Note: This page contains sample records for the topic splice donor site from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: November 12, 2013.
1

Aberrant splicing caused by a MLH1 splice donor site mutation found in a young Japanese patient with Lynch syndrome.  

PubMed

Lynch syndrome, also known as hereditary non-polyposis colorectal cancer, characterized by predisposition to colorectal cancer and other associated cancers, is an autosomal-dominant disorder mainly caused by germline mutations in DNA mismatch repair (MMR) genes such as MLH1, MSH2, and MSH6. Some mutations that disrupt splice donor or acceptor sites cause aberrant mRNA splicing. These mutations are generally considered as pathogenic ones, however, it is sometimes uneasy to accurately predict their pathogenicity without functional assays, particularly when the mutation is a single nucleotide substitution. In this report, we describe a 25-year-old patient with Lynch syndrome who carries a germline variant in a splice donor site of the MLH1 gene (c.790 + 5 G > T), which was first detected among Asian populations. The immunohistochemical analysis revealed loss of MLH1 protein expression in the tumor. Our splicing assay confirmed that the intronic MLH1 variant actually caused aberrant splicing, supporting its pathogenic effect. Our data accumulate more information on the genotype-phenotype relationships in patients with Lynch syndrome. PMID:22766992

Takahashi, Masanobu; Furukawa, Yoichi; Shimodaira, Hideki; Sakayori, Masato; Moriya, Takuya; Moriya, Yoshihiro; Nakamura, Yusuke; Ishioka, Chikashi

2012-12-01

2

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

Microsoft Academic Search

X-linked spondyloepiphyseal dysplasia tarda can be caused by mutations in the SEDL gene. This study describes an interesting novel mutation (IVS4+1A>G) located exactly at the rare noncanonical AT–AC consensus splicing donor point of SEDL, which regained the canonical GT–AG consensus splicing junction in addition to several other rarer noncanonical splice patterns. The mutation activated several cryptic splice sites and generated

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

2009-01-01

3

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene.  

PubMed

Hemophilia A, an X-linked disease caused by deficiency of factor VIII, is characterized by variation in clinical severity and coagulation activity. This variation is though to reflect heterogeneity of mutations in the factor VIII gene. Here we describe a CG-to-CA mutation within a potential cryptic donor splice site in intron 4 of the factor VIII gene from a patient with mild disease. This mutation makes the cryptic sequence resemble more closely the consensus sequence for donor splice sites. We infer that the mutation activates the cryptic donor splice site, which in turn causes a defect in RNA processing. PMID:2838411

Youssoufian, H; Kazazian, H H; Patel, A; Aronis, S; Tsiftis, G; Hoyer, L W; Antonarakis, S E

1988-01-01

4

Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene.  

PubMed Central

Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation. Images

Belaguli, N S; Pater, M M; Pater, A

1992-01-01

5

Characterisation of a novel minisatellite that provides multiple splice donor sites in an interferon-induced transcript.  

PubMed Central

Nucleotide sequence features of the human interferon-inducible gene 6-16 are described and include, within a CpG island, a partially expressed minisatellite consisting of 26 tandemly repeated dodecanucleotides. The repeat unit consensus sequence (CAGGTAAGGGTG) is similar to the mammalian splice donor consensus sequence [(A/C)AGGT(A/G)AGT]. The splice donor site of exon 2, as determined previously, forms part of the most upstream of the repeat units. We show that the two neighbouring repeat units also provide functional splice donor sites effectively extending exon 2 by 12 or 24 nt and inserting four or eight amino acids respectively into the predicted gene product. A similar pattern of differently spliced transcripts is detected in several human cell types. Both the number of repeat units per allele and the nucleotide sequence itself show limited polymorphism within the human population. Similar minisatellites from nonhuman primates are described and also appear to modulate splicing of a 6-16 transcript. The 6-16 minisatellite is therefore an example of tandemly repeated DNA that has a role in gene expression and may provide a useful in vivo system for the analysis of 5' splice site choice and minisatellite biology. Images

Turri, M G; Cuin, K A; Porter, A C

1995-01-01

6

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

PubMed Central

The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastin-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?C transversion at the +1 consensus donor splice site. ImagesFigure 3Figure 4Figure 5Figure 6Figure 7

Godfrey, Maurice; Vandemark, Natalie; Wang, Mei; Velinov, Milen; Wargowski, David; Tsipouras, Petros; Han, Jenny; Becker, Joanne; Robertson, Wendy; Droste, Sabine; Rao, Velidi H.

1993-01-01

7

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

SciTech Connect

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.

Godfrey, M.; Vandemark, N.; Wang, M.; Han, J.; Rao, V.H. (Univ. of Nebraska Medical Center, Omaha (United States)); Velinov, M.; Tsipouras, P. (Univ. of Connecticut Health Sciences Center, Farmington (United States)); Wargowski, D.; Becker, J.; Robertson, W.; Droste, S. (Univ. of Wisconsin, Madison (United States))

1993-08-01

8

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

PubMed

Synpolydactyly 1(SPD1) is a dominantly inherited distal limb anomaly that is characterized by incomplete digit separation and increased number of digits. SPD1 is most commonly caused by polyalanine repeat expansions and mutations in the homeodomain of the HOXD13. We report a splice donor site mutation in HOXD13 associated in most cases with cortical bone thinning. In vitro study of transcripts and truncated protein analysis indicated that c.781+1G>A mutation results in truncated HOXD13 protein p.G190fsX4. Luciferase assay indicated that the truncated HOXD13 protein failed to bind to DNA. The mechanism for this phenotype was truncated protein loss of function. PMID:24055421

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

2013-09-18

9

Genome-wide activation of latent donor splice sites in stress and disease  

PubMed Central

Sequences that conform to the 5? splice site (5?SS) consensus are highly abundant in mammalian introns. Most of these sequences are preceded by at least one in-frame stop codon; thus, their use for splicing would result in pre-maturely terminated aberrant mRNAs. In normally grown cells, such intronic 5?SSs appear not to be selected for splicing. However, under heat shock conditions aberrant splicing involving such latent 5?SSs occurred in a number of specific gene transcripts. Using a splicing-sensitive microarray, we show here that stress-induced (e.g. heat shock) activation of latent splicing is widespread across the human transcriptome, thus highlighting the possibility that latent splicing may underlie certain diseases. Consistent with this notion, our analyses of data from the Gene Expression Omnibus (GEO) revealed widespread activation of latent splicing in cells grown under hypoxia and in certain cancers such as breast cancer and gliomas. These changes were found in thousands of transcripts representing a wide variety of functional groups; among them are genes involved in cell proliferation and differentiation. The GEO analysis also revealed a set of gene transcripts in oligodendroglioma, in which the level of activation of latent splicing increased with the severity of the disease.

Nevo, Yuval; Kamhi, Eyal; Jacob-Hirsch, Jasmine; Amariglio, Ninette; Rechavi, Gideon; Sperling, Joseph; Sperling, Ruth

2012-01-01

10

Genome-