNA trans-Splicing in Vertebrates

    PubMed Central

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-01-01

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

  10. Splice-switching antisense oligonucleotides as therapeutic drugs

    PubMed Central

    Havens, Mallory A.; Hastings, Michelle L.

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  11. Evolutionary Insights into RNA trans-Splicing in Vertebrates.

    PubMed

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-03-10

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint.

  12. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  13. Some characteristics of probabilistic one-sided splicing systems

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    A theoretical model for DNA computing using the recombination behavior of DNA molecules known as asplicing system has been introduced in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings at the specific places 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 yielding the new strings. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions for splicing systems have been considered to increase the computational power of the languages generated. 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 one-sided splicing systems, which are special types of probabilistic splicing systems, are investigated. We prove that probabilistic one-sided splicing systems can also increase the computational power of the languages generated.

  14. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-05

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease.

  15. Donor states in inverse opals

    SciTech Connect

    Mahan, G. D.

    2014-09-21

    We calculate the binding energy of an electron bound to a donor in a semiconductor inverse opal. Inverse opals have two kinds of cavities, which we call octahedral and tetrahedral, according to their group symmetry. We put the donor in the center of each of these two cavities and obtain the binding energy. The binding energies become very large when the inverse opal is made from templates with small spheres. For spheres less than 50 nm in diameter, the donor binding can increase to several times its unconfined value. Then electrons become tightly bound to the donor and are unlikely to be thermally activated to the semiconductor conduction band. This conclusion suggests that inverse opals will be poor conductors.

  16. Donor states in inverse opals

    NASA Astrophysics Data System (ADS)

    Mahan, G. D.

    2014-09-01

    We calculate the binding energy of an electron bound to a donor in a semiconductor inverse opal. Inverse opals have two kinds of cavities, which we call octahedral and tetrahedral, according to their group symmetry. We put the donor in the center of each of these two cavities and obtain the binding energy. The binding energies become very large when the inverse opal is made from templates with small spheres. For spheres less than 50 nm in diameter, the donor binding can increase to several times its unconfined value. Then electrons become tightly bound to the donor and are unlikely to be thermally activated to the semiconductor conduction band. This conclusion suggests that inverse opals will be poor conductors.

  17. Donor selection in heart transplantation

    PubMed Central

    Emani, Sitaramesh; Sai-Sudhakar, Chittoor B.; Higgins, Robert S. D.; Whitson, Bryan A.

    2014-01-01

    There is increased scrutiny on the quality in health care with particular emphasis on institutional heart transplant survival outcomes. An important aspect of successful transplantation is appropriate donor selection. We review the current guidelines as well as areas of controversy in the selection of appropriate hearts as donor organs to ensure optimal outcomes. This decision is paramount to the success of a transplant program as well as recipient survival and graft function post-transplant. PMID:25132976

  18. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    PubMed Central

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-01-01

    Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

  19. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process.

    PubMed

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-07-24

    Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  20. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    PubMed

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  1. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

    PubMed

    Berkers, Celia R; de Jong, Annemieke; Schuurman, Karianne G; Linnemann, Carsten; Meiring, Hugo D; Janssen, Lennert; Neefjes, Jacques J; Schumacher, Ton N M; Rodenko, Boris; Ovaa, Huib

    2015-11-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

  2. Motivations for Giving of Alumni Donors, Lapsed Donors and Non-Donors: Implications for Christian Higher Education

    ERIC Educational Resources Information Center

    Rugano, Emilio Kariuki

    2011-01-01

    This descriptive and causal comparative study sought to identify motivations for alumni donor acquisition and retention in Christian institutions of higher learning. To meet this objective, motivations for alumni donors, lapsed donors, and non-donors were analyzed and compared. Data was collected through an electronic survey of a stratified sample…

  3. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

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

  4. Cotranscriptional splicing efficiency differs dramatically between Drosophila and mouse.

    PubMed

    Khodor, Yevgenia L; Menet, Jerome S; Tolan, Michael; Rosbash, Michael

    2012-12-01

    Spliceosome assembly and/or splicing of a nascent transcript may be crucial for proper isoform expression and gene regulation in higher eukaryotes. We recently showed that cotranscriptional splicing occurs efficiently in Drosophila, but there are not comparable genome-wide nascent splicing data from mammals. To provide this comparison, we analyze a recently generated, high-throughput sequencing data set of mouse liver nascent RNA, originally studied for circadian transcriptional regulation. Cotranscriptional splicing is approximately twofold less efficient in mouse liver than in Drosophila, i.e., nascent intron levels relative to exon levels are ∼0.55 in mouse versus 0.25 in the fly. An additional difference between species is that only mouse cotranscriptional splicing is optimal when 5'-exon length is between 50 and 500 bp, and intron length does not correlate with splicing efficiency, consistent with exon definition. A similar analysis of intron and exon length dependence in the fly is more consistent with intron definition. Contrasted with these differences are many similarities between the two systems: Alternatively annotated introns are less efficiently spliced cotranscriptionally than constitutive introns, and introns of single-intron genes are less efficiently spliced than introns from multi-intron genes. The most striking common feature is intron position: Cotranscriptional splicing is much more efficient when introns are far from the 3' ends of their genes. Additionally, absolute gene length correlates positively with cotranscriptional splicing efficiency independently of intron location and position, in flies as well as in mice. The gene length and distance effects indicate that more "nascent time" gives rise to greater cotranscriptional splicing efficiency in both systems.

  5. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy. 

  6. Splice connector with internal heat transfer jacket

    DOEpatents

    Silva, Frank A.; Mayer, Robert W.

    1977-01-01

    A heat transfer jacket is placed over the terminal portions of the conductors of a pair of high voltage cables which are connected in a splice connection wherein a housing surrounds the connected conductor portions, the heat transfer jacket extending longitudinally between the confronting ends of a pair of adaptor sleeves placed upon the insulation of the cables to engage and locate the adaptor sleeves relative to one another, and laterally between the conductors and the housing to provide a path of relatively high thermal conductivity between the connected conductor portions and the housing.

  7. Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns.

    PubMed

    Asakura, Yukari; Barkan, Alice

    2006-12-01

    Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.

  8. Splicing enhancement in the yeast rp51b intron.

    PubMed Central

    Libri, D; Lescure, A; Rosbash, M

    2000-01-01

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

  9. A Broad Set of Chromatin Factors Influences Splicing

    PubMed Central

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  10. RNA splicing in human disease and in the clinic.

    PubMed

    Baralle, Diana; Buratti, Emanuele

    2017-03-01

    Defects at the level of the pre-mRNA splicing process represent a major cause of human disease. Approximately 15-50% of all human disease mutations have been shown to alter functioning of basic and auxiliary splicing elements. These elements are required to ensure proper processing of pre-mRNA splicing molecules, with their disruption leading to misprocessing of the pre-mRNA molecule and disease. The splicing process is a complex process, with much still to be uncovered before we are able to accurately predict whether a reported genomic sequence variant (GV) represents a splicing-associated disease mutation or a harmless polymorphism. Furthermore, even when a mutation is correctly identified as affecting the splicing process, there still remains the difficulty of providing an exact evaluation of the potential impact on disease onset, severity and duration. In this review, we provide a brief overview of splicing diagnostic methodologies, from in silico bioinformatics approaches to wet lab in vitro and in vivo systems to evaluate splicing efficiencies. In particular, we provide an overview of how the latest developments in high-throughput sequencing can be applied to the clinic, and are already changing clinical approaches.

  11. Scattering of acoustic duct modes by axial liner splices

    NASA Astrophysics Data System (ADS)

    Tam, Christopher K. W.; Ju, Hongbin; Chien, Eugene W.

    2008-03-01

    Recent engine test data and results of computational analysis show that the engine inlet acoustic liner splices have a significant impact on aircraft flight noise certification and cabin noise levels. The phenomenon of scattering of acoustic duct modes by axial liner splices is investigated. Previous studies, invariably, follow the frequency-domain approach. The present study, however, uses the time-domain approach. It is demonstrated that time-domain computation yields results that are in close agreement with frequency-domain results. The scattering phenomenon under consideration is very complex. This study concentrates on the effects of four parameters. They are the width of the splices, the frequency of the incident duct mode, the number of splices and the length of splices. Based on the computed results, the conditions under which scattered wave modes would significantly increase the intensity of transmitted waves are identified. It is also found that surface scattering by liner splices has the tendency to distribute energy equally to all the cut-on scattered azimuthal modes. On the other hand, for each scattered azimuthal mode, the high-order cut-on radial mode, generally, has the highest intensity. Moreover, scattering by liner splices is a local phenomenon. It is confined primarily to an area of the duct adjacent to the junction between the hard wall near the fan face and the spliced liner.

  12. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  13. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  14. Alternative Splicing of STAT3 Is Affected by RNA Editing.

    PubMed

    Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

    2017-03-09

    A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

  15. A novel splice site mutation in a Becker muscular dystrophy patient.

    PubMed

    Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Hall, C D; Mendell, J R; Prior, T W

    1996-04-01

    A Becker muscular dystrophy patient was found to have a single base substitution at the 5' end of intron 54. This single base substitution disrupts the invariant GT dinucleotide within the 5' donor splice site and was shown to cause an out of frame deletion of exon 54 during mRNA processing. This is predicted to produce a truncated dystrophin protein which is more consistent with a DMD phenotype. However, small quantities of normal mRNA are also transcribed and these are sufficient to produce a reduced amount of normal molecular weight dystrophin and give rise to a milder BMD phenotype. This indicates that a single base substitution at an invariant dinucleotide of the splice site consensus sequence may still allow read through of the message and allow the production of some normal protein. This shows that there are a greater number of possible intronic mutations that can lead to a mild phenotype and it also underlines the importance of performing cDNA analysis when screening for small gene alterations in the BMD patient population.

  16. A novel splice site mutation in a Becker muscular dystrophy patient.

    PubMed Central

    Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Hall, C D; Mendell, J R; Prior, T W

    1996-01-01

    A Becker muscular dystrophy patient was found to have a single base substitution at the 5' end of intron 54. This single base substitution disrupts the invariant GT dinucleotide within the 5' donor splice site and was shown to cause an out of frame deletion of exon 54 during mRNA processing. This is predicted to produce a truncated dystrophin protein which is more consistent with a DMD phenotype. However, small quantities of normal mRNA are also transcribed and these are sufficient to produce a reduced amount of normal molecular weight dystrophin and give rise to a milder BMD phenotype. This indicates that a single base substitution at an invariant dinucleotide of the splice site consensus sequence may still allow read through of the message and allow the production of some normal protein. This shows that there are a greater number of possible intronic mutations that can lead to a mild phenotype and it also underlines the importance of performing cDNA analysis when screening for small gene alterations in the BMD patient population. Images PMID:8730289

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

    PubMed Central

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

    2015-01-01

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

  18. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    PubMed Central

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  19. A novel ARH splice site mutation in a Mexican kindred with autosomal recessive hypercholesterolemia.

    PubMed

    Canizales-Quinteros, Samuel; Aguilar-Salinas, Carlos A; Huertas-Vázquez, Adriana; Ordóñez-Sánchez, María L; Rodríguez-Torres, Maribel; Venturas-Gallegos, José L; Riba, Laura; Ramírez-Jimenez, Salvador; Salas-Montiel, Rocío; Medina-Palacios, Giovani; Robles-Osorio, Ludivina; Miliar-García, Angel; Rosales-León, Luis; Ruiz-Ordaz, Blanca H; Zentella-Dehesa, Alejandro; Ferré-D'Amare, Adrian; Gómez-Pérez, Francisco J; Tusié-Luna, Ma Teresa

    2005-01-01

    Autosomal recessive hypercholesterolemia (ARH) is characterized by elevated LDL serum levels, xanthomatosis, and premature coronary artery disease. Three loci have been described for this condition (1p35, 15q25-q26 and 13q). Recently, the responsible gene at the 1p35 locus, encoding an LDL receptor adaptor protein (ARH) has been identified. We studied a Mexican ARH family with two affected siblings. Sequence analysis of the ARH gene (1p35 locus) revealed that the affected siblings are homozygous for a novel mutation (IVS4+2T>G) affecting the donor splice site in intron 4, whereas both the parents and an unaffected sister are heterozygous for this mutation. The IVS4+2T>G mutation results in a major alternative transcript derived from a cryptic splice site, which carries an in-frame deletion of 78 nucleotides in the mature mRNA. The translation of this mRNA yields a mutant protein product (ARH-26) lacking 26 amino acids, resulting in the loss of beta-strands beta6 and beta7 from the PTB domain. This is the first case where a naturally occurring mutant with an altered PTB domain has been identified.

  20. A functional splice variant of the human Golgi CMP-sialic acid transporter.

    PubMed

    Salinas-Marín, Roberta; Mollicone, Rosella; Martínez-Duncker, Iván

    2016-12-01

    The human Golgi Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Le(x) antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

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

    PubMed Central

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

    1991-01-01

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

  2. 5 prime -Azido-(3,6- sup 3 H sub 2 )-1-naphthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: Identification of a 23-kDa protein from maize coleoptile plasma membranes

    SciTech Connect

    Zettl, R.; Feldwisch, J.; Schell, J.; Palme, K. ); Boland, W. )

    1992-01-15

    1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, the authors have synthesized a tritiated, photolabile NPA analogue, 5{prime}-azido-(3,6-{sup 3}H{sub 2})NPA (({sup 3}H{sub 2})N{sub 3}NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of ({sup 3}H{sub 2})N{sub 3}NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with ({sup 3}H{sub 2})N{sub 3}NPA and exposure to UV light, they observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-(7-{sup 3}H)indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

  3. Functional impact of splice isoform diversity in individual cells

    PubMed Central

    Yap, Karen; Makeyev, Eugene V.

    2016-01-01

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

  4. [Alternative splicing regulation: implications in cancer diagnosis and treatment].

    PubMed

    Martínez-Montiel, Nancy; Rosas-Murrieta, Nora; Martínez-Contreras, Rebeca

    2015-04-08

    The accurate expression of the genetic information is regulated by processes like mRNA splicing, proposed after the discoveries of Phil Sharp and Richard Roberts, who demonstrated the existence of intronic sequences, present in almost every structural eukaryotic gene, which should be precisely removed. This intron removal is called "splicing", which generates different proteins from a single mRNA, with different or even antagonistic functions. We currently know that alternative splicing is the most important source of protein diversity, given that 70% of the human genes undergo splicing and that mutations causing defects in this process could originate up to 50% of genetic diseases, including cancer. When these defects occur in genes involved in cell adhesion, proliferation and cell cycle regulation, there is an impact on cancer progression, rising the opportunity to diagnose and treat some types of cancer according to a particular splicing profile.

  5. Differential Impacts of Alternative Splicing Networks on Apoptosis

    PubMed Central

    Lin, Jung-Chun; Tsao, Mei-Fen; Lin, Ying-Ju

    2016-01-01

    Apoptosis functions as a common mechanism to eliminate unnecessary or damaged cells during cell renewal and tissue development in multicellular organisms. More than 200 proteins constitute complex networks involved in apoptotic regulation. Imbalanced expressions of apoptosis-related factors frequently lead to malignant diseases. The biological functions of several apoptotic factors are manipulated through alternative splicing mechanisms which expand gene diversity by generating discrete variants from one messenger RNA precursor. It is widely observed that alternatively-spliced variants encoded from apoptosis-related genes exhibit differential effects on apoptotic regulation. Alternative splicing events are meticulously regulated by the interplay between trans-splicing factors and cis-responsive elements surrounding the regulated exons. The major focus of this review is to highlight recent studies that illustrate the influences of alternative splicing networks on apoptotic regulation which participates in diverse cellular processes and diseases. PMID:27983653

  6. RNA splicing factors as oncoproteins and tumor suppressors

    PubMed Central

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

    2016-01-01

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

  7. Detecting Image Splicing Using Merged Features in Chroma Space

    PubMed Central

    Liu, Guangjie; Dai, Yuewei

    2014-01-01

    Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature. PMID:24574877

  8. Detecting image splicing using merged features in chroma space.

    PubMed

    Xu, Bo; Liu, Guangjie; Dai, Yuewei

    2014-01-01

    Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature.

  9. Functional impact of splice isoform diversity in individual cells.

    PubMed

    Yap, Karen; Makeyev, Eugene V

    2016-08-15

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities.

  10. Positive control of pre-mRNA splicing in vitro.

    PubMed

    Tian, M; Maniatis, T

    1992-04-10

    Positive control of the sex-specific alternative splicing of doublesex (dsx) precursor messenger RNA (pre-mRNA) in Drosophila melanogaster involves the activation of a female-specific 3' splice site by the products of the transformer (tra) and transformer-2 (tra-2) genes. The mechanisms of this process were investigated in an in vitro system in which the female-specific 3' splice site could be activated by recombinant Tra or Tra-2 (or both). An exon sequence essential for regulation in vivo was shown to be both necessary and sufficient for activation in vitro. Nuclear proteins in addition to Tra and Tra-2 were found to bind specifically to this exon sequence. Therefore, Tra and Tra-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery.

  11. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  12. Cadaveric donor selection and management.

    PubMed

    Studer, Sean M; Orens, Jonathan B

    2004-12-01

    The current availability of lung donors is far exceeded by the number of potential transplant recipients who are waiting for an organ. This disparity results in significant morbidity and mortality for those on the waiting list. Although it is desirable to increase overall consent rates for organ donation, doing so requires an intervention to affect societal response. In contrast, increased procurement of organs from marginal donors and improved donor management may be realized through increased study and practice changes within the transplant community. Transplantation of organs from marginal or extended-criteria donors may result in some increase in complications or mortality, but this possibility must be weighed against the morbidity and risk of death risk faced by individuals on the waiting list. The effects of this trade-off are currently being studied in kidney transplantation, and perhaps in the near future lung transplantation may benefit from a similar analysis. Until that time, the limited data regarding criteria for donor acceptability must be incorporated into practice to maximize the overall benefits of lung transplantation.

  13. Dengue antibodies in blood donors

    PubMed Central

    Ribas-Silva, Rejane Cristina; Eid, Andressa Ahmad

    2012-01-01

    Background Dengue is an urban arbovirus whose etiologic agent is a virus of the genus Flavorius with four distinct antigen serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that is transmitted to humans through the bite of the mosquito Aedes aegypti. The Campo Mourão region in Brazil is endemic for dengue fever. Obtective The aim of this study was to evaluate the presence of IgG and IgM antibodies specific to the four serotypes of dengue in donors of the blood donor service in the city of Campo Mourão. Methods Epidemiological records were evaluated and 4 mL of peripheral blood from 213 blood donors were collected in tubes without anticoagulant. Serum was then obtained and immunochromatographic tests were undertaken (Imuno-Rápido Dengue IgM/IgGTM). Individuals involved in the study answered a social and epidemiological questionnaire on data which included age, gender and diagnosis of dengue. Results Only three (1.4%) of the 213 blood tests were positive for IgG anti-dengue antibodies. No donors with IgM antibody, which identifies acute infection, were identified. Conclusions The results of the current analysis show that the introduction of quantitative or molecular serological methods to determine the presence of anti-dengue antibodies or the detection of the dengue virus in blood donors in endemic regions should be established so that the quality of blood transfusions is guaranteed. PMID:23049418

  14. Structural analysis of Aircraft fuselage splice joint

    NASA Astrophysics Data System (ADS)

    Udaya Prakash, R.; Kumar, G. Raj; Vijayanandh, R.; Senthil Kumar, M.; Ramganesh, T.

    2016-09-01

    In Aviation sector, composite materials and its application to each component are one of the prime factors of consideration due to the high strength to weight ratio, design flexibility and non-corrosive so that the composite materials are widely used in the low weight constructions and also it can be treated as a suitable alternative to metals. The objective of this paper is to estimate and compare the suitability of a composite skin joint in an aircraft fuselage with different joints by simulating the displacement, normal stress, vonmises stress and shear stress with the help of numerical solution methods. The reference Z-stringer component of this paper is modeled by CATIA and numerical simulation is carried out by ANSYS has been used for splice joint presents in the aircraft fuselage with three combinations of joints such as riveted joint, bonded joint and hybrid joint. Nowadays the stringers are using to avoid buckling of fuselage skin, it has joined together by rivets and they are connected end to end by splice joint. Design and static analysis of three-dimensional models of joints such as bonded, riveted and hybrid are carried out and results are compared.

  15. Alternatively spliced transcripts of the thymus-specific protease PRSS16 are differentially expressed in human thymus.

    PubMed

    Luther, C; Wienhold, W; Oehlmann, R; Heinemann, M K; Melms, A; Tolosa, E

    2005-02-01

    The putative serine protease PRSS16 is abundantly expressed in the thymic cortex and the gene is encoded within the HLA I complex. Although its function is not yet defined, the very restricted expression points to a role in T-cell development in the thymus. In this study, we show that the PRSS16 mRNA is alternatively spliced to generate at least five transcripts. Apart from the full-length sequence, we found two other isoforms with all putative active site residues of the serine protease, suggesting that those variants may also be functional. Semi-quantitative analysis of the splice variants in different tissue samples revealed a strong correlation between the specific formation of alternatively spliced PRSS16 transcripts and the age and thymus pathology status of the donor. Newborn thymi express mostly the PRSS16-4 and -5 isoforms and lack the PRSS16-1 transcript, which appears around 2 years of age and stays until adulthood. Incidentally, thymi from myasthenia gravis (MG) patients with thymoma showed a marked decrease in the expression of the full-length PRSS16-1 and increased expression of the smaller isoforms. The data suggest a potential role of the PRSS16 isoforms in the postnatal morphogenesis of the thymus and in the thymus pathology related to MG.

  16. The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo.

    PubMed Central

    Yang, X; Bani, M R; Lu, S J; Rowan, S; Ben-David, Y; Chabot, B

    1994-01-01

    Recent in vitro results suggest that the heterogeneous nuclear ribonucleoparticle (hnRNP) A1 protein modulates alternative splicing by favoring distal 5' splice site (5'SS) selection and exon skipping. We used a mouse erythroleukemia (MEL) cell line (CB3C7) deficient in the expression of hnRNP A1 to test whether variations in hnRNP A1 and AlB protein levels affected alternative splicing in vivo. In contrast to A1-expressing MEL cell lines, CB3C7 cells preferentially selected the proximal 13S and 12S 5'SS on the adenovirus E1A pre-mRNA. Transiently expressing the A1 or A1B cDNA in CB3C7 cells shifted 5'SS selection toward the more distal 9S donor site. A1 protein synthesis was required for this effect since the expression of a mutated A1 cDNA did not affect 5'SS selection. These results demonstrate that in vivo variations in hnRNP A1 protein levels can influence 5'SS selection. Images PMID:8041722

  17. 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

  18. Influence of weather conditions on splicing process and parameters of splicing single-mode telecommunication fibers of different types

    NASA Astrophysics Data System (ADS)

    Ratuszek, Marek; Zakrzewski, Zbigniew; Majewski, Jacek; Strozecki, Stefan; Zalewski, Jozef; Konefal, Tadeusz; Kula, Witold

    1999-05-01

    Results of research on the influence of weather conditions (t equals 10 divided by 27 degree(s)C; H equals 30 divided by 90%) on the process of splicing of standard single mode fibers SM (G.652) and fibers with dispersion shifted DS (G.653) have been presented as well as the results of optimization of splicing SM and DS fibers.

  19. Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

    PubMed Central

    Schmidt, Ute; Robert, Marie-Cécile; Yoshida, Minoru; Villemin, Jean-Philippe; Auboeuf, Didier; Aitken, Stuart

    2011-01-01

    Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules. PMID:21624952

  20. 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.

  1. The use of neoplastic donors to increase the donor pool.

    PubMed

    Fiaschetti, P; Pretagostini, R; Stabile, D; Peritore, D; Oliveti, A; Gabbrielli, F; Cenci, S; Ricci, A; Vespasiano, F; Grigioni, W F

    2012-09-01

    The aim of the study was to evaluate the experience of the Centre-Sud Transplant Organization (OCST) area using cadaveric donor with neoplastic diseases to evaluate the possibility of transmission to recipients. From January 1, 2003, to December 31, 2010, the neoplastic risk has been reported to be 5.4% (377/4654 referred donors). In 2003, the number of donors with a tumor and their mean age were respectively: 60 (10.3%) and 59.6 ± 19.9; 2004: 33 (5.2%) and 61.4 ± 15.9; 2005: 32 (6%) and 62.8 ± 15.5; 2006: 46 (7%) and 60.7 ± 19.1; 2007: 51 (7%) and 58.9 ± 16; in 2008: 58 (7%) and 59.7 ± 19.6; 2009: 47 (7%) and 57 ± 26; 2010: 49 (7%) and 64 ± 16. The organ most affected by tumor has been the central nervous system (18%). The tumor was diagnosed before in 325 (86%) cases, versus during organ retrieval in 48 (12.7%) donor operations but before, which four cases (1%) occured after transplantation. According to the histological types and grades, 28 evaluated donors (8.2%) were suitable for transplantation. The histological types were: thyroid carcinoma (n = 3); prostate carcinoma (n = 8), renal clear cell carcinoma (n = 7), oncocytoma (n = 1), meningiomas (n = 2), dermofibrosarcoma (n = 1); verrucous carcinoma of the vulva (n = 1), colon adenocarcinoma (n = 1), grade II astrocytoma (n = 1), adrenal gland tumor (n = 1), gastric GIST (n = 1), oligodendroglioma (n = 1). Forty-five organs were retrieved (22 livers, 19 kidneys, 3 hearts, and 1 pancreas) and transplanted into 44 recipients with 1 liver-kidney combined transplantation. Four recipients died due to causes not related to the tumor. No donor-transmitted tumor was detected among the recipients. Donation is absolutely not indicated in cases of tumors with high metastatic potential and high grades. Performing an accurate evaluation of the donor, taking into account the histological grade, currently can allow, organ retrieval and transplantation with an acceptable risk.

  2. The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

    PubMed Central

    Österman, Lennart; Lindsten, Hans; Holmberg, Monica

    2016-01-01

    Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML. PMID:27783661

  3. 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

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

    NASA Astrophysics Data System (ADS)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

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

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

    PubMed

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

    2016-01-01

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

  6. Aberrant and alternative splicing in skeletal system disease.

    PubMed

    Fan, Xin; Tang, Liling

    2013-10-01

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

  7. Tau mis-splicing in the pathogenesis of neurodegenerative disorders

    PubMed Central

    Park, Sun Ah; Ahn, Sang Il; Gallo, Jean-Marc

    2016-01-01

    Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413] PMID:27222125

  8. Peptidic tools applied to redirect alternative splicing events.

    PubMed

    Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

    2015-05-01

    Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases.

  9. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    PubMed

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues.

  10. An alternative splicing program promotes adipose tissue thermogenesis

    PubMed Central

    Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

    2016-01-01

    Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

  11. In silico prediction of splice-affecting nucleotide variants.

    PubMed

    Houdayer, Claude

    2011-01-01

    It appears that all types of genomic nucleotide variations can be deleterious by affecting normal pre-mRNA splicing via disruption/creation of splice site consensus sequences. As it is neither pertinent nor realistic to perform functional testing for all of these variants, it is important to identify those that could lead to a splice defect in order to restrict experimental transcript analyses to the most appropriate cases. In silico tools designed to provide this type of prediction are available. In this chapter, we present in silico splice tools integrated in the Alamut (Interactive Biosoftware) application and detail their use in routine diagnostic applications. At this time, in silico predictions are useful for variants that decrease the strength of wild-type splice sites or create a cryptic splice site. Importantly, in silico predictions are not sufficient to classify variants as neutral or deleterious: they should be used as part of the decision-making process to detect potential candidates for splicing anomalies, prompting molecular geneticists to carry out transcript analyses in a limited and pertinent number of cases which could be managed in routine settings.

  12. Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

    PubMed Central

    Reid, Daniel C.; Chang, Brian L.; Gunderson, Samuel I.; Alpert, Lauren; Thompson, William A.; Fairbrother, William G.

    2009-01-01

    Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified by a two-color microarray. We applied this approach to locating splicing factor binding sites in and around ∼4000 exons. We also quantified the effect of secondary structure on binding. The method is validated by the finding that U1snRNP binds at the 5′ splice site (5′ss) with a specificity that is nearly identical to the splice donor motif. In agreement with prior reports, we also show that U1snRNP appears to have some affinity for intronic G triplets that are proximal to the 5′ss. Both U1snRNP and the polypyrimidine tract binding protein (PTB) avoid exonic binding, and the PTB binding map shows increased enrichment at the polypyrimidine tract. For PTB, we confirm polypyrimidine specificity and are also able to identify structural determinants of PTB binding. We detect multiple binding motifs enriched in the PTB bound fraction of oligonucleotides. These motif combinations augment binding in vitro and are also enriched in the vicinity of exons that have been determined to be in vivo targets of PTB. PMID:19861426

  13. Single-Donor Leukophoretic Technique

    NASA Technical Reports Server (NTRS)

    Eberhardt, R. N.

    1977-01-01

    Leukocyte separation-and-retrieval device utilizes granulocyte and monocyte property of leukoadhesion to glass surfaces as basis of their separation from whole blood. Device is used with single donor technique and has application in biological and chemical processing, veterinary research and clinical care.

  14. Splicing life, with scalpel and scythe.

    PubMed

    Boone, C K

    1983-04-01

    Two recent reports on the social and moral implications of recombinant DNA research and "genetic engineering" are compared: Splicing Life: A Report on the Social and Ethical Issues of Genetic Engineering with Human Beings, by the President's Commission for the Study of Ethical Issues in Medicine and Biomedical and Behavioral Research, and a shorter Study Paper of Bioethical Concerns, by the Panel on Bioethical Concerns of the National Council of Churches of Christ (NCC). The President's Commission report is seen as more sophisticated, better organized, carefully reasoned, and clearly focused on imminent clinical uses of genetic research. The NCC report is described as more diffuse, concerned with remote and dramatic applications, and heavily exhortatory.

  15. Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African-American Prostate Cancer

    DTIC Science & Technology

    2015-10-01

    RNA and annotated. In addition, we have developed SSOs to manipulate PIK3CD alternative splicing, to correct aberrant splicing leading to production...molecular mechanisms, differential gene expression, alternative RNA splicing, epigenetic alterations, clinical tumor aggressiveness 16. SECURITY...words): Prostate cancer, health disparities among racial groups, molecular mechanisms, differential gene expression, alternative RNA splicing

  16. Dietary uptake efficiency of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl in yellow perch and rainbow trout: Role of dietary and body lipids

    SciTech Connect

    Dabrowska, H.; Fisher, S.W.; Dabrowski, K.; Staubus, A.E.

    1999-05-01

    Dietary uptake efficiency ({alpha}) and eliminate rate constants (k{sub d}) of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (HCBP) were determined in two fish species, yellow perch and rainbow trout, to investigate the influence of dietary and body lipid levels on bioaccumulation. Groups of juvenile fish with significant differences in percent body lipid were fed with a low-fat(LF) or high-fat(HF) diet spiked with 5 or 50 ppb of {sup 14}C-HCBP for 32 d. Thereafter, fish were fed an uncontaminated LF or HF diet to allow for elimination of HCBP. Feeding and growth rates were quantified. There were eight fish lipid-dietary lipid-HCBP concentration exposure combinations for each species. Four fish from each exposure were collected at the beginning of the study and at 10--17-d intervals during exposure and elimination periods for lipid and {sup 14}C-HCBP analysis. The {alpha} values ranged from 74 to 95% in yellow perch and from 79 to 99% in rainbow trout. The greatest {alpha} values, of 95 to 99%, were found in fish given diets with 5 ppb HCBP. Uptake of HCBP was influenced by both dietary and body lipids and depended on the current status of both lipid pools. The elimination rate constants were in the range of 0.000 to 0.004 d{sup {minus}1} in yellow perch and 0.003 to 0.010 d{sup {minus}1} in rainbow trout. No significant differences in elimination rate constants between HF and LF fish groups were found. In fish on a constant dietary lipid regime, the k{sub d} values tended to be less in HF than in LF fish. However, in fish groups offered diets with a change in lipid regime. The k{sub d} tended to be greater. Lipid x time interactions in the HF and LF fish groups undergoing a change in lipid regime indicated that the k{sub d} values, like the {alpha} values, were influenced by both lipid pools. Changes in elimination rates due to dietary/body lipid status impacted BAFs more strongly than changes in uptake efficiencies. The BAFs were in the range of 1.11 to 2

  17. The Resistance and Strength of Soft Solder Splices between Conductors in MICE Coils

    SciTech Connect

    Wu, Hong; Pan, Heng; Green, Michael A; Dietderich, Dan; Gartner, T. E.; Higley, Hugh C; Mentink, M.; Xu, FengYu; Trillaud, F.; Liu, X. K.; Wang, Li; Zheng, S. X.; Tam, D.G.

    2010-08-03

    Two of the three types of MICE magnets will have splices within their coils. The MICE coupling coils may have as many as fifteen one-meter long splices within them. Each of the MICE focusing coils may have a couple of 0.25-meter long conductor splices. Equations for the calculation of resistance of soldered lap splices of various types are presented. This paper presents resistance measurements of soldered lap splices of various lengths. Measured splice resistance is shown for one-meter long splices as a function of the fabrication method. Another important consideration is the strength of the splices. The measured breaking stress of splices of various lengths is presented in this paper. Tin-lead solders and tin-silver solders were used for the splices that were tested. From the data given in this report, the authors recommend that the use of lead free solders be avoided for low temperature coils.

  18. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

    PubMed Central

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations. PMID:25692239

  19. Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

    PubMed Central

    Cote, Gilbert J.; Zhu, Wen; Thomas, Anthony; Martin, Emil; Murad, Ferid; Sharina, Iraida G.

    2012-01-01

    Background Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER). Methodology/Principal Findings Here we report that splicing of C-α1 sGC can be modulated by H2O2 treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H2O2 treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H2O2 occurs at the protein level with variable regulation observed in different breast cancer cells. Conclusions/Significance Our data demonstrate that H2O2 regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H2O2 treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress. PMID:22911749

  20. TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs.

    PubMed

    Le Guiner, C; Lejeune, F; Galiana, D; Kister, L; Breathnach, R; Stévenin, J; Del Gatto-Konczak, F

    2001-11-02

    TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.

  1. Defective control of pre–messenger RNA splicing in human disease

    PubMed Central

    Shkreta, Lulzim

    2016-01-01

    Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies. PMID:26728853

  2. Defective control of pre-messenger RNA splicing in human disease.

    PubMed

    Chabot, Benoit; Shkreta, Lulzim

    2016-01-04

    Examples of associations between human disease and defects in pre-messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies.

  3. Live donor transplantation--the incompetent donor: comparative law.

    PubMed

    Wolfman, Samuel; Shaked, Tali

    2008-12-01

    Informed consent of the patient to medical treatment is an essential prerequisite for any invasive medical procedure. However in emergency cases, when the patient is unable to sign a consent form due to unconsciousness or to psychotic state, than the primary medical consideration shall take place. In such a case, in order to save life or even prevent a major medical hazard to the patient, doctors are allowed, in certain cases and in accordance with well accepted medical practice, to perform invasive procedures, major surgery or risky pharmacological treatment, without the explicit consent of the patient. All the above refers to the cases when avoidance of such non-consented treatment may harm severely the health and wellbeing of the patient and there is no doubt that such treatment is for the ultimate benefit of the patient. The question, however, shall arise when such a medical procedure is not necessarily for the benefit of the patient, but rather for the benefit of somebody else. Such is the case in the transplantation area and the question of living donor-donee relationship. This paper shall analyze the legal situation in cases of non competent donors whose consent cannot be considered legal consent given in full understanding and out of free will. It will also compare three legal systems, the Israeli, the American and the traditional Jewish law, with regard to the different approaches to this human problem, where the autonomy of the donor may be sacrificed for the purpose of saving life of another person.

  4. Characterization of the Wilson disease gene: Genomic organization; alternative splicing; structure/function predictions; and population frequencies of disease-specific mutations

    SciTech Connect

    Petrukhin, K.; Chernov, I.; Ross, B.M.

    1994-09-01

    The Wilson disease (WD) gene has recently been identified as a putative copper-transporting ATPase with high amino acid similarity with the Menkes disease (MNK) gene. We have further characterized the WD gene by extending the 5{prime}-coding and non-coding DNA sequence and elucidating the intron/exon structure and genomic organization. Analysis of RNA transcripts from liver, brain, kidney and placenta reveals extensive alternative splicing which may provide a mechanism to regulate the quantity of functional protein product. Comparative sequence analysis shows that WD and MNK belong to the sub-family of heavy metal-transporting ATPases with several characterizing features which include unique amino acid motifs and distinct N-terminal and C-terminal transmembrane structure. Our data indicate that the 600 amino acid metal binding portion of the WD and MNK proteins was formed by gene duplication events and splicing of the 6 metal binding domain segment to a common ancestral protein. We have raised a WD-specific anti-peptide antibody to the N-terminal region and are beginning to explore the cellular and intracellular location of the WD protein. The metal-binding segment of the WD protein has been expressed in E. coli and metal binding assays are underway to characterize this aspect of the protein`s function. We have identified numerous disease-specific mutations and developed a rapid {open_quotes}reverse dot blot{close_quotes} screening protocol to determine mutation frequencies in different populations. The most common mutation disrupts the characteristic SEHP motif and accounts for more than 40% of WD cases in North American, Russian, and Swedish populations. This mutation has not been observed in our limited Sicilian sample.

  5. Long-range RNA pairings contribute to mutually exclusive splicing.

    PubMed

    Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

    2016-01-01

    Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks.

  6. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 25 feet of the machine, except cable reel equipment. Temporary splices in trailing cables shall be... used. As used in this section, the term “splice” means the mechanical joining of one or more...

  7. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 25 feet of the machine, except cable reel equipment. Temporary splices in trailing cables shall be... used. As used in this section, the term “splice” means the mechanical joining of one or more...

  8. Cutaway Isometric, Upper Chord (Compression Joint), Lower Chord (Tension Splice) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Cutaway Isometric, Upper Chord (Compression Joint), Lower Chord (Tension Splice) - McConnell's Mill Bridge, Spanning Slippery Rock Creek at McConnell's Mill Road (Township Route 415), Ellwood City, Lawrence County, PA

  9. A long noncoding way to alternative splicing in plant development.

    PubMed

    Kornblihtt, Alberto R

    2014-07-28

    In this issue of Developmental Cell, Bardou et al. (2014) elucidate how long, highly structured noncoding RNAs control alternative splicing regulators that specifically mediate the action of the hormone auxin in the promotion of lateral root growth in Arabidopsis.

  10. 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.

  11. 3. Detail of beam splice and column capital on the ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Detail of beam splice and column capital on the second floor of the Cloth Room Building/Old Bleach House, Monadnock Mills. Beam and column edges are chamfered. - Monadnock Mills, 15 Water Street, Claremont, Sullivan County, NH

  12. Biochemistry and regulation of pre-mRNA splicing.

    PubMed

    Adams, M D; Rudner, D Z; Rio, D C

    1996-06-01

    During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

  13. Chord Splicing & Joining Detail; Chord & CrossBracing Joint Details; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Chord Splicing & Joining Detail; Chord & Cross-Bracing Joint Details; Cross Bracing Center Joint Detail; Chord & Diagonal Joint Detail - Vermont Covered Bridge, Highland Park, spanning Kokomo Creek at West end of Deffenbaugh Street (moved to), Kokomo, Howard County, IN

  14. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., AND APPROVAL OF MINING PRODUCTS ELECTRIC MOTOR-DRIVEN MINE EQUIPMENT AND ACCESSORIES Construction and Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held...

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

    PubMed

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

    2015-02-01

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

  16. Amphiphilic NO-donor antioxidants.

    PubMed

    Chegaev, Konstantin; Lazzarato, Loretta; Rolando, Barbara; Marini, Elisabetta; Lopez, Gloria V; Bertinaria, Massimo; Di Stilo, Antonella; Fruttero, Roberta; Gasco, Alberto

    2007-02-01

    Models of amphiphilic NO-donor antioxidants 24-26 were designed and synthesized. The products were obtained by linking a lipophilic tail (C(6), C(8), C(10)) with a polar head constituted by the 2,6-dimethoxyphenol antioxidant joined to the NO-donor 3-furoxancarboxamide substructure through a bridge containing a quaternary ammonium group. Compound 23, containing the shortest C(2)-alkyl chain, was also studied as a reference. The antioxidant properties (TBARS and LDL oxidation assays) and the vasodilator properties of the compounds were studied in vitro. The ability of these products to interact with phospholipid vesicles was also investigated by NMR techniques. The results indicate that both activities are modulated by the ability of the compounds to accumulate on phospholipid layers.

  17. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Donor notification. 630.6 Section 630.6 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification. (a) Notification of donors. You, an...

  18. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  19. Modulators of alternative splicing as novel therapeutics in cancer.

    PubMed

    Oltean, Sebastian

    2015-10-10

    Alternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g., pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics.

  20. Alternative Splicing of Type II Procollagen: IIB or not IIB?

    PubMed Central

    McAlinden, Audrey

    2015-01-01

    Over two decades ago, two isoforms of the type II procollagen gene (COL2A1) were discovered. These isoforms, named IIA and IIB, are generated in a developmentally-regulated manner by alternative splicing of exon 2. Chondroprogenitor cells synthesize predominantly IIA isoforms (containing exon 2) while differentiated chondrocytes produce mainly IIB transcripts (devoid of exon 2). Importantly, this IIA-to-IIB alternative splicing switch occurs only during chondrogenesis. More recently, two other isoforms have been reported (IIC and IID) that also involve splicing of exon 2; these findings highlight the complexities involving regulation of COL2A1 expression. The biological significance of why different isoforms of COL2A1 exist within the context of skeletal development and maintenance is still not completely understood. This review will provide current knowledge on COL2A1 isoform expression during chondrocyte differentiation and what is known about some of the mechanisms that control exon 2 alternative splicing. Utilization of mouse models to address the biological significance of Col2a1 alternative splicing in vivo will also be discussed. From the knowledge acquired to date, some new questions and concepts are now being proposed on the importance of Col2a1 alternative splicing in regulating extracellular matrix assembly and how this may subsequently affect cartilage and endochondral bone quality and function. PMID:24669942

  1. Reflections on protein splicing: structures, functions and mechanisms

    PubMed Central

    Anraku, Yasuhiro; Satow, Yoshinori

    2009-01-01

    Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. PMID:19907126

  2. Functional studies on the ATM intronic splicing processing element.

    PubMed

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

    2005-01-01

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

  3. Different roles played by periostin splice variants in retinal neovascularization.

    PubMed

    Nakama, Takahito; Yoshida, Shigeo; Ishikawa, Keijiro; Kobayashi, Yoshiyuki; Abe, Takaya; Kiyonari, Hiroshi; Shioi, Go; Katsuragi, Naruto; Ishibashi, Tatsuro; Morishita, Ryuichi; Taniyama, Yoshiaki

    2016-12-01

    Retinal neovascularization (NV) due to retinal ischemia is one of the major causes of vision reduction in patients with different types of retinal diseases although anti-vascular endothelial growth factor (anti-VEGF) therapy can partially reduce the size of the retinal NV. We recently reported that periostin plays an important role in the development of NV and the formation of preretinal fibrovascular membranes, but the role of the splice variants of periostin on retinal NV has not been determined. We examined the expressions of periostin splice variants in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of periostin splice variants on retinal NV using periostin knock out mice, and the effects of anti-periostin antibodies on retinal NV. Our results showed that the expressions of the periostin splice variants were increased in ischemic retinas. The degree of increase of periostin lacking exon 17 was the highest among the periostin splice variants examined. Both genetic ablation of periostin exons 17 and 21 and antibodies for periostin exons 17 and 21 affected preretinal pathological NV. Inhibition of exon 17 of periostin had the greatest effect in reducing preretinal pathological NV. These findings suggest a causal link between periostin splice variants and retinal NV, and an intravitreal injection of antibody for exon 17 and exon 21 of periostin should be considered to inhibit preretinal pathological NV.

  4. Functional studies on the ATM intronic splicing processing element

    PubMed Central

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

    2005-01-01

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

  5. Regulation of chemoresistance via alternative messenger RNA splicing.

    PubMed

    Eblen, Scott T

    2012-04-15

    The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.

  6. Designing shallow donors in diamond

    NASA Astrophysics Data System (ADS)

    Moussa, Jonathan

    2015-03-01

    The production of n-type semiconducting diamond has been a long-standing experimental challenge. The first-principles simulation of shallow dopants in semiconductors has been a long-standing theoretical challenge. A desirable theoretical goal is to identify impurities that will act as shallow donors in diamond and assess their experimental viability. I will discuss this identification process for the LiN4 donor complex. It builds a scientific argument from several models and computational results in the absence of computational tools that are both trustworthy and computationally tractable for this task. I will compare the theoretical assessment of viability with recent experimental efforts to co-dope diamond with lithium and nitrogen. Finally, I discuss the computational tools needed to facilitate future work on this problem and some preliminary simulations of donors near diamond surfaces. Sandia National Laboratories is a multi-program lab managed and operated by Sandia Corp., a wholly owned subsidiary of Lockheed Martin Corp., for the U.S. Department of Energy's National Nuclear Security Administration under Contract DE-AC04-94AL85000.

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

    PubMed Central

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

    2009-01-01

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

  8. Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system.

    PubMed

    Johnson, T L; Abelson, J

    2001-08-01

    Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA.

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

    PubMed

    Xu, Dong-Qing; Mattox, William

    2006-01-15

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

  10. The Involvement of Splicing Factor hnRNP A1 in UVB-Induced Alternative Splicing of hdm2.

    PubMed

    Feng, Jianguo; Li, Li; Tong, Lingying; Tang, Liling; Wu, Shiyong

    2016-01-12

    Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 via increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA. This article is protected by copyright. All rights reserved.

  11. Heteroaromatic donors in donor-acceptor-donor based fluorophores facilitate zinc ion sensing and cell imaging.

    PubMed

    Sreejith, Sivaramapanicker; Divya, Kizhumuri P; Jayamurthy, Purushothaman; Mathew, Jomon; Anupama, V N; Philips, Divya Susan; Anees, Palappuravan; Ajayaghosh, Ayyappanpillai

    2012-11-01

    The excited state intra molecular charge transfer (ICT) property of fluorophores has been extensively used for the design of fluorescent chemosensors. Herein, we report the synthesis and properties of three donor–π-acceptor–π-donor (D–π-A–π-D) based molecular probes BP, BT and BA. Two heteroaromatic rings, pyrrole (BP), and thiophene (BT) and a non-heteroaromatic ring N-alkoxy aniline (BA) were selected as donor moieties which were linked to a bipyridine binding site through a vinylic linkage. The heteroaromatic systems BP and BT perform selective and ratiometric emission signalling for zinc ions whereas the non-heteroaromatic probe BA does not. The advantages of the D–π-A–π-D design strategy in the design of ICT based probes for the selective fluorescent ratiometric signalling of zinc ions in biological media is discussed. Further, the use of BP, BT and BA for imaging Zn(2+) ions from MCF-7 cell lines is demonstrated.

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

    PubMed Central

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

    2015-01-01

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

  13. The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

    NASA Astrophysics Data System (ADS)

    Choudhury, Rajarshi; Roy, Sreerupa Ghose; Tsai, Yihsuan S.; Tripathy, Ashutosh; Graves, Lee M.; Wang, Zefeng

    2014-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.

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

    PubMed Central

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

    2016-01-01

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

  15. Evolutionary Character of Alternative Splicing in Plants

    PubMed Central

    Zhang, Chengjun; Yang, Hong; Yang, Huizhao

    2015-01-01

    Alternative splicing (AS) is one of the most important ways to enhance the functional diversity of genes. Huge amounts of data have been produced by microarray, expressed sequence tag, and RNA-seq, and plenty of methods have been developed specifically for this task. The most frequently asked questions in previous research were as follows. What is the content rate of AS genes among the whole gene set? How many AS types are presented in the genome, and which type is dominant? How about the conservation ability of AS among different species? Which kinds of isoforms from some genes have the environmental response to help individual adaptation? Based on this background, we collected analysis results from 17 species to try to map out the landscape of AS studies in plants. We have noted the shortages of previous results, and we appeal to all scientists working in the AS field to make a standard protocol so that analyses between different projects are comparable. PMID:26819552

  16. Cytoplasmic Drosha activity generated by alternative splicing

    PubMed Central

    Dai, Lisheng; Chen, Kevin; Youngren, Brenda; Kulina, Julia; Yang, Acong; Guo, Zhengyu; Li, Jin; Yu, Peng; Gu, Shuo

    2016-01-01

    RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha. PMID:27471035

  17. 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

  18. [Serum ferritin in donors with regular plateletpheresis].

    PubMed

    Ma, Chun-Hui; Guo, Ru-Hua; Wu, Wei-Jian; Yan, Jun-Xiong; Yu, Jin-Lin; Zhu, Ye-Hua; He, Qi-Tong; Luo, Yi-Hong; Huang, Lu; Ye, Rui-Yun

    2011-04-01

    This study was aimed to evaluate the impact of regular donating platelets on serum ferritin (SF) of donors. A total of 93 male blood donors including 24 initial plateletpheresis donors and 69 regular plateletpheresis donors were selected randomly. Their SF level was measured by ELISA. The results showed that the SF level of initial plateletpheresis donors and regular plateletpheresis donors were 91.08 ± 23.38 µg/L and 57.16 ± 35.48 µg/L respectively, and all were in normal levels, but there was significant difference between the 2 groups (p < 0.05). The SF level decreased when the donation frequency increased, there were no significant differences between the groups with different donation frequency. Correlation with lifetime donations of platelets was not found. It is concluded that regular plateletpheresis donors may have lower SF level.

  19. JAK2 Exon 14 Skipping in Patients with Primary Myelofibrosis: A Minor Splice Variant Modulated by the JAK2-V617F Allele Burden

    PubMed Central

    Catarsi, Paolo; Rosti, Vittorio; Morreale, Giacomo; Poletto, Valentina; Villani, Laura; Bertorelli, Roberto; Pedrazzini, Matteo; Zorzetto, Michele; Barosi, Giovanni

    2015-01-01

    Background Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases. Materials and Methods By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors. Results We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence. Conclusions Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF. PMID:25617626

  20. Publisher's note. Identification of a novel synonymous mutation in the human β-ureidopropionase gene UPB1 affecting pre-mRNA splicing.

    PubMed

    Meijer, J; Nakajima, Y; Zhang, C; Meinsma, R; Ito, T; Van Kuilenburg, A B P

    2014-01-01

    β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO2. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C>T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G>A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C>T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92 104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.

  1. Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

    PubMed Central

    Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

    2009-01-01

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

  2. Simulation shows that HLA-matched stem cell donors can remain unidentified in donor searches

    PubMed Central

    Sauter, Jürgen; Solloch, Ute V.; Giani, Anette S.; Hofmann, Jan A.; Schmidt, Alexander H.

    2016-01-01

    The heterogeneous nature of HLA information in real-life stem cell donor registries may hamper unrelated donor searches. It is even possible that fully HLA-matched donors with incomplete HLA information are not identified. In our simulation study, we estimated the probability of these unnecessarily failed donor searches. For that purpose, we carried out donor searches in several virtual donor registries. The registries differed by size, composition with respect to HLA typing levels, and genetic diversity. When up to three virtual HLA typing requests were allowed within donor searches, the share of unnecessarily failed donor searches ranged from 1.19% to 4.13%, thus indicating that non-identification of completely HLA-matched stem cell donors is a problem of practical relevance. The following donor registry characteristics were positively correlated with the share of unnecessarily failed donor searches: large registry size, high genetic diversity, and, most strongly correlated, large fraction of registered donors with incomplete HLA typing. Increasing the number of virtual HLA typing requests within donor searches up to ten had a smaller effect. It follows that the problem of donor non-identification can be substantially reduced by complete high-resolution HLA typing of potential donors. PMID:26876789

  3. scaRNAs regulate splicing and vertebrate heart development.

    PubMed

    Patil, Prakash; Kibiryeva, Nataliya; Uechi, Tamayo; Marshall, Jennifer; O'Brien, James E; Artman, Michael; Kenmochi, Naoya; Bittel, Douglas C

    2015-08-01

    Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.

  4. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  5. Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis

    PubMed Central

    Ramprasad, Vedam Lakshmi; Soumittra, Nagasamy; Nancarrow, Derek; Sen, Parveen; McKibbin, Martin; Williams, Grange A; Arokiasamy, Tharigopala; Lakshmipathy, Praveena; Inglehearn, Chris F

    2008-01-01

    Purpose Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a “ciliopathy.” Methods Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation. Results A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation. Conclusions We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The

  6. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature.

    PubMed

    Jiang, Jianfu; Liu, Xinna; Liu, Chonghuai; Liu, Guotian; Li, Shaohua; Wang, Lijun

    2017-02-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels.

  7. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature1[OPEN

    PubMed Central

    Jiang, Jianfu; Liu, Xinna; Liu, Guotian; Li, Shaohua

    2017-01-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels. PMID:28049741

  8. Donor free radical explosive composition

    DOEpatents

    Walker, Franklin E. [15 Way Points Rd., Danville, CA 94526; Wasley, Richard J. [4290 Colgate Way, Livermore, CA 94550

    1980-04-01

    An improved explosive composition is disclosed and comprises a major portion of an explosive having a detonation velocity between about 1500 and 10,000 meters per second and a minor amount of a donor additive comprising an organic compound or mixture of organic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and which is not an explosive, or an inorganic compound or mixture of inorganic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and selected from ammonium or alkali metal persulfates.

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

    PubMed

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

    2010-04-01

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

  10. Long-time dynamics through parallel trajectory splicing

    DOE PAGES

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; ...

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategymore » whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.« less

  11. Human NDE1 splicing and mammalian brain development.

    PubMed

    Mosca, S; Raponi, M; Meneghello, A; Buratti, E; Woods, C G; Baralle, D

    2017-03-07

    Exploring genetic and molecular differences between humans and other close species may be the key to explain the uniqueness of our brain and the selective pressures under which it evolves. Recent discoveries unveiled the involvement of Nuclear distribution factor E-homolog 1 (NDE1) in human cerebral cortical neurogenesis and suggested a role in brain evolution; however the evolutionary changes involved have not been investigated. NDE1 has a different gene structure in human and mouse resulting in the production of diverse splicing isoforms. In particular, mouse uses the terminal exon 8 T, while Human uses terminal exon 9, which is absent in rodents. Through chimeric minigenes splicing assay we investigated the unique elements regulating NDE1 terminal exon choice. We found that selection of the terminal exon is regulated in a cell dependent manner and relies on gain/loss of splicing regulatory sequences across the exons. Our results show how evolutionary changes in cis as well as trans acting signals have played a fundamental role in determining NDE1 species specific splicing isoforms supporting the notion that alternative splicing plays a central role in human genome evolution, and possibly human cognitive predominance.

  12. The bromodomain protein BRD4 regulates splicing during heat shock

    PubMed Central

    Hussong, Michelle; Kaehler, Christian; Kerick, Martin; Grimm, Christina; Franz, Alexandra; Timmermann, Bernd; Welzel, Franziska; Isensee, Jörg; Hucho, Tim; Krobitsch, Sylvia; Schweiger, Michal R.

    2017-01-01

    The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies. PMID:27536004

  13. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

    PubMed Central

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-01-01

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts. PMID:23892457

  14. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  15. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

    PubMed

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-08-14

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

  16. Long-time dynamics through parallel trajectory splicing

    SciTech Connect

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; Kaxiras, Efthimios; Voter, Arthur F.

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategy whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.

  17. Coexpression of neuronatin splice forms promotes medulloblastoma growth.

    PubMed

    Siu, I-Mei; Bai, Renyuan; Gallia, Gary L; Edwards, Jennifer B; Tyler, Betty M; Eberhart, Charles G; Riggins, Gregory J

    2008-10-01

    Medulloblastoma (MB) is the most common pediatric brain cancer. Several important developmental pathways have been implicated in MB formation, but fewer therapeutic targets have been identified. To locate frequently overexpressed genes, we performed a comprehensive gene expression survey of MB. Our comparison of 20 primary tumors to normal cerebellum identified neuronatin (NNAT) as the most frequently overexpressed gene in our analysis. NNAT is a neural-specific developmental gene with alpha and beta splice forms. Functional evaluation revealed that RNA interference knockdown of NNAT causes a significant decrease in proliferation. Conversely, coexpression of both splice forms in NNAT-negative MB cell lines increased proliferation, caused a significant shift from G(1) to G(2)/M, and increased soft agar colony formation and size. When expressed individually, each NNAT splice form had much less effect on these in vitro oncogenic predictors. In an in vivo model, the coexpression of both splice forms conferred the ability of xenograft formation to human MB cells that do not normally form xenografts, whereas a control gene had no effect. Our findings suggest that the frequently observed overexpression of both NNAT splice forms in MB enhances growth in this cancer.

  18. Splign: algorithms for computing spliced alignments with identification of paralogs

    PubMed Central

    Kapustin, Yuri; Souvorov, Alexander; Tatusova, Tatiana; Lipman, David

    2008-01-01

    Background The computation of accurate alignments of cDNA sequences against a genome is at the foundation of modern genome annotation pipelines. Several factors such as presence of paralogs, small exons, non-consensus splice signals, sequencing errors and polymorphic sites pose recognized difficulties to existing spliced alignment algorithms. Results We describe a set of algorithms behind a tool called Splign for computing cDNA-to-Genome alignments. The algorithms include a high-performance preliminary alignment, a compartment identification based on a formally defined model of adjacent duplicated regions, and a refined sequence alignment. In a series of tests, Splign has produced more accurate results than other tools commonly used to compute spliced alignments, in a reasonable amount of time. Conclusion Splign's ability to deal with various issues complicating the spliced alignment problem makes it a helpful tool in eukaryotic genome annotation processes and alternative splicing studies. Its performance is enough to align the largest currently available pools of cDNA data such as the human EST set on a moderate-sized computing cluster in a matter of hours. The duplications identification (compartmentization) algorithm can be used independently in other areas such as the study of pseudogenes. Reviewers This article was reviewed by: Steven Salzberg, Arcady Mushegian and Andrey Mironov (nominated by Mikhail Gelfand). PMID:18495041

  19. Cutting a Long Intron Short: Recursive Splicing and Its Implications

    PubMed Central

    Georgomanolis, Theodore; Sofiadis, Konstantinos; Papantonis, Argyris

    2016-01-01

    Over time eukaryotic genomes have evolved to host genes carrying multiple exons separated by increasingly larger intronic, mostly non-protein-coding, sequences. Initially, little attention was paid to these intronic sequences, as they were considered not to contain regulatory information. However, advances in molecular biology, sequencing, and computational tools uncovered that numerous segments within these genomic elements do contribute to the regulation of gene expression. Introns are differentially removed in a cell type-specific manner to produce a range of alternatively-spliced transcripts, and many span tens to hundreds of kilobases. Recent work in human and fruitfly tissues revealed that long introns are extensively processed cotranscriptionally and in a stepwise manner, before their two flanking exons are spliced together. This process, called “recursive splicing,” often involves non-canonical splicing elements positioned deep within introns, and different mechanisms for its deployment have been proposed. Still, the very existence and widespread nature of recursive splicing offers a new regulatory layer in the transcript maturation pathway, which may also have implications in human disease. PMID:27965595

  20. BRR2a Affects Flowering Time via FLC Splicing

    PubMed Central

    Mahrez, Walid; Shin, Juhyun; Exner, Vivien; Siretskiy, Alexey; Köhler, Claudia

    2016-01-01

    Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects. PMID:27100965

  1. Oncogenes and RNA splicing of human tumor viruses.

    PubMed

    Ajiro, Masahiko; Zheng, Zhi-Ming

    2014-09-01

    Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis.

  2. Human NDE1 splicing and mammalian brain development

    PubMed Central

    Mosca, S.; Raponi, M.; Meneghello, A.; Buratti, E.; Woods, C. G.; Baralle, D.

    2017-01-01

    Exploring genetic and molecular differences between humans and other close species may be the key to explain the uniqueness of our brain and the selective pressures under which it evolves. Recent discoveries unveiled the involvement of Nuclear distribution factor E-homolog 1 (NDE1) in human cerebral cortical neurogenesis and suggested a role in brain evolution; however the evolutionary changes involved have not been investigated. NDE1 has a different gene structure in human and mouse resulting in the production of diverse splicing isoforms. In particular, mouse uses the terminal exon 8 T, while Human uses terminal exon 9, which is absent in rodents. Through chimeric minigenes splicing assay we investigated the unique elements regulating NDE1 terminal exon choice. We found that selection of the terminal exon is regulated in a cell dependent manner and relies on gain/loss of splicing regulatory sequences across the exons. Our results show how evolutionary changes in cis as well as trans acting signals have played a fundamental role in determining NDE1 species specific splicing isoforms supporting the notion that alternative splicing plays a central role in human genome evolution, and possibly human cognitive predominance. PMID:28266585

  3. Laparoscopic donor nephrectomy versus open donor nephrectomy: recipient's perspective.

    PubMed

    Jamale, Tukaram E; Hase, Niwrutti K; Iqbal, Anwar M

    2012-11-01

    Effects of laparoscopic donor nephrectomy (LDN) on graft function, especially early post-transplant, have been controversial. To assess and compare early and late graft function in kidneys procured by open and laparoscopic methods, a retrospective observational study was carried out on 37 recipients-donors who underwent LDN after introduction of this technique in February 2007 at our center, a tertiary care nephrology referral center. Demographic, immunological and intraoperative variables as well as immunosuppressive protocols and number of human leukocyte antigen (HLA) mismatches were noted. Early graft function was assessed by serum creatinine on Days two, five, seven, 14 and 28 and at the time of discharge. Serum creatinine values at three months and at one year post-transplant were considered as the surrogates of late graft function. Data obtained were compared with the data from 33 randomly selected kidney transplants performed after January 2000 by the same surgical team, in whom open donor nephrectomy was used. Pearson's chi square test, Student's t test and Mann-Whitney U test were used for statistical analysis. Early graft function (serum creatinine on Day five 2.15 mg/dL vs 1.49 mg/dL, P = 0.027) was poorer in the LDN group. Late graft function as assessed by serum creatinine at three months (1.45 mg/dL vs 1.31 mg/dL, P = 0.335) and one year (1.56 mg/dL vs 1.34 mg/dL, P = 0.275) was equivalent in the two groups. Episodes of early acute graft dysfunction due to acute tubular necrosis were significantly higher in the LDN group (37.8% vs 12.1%, Z score 2.457, P = 0.014). Warm ischemia time was significantly prolonged in the LDN group (255 s vs 132.5 s, P = 0.002). LDN is associated with slower recovery of graft function and higher incidence of early acute graft dysfunction due to acute tubular necrosis. Late graft function at one year is however comparable.

  4. Can the HIV-1 splicing machinery be targeted for drug discovery?

    PubMed

    Dlamini, Zodwa; Hull, Rodney

    2017-01-01

    HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed.

  5. Genome-wide identification of zero nucleotide recursive splicing in Drosophila.

    PubMed

    Duff, Michael O; Olson, Sara; Wei, Xintao; Garrett, Sandra C; Osman, Ahmad; Bolisetty, Mohan; Plocik, Alex; Celniker, Susan E; Graveley, Brenton R

    2015-05-21

    Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points--5' splice sites recreated after splicing. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identify four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together, these results indicate that recursive splicing is commonly used in Drosophila, occurs in humans, and provides insight into the mechanisms by which some large introns are removed.

  6. Can the HIV-1 splicing machinery be targeted for drug discovery?

    PubMed Central

    Dlamini, Zodwa; Hull, Rodney

    2017-01-01

    HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. PMID:28331370

  7. Cadmium restores in vitro splicing activity inhibited by zinc-depletion.

    PubMed

    Lee, Myeong Jin; Ayaki, Hitoshi; Goji, Junko; Kitamura, Keiko; Nishio, Hisahide

    2006-10-01

    Zinc (Zn)-depletion inhibits the second step of RNA splicing, namely exon-ligation. To investigate the effects of cadmium (Cd) and other metal ions on RNA splicing inhibited by Zn-depletion, we measured in vitro splicing activities in the presence of these metals. Zn-depletion in the splicing reaction mixture was achieved by addition of a Zn-chelator, 1,10-phenanthroline. Cd(II) at 1, 5 and 10 microM restored the splicing activity to 2, 24 and 72% of that in the control reaction mixture, while higher concentrations of Cd(II) decreased the splicing activity, and more than 50 microM Cd(II) showed a complete absence of spliced products. Hg(II) also restored the splicing activity, albeit to a lesser extent, since 5 and 10 microM Hg(II) restored the splicing activity to 3 and 4% of the control value. The other metal ions examined in this study, Co(II), Cu(II), Mg(II) and Mn(II), did not show any restoration of the splicing activity. We concluded that Cd(II) could restore the in vitro splicing activity inhibited by Zn-depletion, although higher concentrations of Cd(II) prevented progress of the RNA splicing reaction. These results suggest that Cd(II) has a bifunctional property regarding RNA splicing, and is stimulatory at low concentrations and inhibitory at high concentrations.

  8. 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

  9. An exonic splicing silencer in the testes-specific DNA ligase III β exon

    PubMed Central

    Chew, Shern L.; Baginsky, Lysa; Eperon, Ian C.

    2000-01-01

    Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation. PMID:10606636

  10. Hydroperoxides as Hydrogen Bond Donors

    NASA Astrophysics Data System (ADS)

    Møller, Kristian H.; Tram, Camilla M.; Hansen, Anne S.; Kjaergaard, Henrik G.

    2016-06-01

    Hydroperoxides are formed in the atmosphere following autooxidation of a wide variety of volatile organics emitted from both natural and anthropogenic sources. This raises the question of whether they can form hydrogen bonds that facilitate aerosol formation and growth. Using a combination of Fourier transform infrared spectroscopy, FT-IR, and ab initio calculations, we have compared the gas phase hydrogen bonding ability of tert-butylhydroperoxide (tBuOOH) to that of tert-butanol (tBuOH) for a series of bimolecular complexes with different acceptors. The hydrogen bond acceptor atoms studied are nitrogen, oxygen, phosphorus and sulphur. Both in terms of calculated redshifts and binding energies (BE), our results suggest that hydroperoxides are better hydrogen bond donors than the corresponding alcohols. In terms of hydrogen bond acceptor ability, we find that nitrogen is a significantly better acceptor than the other three atoms, which are of similar strength. We observe a similar trend in hydrogen bond acceptor ability with other hydrogen bond donors including methanol and dimethylamine.

  11. [Clinical selection of blood donors].

    PubMed

    Danic, B

    2003-06-01

    For 20 years, the organization set up to insure the blood transfusion safety has never stopped strengthening. It is based on clinical and epidemiological selection of the blood donation candidates, biologic selection of blood donations and different physico-chemical techniques for pathogens reduction or inactivation in blood products. In France, this organization is optimized by the assertion of the voluntary and non-remunerated character of blood donation registered in the law of January 4th, 1993. The blood donors selection is structured in three successive stages. The first stage is the pre-donation information. The second stage begins with reading and filling out an info-questionnaire which prepare for an interview with a physician. This interview is specially directed to prevention of transfusion-transmitted infections and the prevention of adverse reactions after a 400 to 600 mL collection of whole blood or components. Finally, the third stage is the delivery of a post-donation information which invites the donor to contact the "établissement français du sang" (EFS) in case of a new event arisen after the donation or in case of reviewing of its own answers during the medical interview.

  12. Gamete donors' expectations and experiences of contact with their donor offspring

    PubMed Central

    Kirkman, Maggie; Bourne, Kate; Fisher, Jane; Johnson, Louise; Hammarberg, Karin

    2014-01-01

    STUDY QUESTION What are the expectations and experiences of anonymous gamete donors about contact with their donor offspring? SUMMARY ANSWER Rather than consistently wanting to remain distant from their donor offspring, donors' expectations and experiences of contact with donor offspring ranged from none to a close personal relationship. WHAT IS KNOWN ALREADY Donor conception is part of assisted reproduction in many countries, but little is known about its continuing influence on gamete donors' lives. STUDY DESIGN, SIZE, DURATION A qualitative research model appropriate for understanding participants' views was employed; semi-structured interviews were conducted during January–March 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS Before 1998, gamete donors in Victoria, Australia, were subject to evolving legislation that allowed them to remain anonymous or (from 1988) to consent to the release of identifying information. An opportunity to increase knowledge of donors' expectations and experiences of contact with their donor offspring recently arose in Victoria when a recommendation was made to introduce mandatory identification of donors on request from their donor offspring, with retrospective effect. Pre-1998 donors were invited through an advertising campaign to be interviewed about their views, experiences and expectations; 36 sperm donors and 6 egg donors participated. MAIN RESULTS AND THE ROLE OF CHANCE This research is unusual in achieving participation by donors who would not normally identify themselves to researchers or government inquiries. Qualitative thematic analysis revealed that most donors did not characterize themselves as parents of their donor offspring. Donors' expectations and experiences of contact with donor offspring ranged from none to a close personal relationship. LIMITATIONS, REASONS FOR CAUTION It is not possible to establish whether participants were representative of all pre-1998 donors. WIDER IMPLICATIONS OF THE FINDINGS Anonymous

  13. [Assessment and selection of kidney living donors].

    PubMed

    Gentil Govantes, Miguel Ángel; Pereira Palomo, Porfirio

    2010-01-01

    Donor protection should always be taken account during the selection and assessment of a living donor. On these terms, the evaluation of a potential donor must include these issues: 1) The donor act is altruistic, consciousness and out of coercion; 2) Life expectancy and quality of life of the recipient will improve after the living donor kidney transplantation; 3) The donor has normal renal function and the potential risk of developing nephropathy in the long term follow up is scarce (familiar nephropathies and other processes that may increase the potential risk for renal disease in the future, like severe hypertension, diabetes, etc must be ruled out). The glomerular filtrate should meet criteria for the normal function corresponding to age furthermore the absence of proteinuria and urine smear is normal; 4) The screening in the donor should contemplate those clinical situations or diseases non related to the kidney function but might elevate the surgical and/or anesthesia risk besides disease transmission to the recipient (as neoplasia or infections); 5) The surgical act is possible without technical difficulties and always performed after a negative result of the crossmatch between donor and recipient. The living donor evaluation process will follow a different schedule based on each particular case and the center facilities. Any case, the mentioned process is divided in two parts: The first one contains an initial screening (using non invasive and low cost tests) that allows discarding contraindications for donation (in both donor and recipient). In a second phase the assessment of the donor varies with donor characteristics. However, a test for renal function is mandatory besides imaging techniques (like angioTC), screening for transmissible diseases and a detailed evaluation for psychosocial aspects preferably made by professional. Moreover Spanish policy on living donation requires a report with information about the consent for donation developed by an

  14. Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

    PubMed Central

    Barbazuk, W. Brad

    2017-01-01

    RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

  15. The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

    PubMed Central

    Bebee, Thomas W; Park, Juw Won; Sheridan, Katherine I; Warzecha, Claude C; Cieply, Benjamin W; Rohacek, Alex M; Xing, Yi; Carstens, Russ P

    2015-01-01

    Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo. DOI: http://dx.doi.org/10.7554/eLife.08954.001 PMID:26371508

  16. Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

    PubMed

    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

    2014-06-10

    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.

  17. 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.

  18. 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

  19. Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells

    PubMed Central

    Carter, Gay; Patel, Rekha; Apostolatos, André; Murr, Michel; Cooper, Denise R.

    2014-01-01

    Background Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject. Methods We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student’s t-test. Results We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/β+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression. Conclusions Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC. PMID:27358850

  20. Heat splicing of plastic fibers using a PEEK tube

    NASA Astrophysics Data System (ADS)

    Hara, K.; Horiuchi, K.; Kim, S.; Nakano, I.; Takebayashi, T.; Takikawa, K.; Yasuoka, K.; Koyano, M.; Matsui, S.; Takeuchi, H.

    1994-08-01

    We have developed a device to splice plastic optical fibers by thermal fusion. The device heats the joint of fibers inserted in a PEEK tube, which can help align the fiber axes and protects the fused joint mechanically. We have tuned the device to produce splices with good light transmission and mechanical strength. Light transmission of nearly 95% with an rms variation of 1.5% has been achieved. The joint supports 1.2 kgf in tension and the bending radius can be as small as 3 cm. The mechanical strength can be improved easily by adding epoxy. We describe the optimization of the device and measurement results of 28000 fiber splices for the CDF plug-upgrade tile/fiber calorimeter.

  1. Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

    PubMed

    Han, Hong; Braunschweig, Ulrich; Gonatopoulos-Pournatzis, Thomas; Weatheritt, Robert J; Hirsch, Calley L; Ha, Kevin C H; Radovani, Ernest; Nabeel-Shah, Syed; Sterne-Weiler, Tim; Wang, Juli; O'Hanlon, Dave; Pan, Qun; Ray, Debashish; Zheng, Hong; Vizeacoumar, Frederick; Datti, Alessandro; Magomedova, Lilia; Cummins, Carolyn L; Hughes, Timothy R; Greenblatt, Jack F; Wrana, Jeffrey L; Moffat, Jason; Blencowe, Benjamin J

    2017-02-02

    Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

  2. Alternative splicing and evolution: diversification, exon definition and function.

    PubMed

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

    2010-05-01

    Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how and when did AS evolve? Which AS events are functional? What are the evolutionary forces that shaped, and continue to shape, AS? And what determines whether an exon is spliced in a constitutive or alternative manner? In this Review, we summarize the current knowledge of AS and evolution and provide insights into some of these unresolved questions.

  3. RNA Splicing Factors and RNA-Directed DNA Methylation.

    PubMed

    Huang, Chao-Feng; Zhu, Jian-Kang

    2014-03-26

    RNA-directed histone and/or DNA modification is a conserved mechanism for the establishment of epigenetic marks from yeasts and plants to mammals. The heterochromation formation in yeast is mediated by RNAi-directed silencing mechanism, while the establishment of DNA methylation in plants is through the RNA-directed DNA methylation (RdDM) pathway. Recently, splicing factors are reported to be involved in both RNAi-directed heterochromatin formation in yeast and the RdDM pathway in plants. In yeast, splicing factors may provide a platform for facilitating the siRNA generation through an interaction with RDRC and thereby affect the heterochromatin formation, whereas in plants, various splicing factors seem to act at different steps in the RdDM pathway.

  4. Identifying splicing regulatory elements with de Bruijn graphs.

    PubMed

    Badr, Eman; Heath, Lenwood S

    2014-12-01

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

  5. Alternative splicing of the androgen receptor in polycystic ovary syndrome

    PubMed Central

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C. K.; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ∼62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS. PMID:25825716

  6. Small molecule modulators of pre-mRNA splicing in cancer therapy

    PubMed Central

    Salton, Maayan; Misteli, Tom

    2015-01-01

    Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. RNA splicing events are key to the pathology of numerous diseases, including cancers. Some tumors are molecularly addicted to specific RNA splicing isoforms making interference with pre-mRNA processing a viable therapeutic strategy. Several RNA splicing modulators have been recently characterized showing promise in pre-clinical studies. While the targets of most splicing modulators are constitutive RNA processing components, with undesirable side effects, selectivity for individual splicing events has been observed. Given the high prevalence of splicing defects in cancer, small molecule modulators of RNA processing represent a novel therapeutic strategy in cancer treatment. Here, we review their reported effects, potential mechanisms, and limitations. PMID:26700537

  7. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  8. Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

    PubMed Central

    Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

    2014-01-01

    Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

  9. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGES

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; ...

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  10. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  11. Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein

    SciTech Connect

    Lewin, A.S.; Thomas, J. Jr.; Tirupati, H.K.

    1995-12-01

    This report investigates the coupling between transcription and splicing of a mitochondrial group I intron in Saccharomyces cerevisiae and the effect of the Cbp2 protein on splicing. 65 refs., 7 figs.

  12. Integrative analysis of many RNA-seq datasets to study alternative splicing.

    PubMed

    Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

    2014-06-01

    Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale.

  13. Extended Criteria Donors in Liver Transplantation.

    PubMed

    Vodkin, Irine; Kuo, Alexander

    2017-05-01

    Mortality rates on the liver transplant waiting list are increasing. The shortage of organs has resulted in higher utilization of extended criteria donors (ECDs), with centers pushing the limits of what is acceptable for transplantation. Donor quality is more appropriately represented as a continuum of risk, and careful selection and matching of ECD grafts with recipients may lead to excellent outcomes. Although there is no precise definition for what constitutes an ECD liver, this review focuses on frequently cited characteristics, including donor age, steatosis, donation after cardiac death, and donors with increased risk of disease transmission.

  14. Novel guidelines for organ donor cancer screening.

    PubMed

    Hassanain, Mazen

    2014-05-20

    Donor transmitted malignancy is a real disastrous risk when dealing with expanded criteria donors. As donor age is increasing, guidelines for cancer screening of the elderly brain dead organ donors must be evidence-based but systematic review of such is sparse. Based on a review of published literature and our 20 years' experience, we propose a new series of guidelines concerning screening for the four most common malignancies: breast colon, lung and prostate cancer. Prospective testing of the efficacy of such protocol will then follow.

  15. Nitrogen related shallow thermal donors in silicon

    NASA Astrophysics Data System (ADS)

    Fujita, N.; Jones, R.; Öberg, S.; Briddon, P. R.

    2007-07-01

    In this letter, the authors investigate the electrical properties of nitrogen related shallow thermal donor (STD) candidates and their concentrations under different doping conditions by means of density functional theory. Experimentally, the existence of STDs containing one nitrogen atom and both even and odd numbers of oxygen atoms has been proposed. However, so far first principles studies have not presented a candidate for the latter. Here, they show that the NO defect possesses a shallow donor level. Adding one or two more oxygen atoms results in the donor level to become shallower. The fraction of shallow nitrogen related donors to N dimers increases in material with low concentration of nitrogen.

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

    An, Ping; Burge, Christopher B

    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

  18. Minor class splicing shapes the zebrafish transcriptome during development.

    PubMed

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M; Doggett, Karen; Keightley, Maria-Cristina; Boglev, Yeliz; Trotter, Andrew J; Ng, Annie Y; Wilkins, Simon J; Verkade, Heather; Ober, Elke A; Field, Holly A; Grimmond, Sean M; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2014-02-25

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

  19. Regulation of Chemoresistance Via Alternative Messenger RNA Splicing

    PubMed Central

    Eblen, Scott T.

    2012-01-01

    The acquisition of drug resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that have were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell’s response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype. PMID:22248731

  20. The splicing fate of plant SPO11 genes.

    PubMed

    Sprink, Thorben; Hartung, Frank

    2014-01-01

    Toward the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in Arabidopsis thaliana are essential for the initiation of double strand breaks (DSBs) during the meiotic prophase. In nearly all eukaryotic organisms DSB induction during prophase I by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO) as physical connections between the homologous chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa, and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of alternative splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non-functional as they either showed intron retention (IR) or shortened exons. However, the positional distribution and number of alternative splicing events vary strongly between the different plants. The cDNAs showed in most cases premature termination codons (PTCs) due to frameshift. Nevertheless, in some cases we found alternatively spliced but functional cDNAs. These findings let us suggest that alternative splicing of SPO11 depends on the respective gene sequence and on the plant species. Therefore, this conserved mechanism might play a role concerning regulation of SPO11.

  1. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    SciTech Connect

    Lin, D.; Miller, W.L. ); Chang, Y.J.; Strauss, J.F. III )

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  2. Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.

    PubMed

    Hull, J; Shackleton, S; Harris, A

    1994-07-01

    Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

  3. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

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

    PubMed Central

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

    2014-01-01

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

  5. Differences in social representation of blood donation between donors and non-donors: an empirical study

    PubMed Central

    Guarnaccia, Cinzia; Giannone, Francesca; Falgares, Giorgio; Caligaris, Aldo Ozino; Sales-Wuillemin, Edith

    2016-01-01

    Background Both donors and non-donors have a positive image of blood donation, so donors and non-donors do not differ regarding their views on donation but do differ in converting their opinion into an active deed of donation. Several studies have identified altruism and empathy as the main factors underlying blood donation. However, a mixture of various motivational factors mould the complex behaviour of donation. This paper presents an exploratory study on differences of social representations of blood donation between blood donors and non-donors, in order to understand the reasons that bring someone to take the decision to become a blood donor. Materials and methods Participants filled in the Adapted Self-Report Altruism Scale, Toronto Empathy Questionnaire and answered a test of verbal association. Descriptive and correlation analyses were carried out on quantitative data, while a prototypic analysis was used for qualitative data. Results The study was carried out on a convenience sample of 786 individuals, 583 donors (mean age: 35.40 years, SD: 13.01 years; 39.3% female) and 203 non-donors (mean age: 35.10 years, SD: 13.30 years; 67.5% female). Social representations of donors seem to be more complex and articulated than those of non-donors. The terms that appear to be central were more specific in donors (life, needle, blood, help, altruism were the words most associated by non-donors; life, aid, altruism, solidarity, health, love, gift, generosity, voluntary, control, needed, useful, needle were the words most associated by donors). Furthermore, non-donors associated a larger number of terms referring to negative aspects of blood donation. Discussion Aspects related to training and the accuracy of any information on blood donation seem to be important in the decision to become a donor and stabilise the behaviour of donation over time, thus ensuring the highest levels of quality and safety in blood establishments. PMID:26674814

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

    PubMed Central

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

    2013-01-01

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

  7. Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

    PubMed Central

    Ghosh, Anirban; Kuppusamy, Hemalatha; Pilarski, Linda M.

    2009-01-01

    Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression. PMID:19451652

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

    PubMed Central

    Bergsma, Atze J; in ‘t Groen, Stijn LM; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, WWM Pim

    2016-01-01

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

  9. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the reason for that decision; (ii) Where appropriate, the types of donation of blood or blood... GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification. (a) Notification of donors. You, an establishment that collects blood or blood components, must make...

  10. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the reason for that decision; (ii) Where appropriate, the types of donation of blood or blood... GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification. (a) Notification of donors. You, an establishment that collects blood or blood components, must make...

  11. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the reason for that decision; (ii) Where appropriate, the types of donation of blood or blood... GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification. (a) Notification of donors. You, an establishment that collects blood or blood components, must make...

  12. Fecal microbiota transplantation and donor standardization.

    PubMed

    Owens, Casey; Broussard, Elizabeth; Surawicz, Christina

    2013-09-01

    Clostridium difficile diarrhea is a common and severe infectious disease. Antibiotics, which are standard initial treatment, are less effective for treating refractory or recurrent infection. Fecal microbiota transplantation, where healthy donor stool is transplanted into a patient, is an alternative to antibiotic therapy that requires standardization for donors and patients.

  13. Payment for donor kidneys: pros and cons.

    PubMed

    Friedman, E A; Friedman, A L

    2006-03-01

    Continuous growth of the end stage renal disease population treated by dialysis, outpaces deceased donor kidneys available, lengthens the waiting time for a deceased donor transplant. As estimated by the United States Department of Health & Human Services: '17 people die each day waiting for transplants that can't take place because of the shortage of donated organs.' Strategies to expand the donor pool--public relations campaigns and Drivers' license designation--have been mainly unsuccessful. Although illegal in most nations, and viewed as unethical by professional medical organizations, the voluntary sale of purchased donor kidneys now accounts for thousands of black market transplants. The case for legalizing kidney purchase hinges on the key premise that individuals are entitled to control of their body parts even to the point of inducing risk of life. One approach to expanding the pool of kidney donors is to legalize payment of a fair market price of about 40,000 dollars to donors. Establishing a federal agency to manage marketing and purchase of donor kidneys in collaboration with the United Network for Organ Sharing might be financially self-sustaining as reduction in costs of dialysis balances the expense of payment to donors.

  14. Negotiating boundaries: Accessing donor gametes in India

    PubMed Central

    Widge, A.; Cleland, J.

    2011-01-01

    Background: This paper documents how couples and providers access donor materials for conception in the Indian context and perceptions about using them. The objective is to facilitate understanding of critical issues and relevant concerns. Methods: A postal survey was conducted with a sample of 6000 gynaecologists and in-depth interviews were conducted with 39 gynaecologists in four cities. Results: Donor gametes are relatively more acceptable than a few years ago, especially if confidentiality can be maintained, though lack of availability of donor materials is sometimes an impediment to infertility treatment. Donor sperms are usually accessed from in-house or commercial sperm banks, pathology laboratories, IVF centres, professional donors, relatives or friends. There is scepticism about screening procedures of sperm banks. Donor eggs are usually accessed from voluntary donors, friends, relatives, egg sharing programmes, donation from other patients, advertising and commercial donors. There are several concerns regarding informed consent for using donated gametes, using relatives and friends gametes, the unregulated use of gametes and embryos, record keeping and documentation, unethical and corrupt practices and commercialisation. Conclusion: These issues need to be addressed by patients, providers and regulatory authorities by providing information, counselling, ensuring informed consent, addressing exploitation and commercialisation, ensuring monitoring, proper documentation and transparency. PMID:24753849

  15. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... of donation of blood or blood components that the donor should not donate in the future; (3) Where... the reason for that decision; (ii) Where appropriate, the types of donation of blood or blood... GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification....

  16. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  17. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  18. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  19. The Experience of Living Kidney Donors

    ERIC Educational Resources Information Center

    Brown, Judith Belle; Karley, Mary Lou; Boudville, Neil; Bullas, Ruth; Garg, Amit X.; Muirhead, Norman

    2008-01-01

    This article describes the experiences, feelings, and ideas of living kidney donors. Using a phenomenological, qualitative research approach, the authors interviewed 12 purposefully selected living kidney donors (eight men and four women), who were between four and 29 years since donation. Interviews were audiotaped, and transcribed verbatim, and…

  20. Organ donors: deceased or alive? Quo vadis?

    PubMed

    Rozental, R

    2006-01-01

    Irrespectively of universal shortage of donor organs there is a tendency of increasing the number of transplantations from living and deceased donors. Each of these two methods has positive and negative features. The main obstacles using living donors are health hazard, necessity to solve certain donor's social and psychological problems, possibility of organ trade and moving. The main problems connected with organ retrieval from deceased donors are possible conflicts with public opinion: difficulties in interpretation of brain death, legislation, obtaining of informed consent from donor's relatives, etc. Future progress in organ transplantation may take place through activation of organ retrieval from deceased donors. The most perspective ways are change to presumed consent in all countries, establishing of centralized system of donor detection and registration, intensification of transplant coordination, active contacts with mass-media, etc. It is necessary to increase (enhance) participation of the members of the public in organ donation process, to develop solidarity among the public members and to involve public authorities to deal with this problem. Bioethical standards should be put in accordance with common progress and some ethical traditions should be changed.

  1. Non Heart-Beating Donors in England

    PubMed Central

    Chaib, Eleazar

    2008-01-01

    When transplantation started all organs were retrieved from patients immediately after cardio-respiratory arrest, i.e. from non-heart-beating donors. After the recognition that death resulted from irreversible damage to the brainstem, organ retrieval rapidly switched to patients certified dead after brainstem testing. These heart-beating-donors have become the principal source of organs for transplantation for the last 30 years. The number of heart-beating-donors are declining and this is likely to continue, therefore cadaveric organs from non-heart-beating donor offers a large potential of resources for organ transplantation. The aim of this study is to examine clinical outcomes of non-heart-beating donors in the past 10 years in the UK as an way of decreasing pressure in the huge waiting list for organs transplantation. PMID:18297216

  2. Kinetics of thermal donor generation in silicon

    NASA Technical Reports Server (NTRS)

    Mao, B.-Y.; Lagowski, J.; Gatos, H. C.

    1984-01-01

    The generation kinetics of thermal donors at 450 C in Czochralski-grown silicon was found to be altered by high-temperature preannealing (e.g., 1100 C for 30 min). Thus, when compared with as-grown Si, high-temperature preannealed material exhibits a smaller concentration of generated thermal donors and a faster thermal donor saturation. A unified mechanism of nucleation and oxygen diffusion-controlled growth (based on solid-state plate transformation theory) is proposed to account for generation kinetics of thermal donors at 450 C, in as-grown and high-temperature preannealed Czochralski silicon crystals. This mechanism is consistent with the main features of the models which have been proposed to explain the formation of oxygen thermal donors in silicon.

  3. Leveraging transcript quantification for fast computation of alternative splicing profiles

    PubMed Central

    Alamancos, Gael P.; Pagès, Amadís; Trincado, Juan L.; Bellora, Nicolás; Eyras, Eduardo

    2015-01-01

    Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. PMID:26179515

  4. Deep learning of the tissue-regulated splicing code

    PubMed Central

    Leung, Michael K. K.; Xiong, Hui Yuan; Lee, Leo J.; Frey, Brendan J.

    2014-01-01

    Motivation: Alternative splicing (AS) is a regulated process that directs the generation of different transcripts from single genes. A computational model that can accurately predict splicing patterns based on genomic features and cellular context is highly desirable, both in understanding this widespread phenomenon, and in exploring the effects of genetic variations on AS. Methods: Using a deep neural network, we developed a model inferred from mouse RNA-Seq data that can predict splicing patterns in individual tissues and differences in splicing patterns across tissues. Our architecture uses hidden variables that jointly represent features in genomic sequences and tissue types when making predictions. A graphics processing unit was used to greatly reduce the training time of our models with millions of parameters. Results: We show that the deep architecture surpasses the performance of the previous Bayesian method for predicting AS patterns. With the proper optimization procedure and selection of hyperparameters, we demonstrate that deep architectures can be beneficial, even with a moderately sparse dataset. An analysis of what the model has learned in terms of the genomic features is presented. Contact: frey@psi.toronto.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24931975

  5. Splicing regulator SLU7 is essential for maintaining liver homeostasis

    PubMed Central

    Elizalde, María; Urtasun, Raquel; Azkona, María; Latasa, María U.; Goñi, Saioa; García-Irigoyen, Oihane; Uriarte, Iker; Segura, Victor; Collantes, María; Di Scala, Mariana; Lujambio, Amaia; Prieto, Jesús; Ávila, Matías A.; Berasain, Carmen

    2014-01-01

    A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence. PMID:24865429

  6. The Evolutionary Relationship between Alternative Splicing and Gene Duplication

    PubMed Central

    Iñiguez, Luis P.; Hernández, Georgina

    2017-01-01

    The protein diversity that exists today has resulted from various evolutionary processes. It is well known that gene duplication (GD) along with the accumulation of mutations are responsible, among other factors, for an increase in the number of different proteins. The gene structure in eukaryotes requires the removal of non-coding sequences, introns, to produce mature mRNAs. This process, known as cis-splicing, referred to here as splicing, is regulated by several factors which can lead to numerous splicing arrangements, commonly designated as alternative splicing (AS). AS, producing several transcripts isoforms form a single gene, also increases the protein diversity. However, the evolution and manner for increasing protein variation differs between AS and GD. An important question is how are patterns of AS affected after a GD event. Here, we review the current knowledge of AS and GD, focusing on their evolutionary relationship. These two processes are now considered the main contributors to the increasing protein diversity and therefore their relationship is a relevant, yet understudied, area of evolutionary study. PMID:28261262

  7. RNA catalyzes nuclear pre-mRNA splicing

    PubMed Central

    Fica, Sebastian M.; Tuttle, Nicole; Novak, Thaddeus; Li, Nan-Sheng; Lu, Jun; Koodathingal, Prakash; Dai, Qing; Staley, Jonathan P.; Piccirilli, Joseph A.

    2014-01-01

    SUMMARY In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a multi-megadalton machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, following the discovery of self-splicing group II intron RNAs, the snRNAs were hypothesized to catalyze splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported to date. By using metal rescue strategies, here we show that the U6 snRNA catalyzes both splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Strikingly, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms, and likely common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome. PMID:24196718

  8. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  9. 16. VIEW OF TYPICAL MAIN GIRDER WELDED 'BUTT SPLICE,' SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. VIEW OF TYPICAL MAIN GIRDER WELDED 'BUTT SPLICE,' SHOWING THE 'EYE PLATES' AND THE ATTACHMENT OF THE SIDEWALK CANTILEVER BRACKETS, NORTH GIRDER, PANEL 5, LOOKING SOUTHEAST Ryan & Harms - Benton Street Bridge, Spanning Iowa River at Benton Street, Iowa City, Johnson County, IA

  10. Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin.

    PubMed

    Mano, S; Hayashi, M; Nishimura, M

    1999-02-01

    Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.

  11. Sim4cc: a cross-species spliced alignment program.

    PubMed

    Zhou, Leming; Pertea, Mihaela; Delcher, Arthur L; Florea, Liliana

    2009-06-01

    Advances in sequencing technologies have accelerated the sequencing of new genomes, far outpacing the generation of gene and protein resources needed to annotate them. Direct comparison and alignment of existing cDNA sequences from a related species is an effective and readily available means to determine genes in the new genomes. Current spliced alignment programs are inadequate for comparing sequences between different species, owing to their low sensitivity and splice junction accuracy. A new spliced alignment tool, sim4cc, overcomes problems in the earlier tools by incorporating three new features: universal spaced seeds, to increase sensitivity and allow comparisons between species at various evolutionary distances, and powerful splice signal models and evolutionarily-aware alignment techniques, to improve the accuracy of gene models. When tested on vertebrate comparisons at diverse evolutionary distances, sim4cc had significantly higher sensitivity compared to existing alignment programs, more than 10% higher than the closest competitor for some comparisons, while being comparable in speed to its predecessor, sim4. Sim4cc can be used in one-to-one or one-to-many comparisons of genomic and cDNA sequences, and can also be effectively incorporated into a high-throughput annotation engine, as demonstrated by the mapping of 64,000 Fagus grandifolia 454 ESTs and unigenes to the poplar genome.

  12. Computational definition of sequence motifs governing constitutive exon splicing.

    PubMed

    Zhang, Xiang H-F; Chasin, Lawrence A

    2004-06-01

    We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.

  13. The Role of Alternative Splicing in Breast Cancer Progression

    DTIC Science & Technology

    2007-09-01

    tumorigenesis Reportable Outcomes: -portions of the work have been chosen for an oral presentation at this year’s Cold Spring Harbor “Eukaryotic mRNA...splicing alterations. References: N/A Appendices: Copy of abstract of the work presented during the Cold Spring Harbor “Eukaryotic mRNA Processing” meeting, August 22-26, 2007

  14. Stiff, Strong Splice For A Composite Sandwich Structure

    NASA Technical Reports Server (NTRS)

    Schmaling, D.

    1991-01-01

    New type of splice for composite sandwich structure reduces peak shear stress in structure. Layers of alternating fiber orientation interposed between thin ears in adhesive joint. Developed for structural joint in spar of helicopter rotor blade, increases precision of control over thickness of adhesive at joint. Joint easy to make, requires no additional pieces, and adds little weight.

  15. In Vivo Analysis of Alternative Pre-mRNA Splicing.

    DTIC Science & Technology

    1996-10-01

    SF2 influences 5’ splice site selection by activating proximal sites. Cell 62: 35-42 Koehorst SG, Jacobs HM, Thijssen JH, Blankenstein MA (1993) Wild...S, Frati L and Gulino A (1995) Estrogen receptors: new perspectives in breast cancer management. J. Ster. Bioch . Mol. Biol. 49:327-331. Pfeffer U

  16. 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.

  17. A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

    PubMed Central

    Esposito, Maria Valeria; Nunziato, Marcella; Starnone, Flavio; Telese, Antonella; Calabrese, Alessandra; D’Aiuto, Giuseppe; Pucci, Pietro; D’Aiuto, Massimiliano; Baralle, Francisco; D’Argenio, Valeria; Salvatore, Francesco

    2016-01-01

    About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset. PMID:28009814

  18. Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

    PubMed

    Aznarez, Isabel; Chan, Elayne M; Zielenski, Julian; Blencowe, Benjamin J; Tsui, Lap-Chee

    2003-08-15

    Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

  19. Donor Conception and "Passing," or; Why Australian Parents of Donor-Conceived Children Want Donors Who Look Like Them.

    PubMed

    Wong, Karen-Anne

    2017-03-01

    This article explores the processes through which Australian recipients select unknown donors for use in assisted reproductive technologies and speculates on how those processes may affect the future life of the donor-conceived person. I will suggest that trust is an integral part of the exchange between donors, recipients, and gamete agencies in donor conception and heavily informs concepts of relatedness, race, ethnicity, kinship, class, and visibility. The decision to be transparent (or not) about a child's genetic parentage affects recipient parents' choices of donor, about who is allowed to "know" children's genetic backgrounds, and how important it is to be able to "pass" as an unassisted conception. In this way, recipients must trust the process, institutions, and individuals involved in their treatment, as well as place trust in the future they imagine for their child. The current market for donor gametes reproduces normative conceptions of the nuclear family, kinship, and relatedness by facilitating "matching" donors to recipients by phenotype and cultural affinities. Recipient parents who choose not to prioritize "matching," and actively disclose the process of children's conceptions, may embark on a project of queering heteronormative family structures and place great trust in both their own children and changing social attitudes to reduce stigma and generate acceptance for non-traditional families.

  20. Some characteristics on the generative power of weighted one-sided splicing systems

    NASA Astrophysics Data System (ADS)

    Gan, Yee Siang; Fong, Wan Heng; Sarmin, Nor Haniza; Turaev, Sherzod

    2015-10-01

    A splicing system is a formal model for DNA based computation using the recombinant behavior of DNA molecules in the presence of enzymes and ligase. Since it was introduced in 1987, several variants with different restrictions and extensions have been developed. In this paper, a restricted variant of splicing systems, called one-sided splicing systems have been studied. The generative capacity of one-sided splicing systems with the presence of weight is investigated. We have also shown that the use of different weighting spaces and weight operations results in weighted one-sided splicing systems with different generative capacities.

  1. Donor, dad, or…? Young adults with lesbian parents' experiences with known donors.

    PubMed

    Goldberg, Abbie E; Allen, Katherine R

    2013-06-01

    In this exploratory qualitative study of 11 young adults, ages 19-29 years, we examine how young people who were raised by lesbian parents make meaning out of and construct their relationships with known donors. In-depth interviews were conducted to examine how participants defined their family composition, how they perceived the role of their donors in their lives, and how they negotiated their relationships with their donors. Findings indicate that mothers typically chose known donors who were family friends, that the majority of participants always knew who their donors were, and that their contact with donors ranged from minimal to involved. Further, participants perceived their donors in one of three ways: as strictly donors and not members of their family; as extended family members but not as parents; and as fathers. The more limited role of donors in participants' construction of family relationships sheds light on how children raised in lesbian, gay, and bisexual families are contributing to the redefinition and reconstruction of complex kinship arrangements. Our findings hold implications for clinicians who work with lesbian-mother families, and suggest that young adulthood is an important developmental phase during which interest in and contact with the donor may shift, warranting a transfer of responsibility from mother to offspring in terms of managing the donor-child relationship.

  2. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

    PubMed Central

    Fu, X D; Mayeda, A; Maniatis, T; Krainer, A R

    1992-01-01

    The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro. Images PMID:1454802

  3. SpliceNet: recovering splicing isoform-specific differential gene networks from RNA-Seq data of normal and diseased samples.

    PubMed

    Yalamanchili, Hari Krishna; Li, Zhaoyuan; Wang, Panwen; Wong, Maria P; Yao, Jianfeng; Wang, Junwen

    2014-09-01

    Conventionally, overall gene expressions from microarrays are used to infer gene networks, but it is challenging to account splicing isoforms. High-throughput RNA Sequencing has made splice variant profiling practical. However, its true merit in quantifying splicing isoforms and isoform-specific exon expressions is not well explored in inferring gene networks. This study demonstrates SpliceNet, a method to infer isoform-specific co-expression networks from exon-level RNA-Seq data, using large dimensional trace. It goes beyond differentially expressed genes and infers splicing isoform network changes between normal and diseased samples. It eases the sample size bottleneck; evaluations on simulated data and lung cancer-specific ERBB2 and MAPK signaling pathways, with varying number of samples, evince the merit in handling high exon to sample size ratio datasets. Inferred network rewiring of well established Bcl-x and EGFR centered networks from lung adenocarcinoma expression data is in good agreement with literature. Gene level evaluations demonstrate a substantial performance of SpliceNet over canonical correlation analysis, a method that is currently applied to exon level RNA-Seq data. SpliceNet can also be applied to exon array data. SpliceNet is distributed as an R package available at http://www.jjwanglab.org/SpliceNet.

  4. Bright Solid-State Emission of Disilane-Bridged Donor-Acceptor-Donor and Acceptor-Donor-Acceptor Chromophores.

    PubMed

    Shimada, Masaki; Tsuchiya, Mizuho; Sakamoto, Ryota; Yamanoi, Yoshinori; Nishibori, Eiji; Sugimoto, Kunihisa; Nishihara, Hiroshi

    2016-02-24

    The development of disilane-bridged donor-acceptor-donor (D-Si-Si-A-Si-Si-D) and acceptor-donor-acceptor (A-Si-Si-D-Si-Si-A) compounds is described. Both types of compound showed strong emission (λem =ca. 500 and ca. 400 nm, respectively) in the solid state with high quantum yields (Φ: up to 0.85). Compound 4 exhibited aggregation-induced emission enhancement in solution. X-ray diffraction revealed that the crystal structures of 2, 4, and 12 had no intermolecular π-π interactions to suppress the nonradiative transition in the solid state.

  5. 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.

  6. 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.

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

    PubMed

    Ramirez-Gomez, Francisco; Greene, Whitney; Rego, Katherine; Hansen, John D; Costa, Greg; Kataria, Priti; Bromage, Erin S

    2012-02-01

    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.

  8. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon

    PubMed Central

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ∼5,655 intronless genes, ∼13,302 constitutively spliced, and ∼7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ≥5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes. PMID:26156297

  9. Variation in sequence and organization of splicing regulatory elements in vertebrate genes

    PubMed Central

    Yeo, Gene; Hoon, Shawn; Venkatesh, Byrappa; Burge, Christopher B.

    2004-01-01

    Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells. PMID:15505203

  10. Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites.

    PubMed

    Shepard, Peter J; Choi, Eun-A; Busch, Anke; Hertel, Klemens J

    2011-11-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.

  11. Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon.

    PubMed

    Mandadi, Kranthi K; Scholthof, Karen-Beth G

    2015-01-01

    Splicing and alternative splicing (AS) are widespread co- and post-transcriptional regulatory processes in plants. Recently, we characterized genome-wide AS landscapes and virus-induced AS patterns in Brachypodium distachyon (Brachypodium), a C3 model grass. Brachypodium plants infected with Panicum mosaic virus (PMV) alone or in mixed infections with its satellite virus (SPMV) were used for high-throughput, paired-end RNA sequencing. Here, using gene attributes of ∼5,655 intronless genes, ∼13,302 constitutively spliced, and ∼7,564 alternatively spliced genes, we analyzed the influence of genomic features on splicing incidence and AS frequency. In Brachypodium, gene length, coding sequence length, and exon and intron number were positively correlated to splicing incidence and AS frequency. In contrast, exon length and the percentage composition of GC (%GC) content were inversely correlated with splicing incidence and AS frequency. Although gene expression status had little correlation with splicing occurrence per se, it negatively correlated to AS frequency: i.e., genes with ≥5 alternatively spliced transcripts were significantly less expressed compared to genes encoding <5 alternative transcripts. Further gene set enrichment analysis uncovered unique functional relationships among nonspliced, constitutively spliced and alternatively spliced genes.

  12. Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing.

    PubMed

    Kong, Ka-Yiu Edwin; Tang, Hei-Man Vincent; Pan, Kewu; Huang, Zhe; Lee, Tsz-Hang Jimmy; Hinnebusch, Alan G; Jin, Dong-Yan; Wong, Chi-Ming

    2014-01-01

    Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.

  13. 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

  14. Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response.

    PubMed

    Corkery, Dale P; Holly, Alice C; Lahsaee, Sara; Dellaire, Graham

    2015-01-01

    Alternative pre-mRNA splicing in higher eukaryotes enhances transcriptome complexity and proteome diversity. Its regulation is mediated by a complex RNA-protein network that is essential for the maintenance of cellular and tissue homeostasis. Disruptions to this regulatory network underlie a host of human diseases and contribute to cancer development and progression. The splicing kinases are an important family of pre-mRNA splicing regulators, , which includes the CDC-like kinases (CLKs), the SRSF protein kinases (SRPKs) and pre-mRNA splicing 4 kinase (PRP4K/PRPF4B). These splicing kinases regulate pre-mRNA splicing via phosphorylation of spliceosomal components and serine-arginine (SR) proteins, affecting both their nuclear localization within nuclear speckle domains as well as their nucleo-cytoplasmic shuttling. Here we summarize the emerging evidence that splicing kinases are dysregulated in cancer and play important roles in both tumorigenesis as well as therapeutic response to radiation and chemotherapy.

  15. RNA splicing regulated by RBFOX1 is essential for cardiac function in zebrafish.

    PubMed

    Frese, Karen S; Meder, Benjamin; Keller, Andreas; Just, Steffen; Haas, Jan; Vogel, Britta; Fischer, Simon; Backes, Christina; Matzas, Mark; Köhler, Doreen; Benes, Vladimir; Katus, Hugo A; Rottbauer, Wolfgang

    2015-08-15

    Alternative splicing is one of the major mechanisms through which the proteomic and functional diversity of eukaryotes is achieved. However, the complex nature of the splicing machinery, its associated splicing regulators and the functional implications of alternatively spliced transcripts are only poorly understood. Here, we investigated the functional role of the splicing regulator rbfox1 in vivo using the zebrafish as a model system. We found that loss of rbfox1 led to progressive cardiac contractile dysfunction and heart failure. By using deep-transcriptome sequencing and quantitative real-time PCR, we show that depletion of rbfox1 in zebrafish results in an altered isoform expression of several crucial target genes, such as actn3a and hug. This study underlines that tightly regulated splicing is necessary for unconstrained cardiac function and renders the splicing regulator rbfox1 an interesting target for investigation in human heart failure and cardiomyopathy.

  16. Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers.

    PubMed

    Goren, Amir; Ram, Oren; Amit, Maayan; Keren, Hadas; Lev-Maor, Galit; Vig, Ida; Pupko, Tal; Ast, Gil

    2006-06-23

    Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between co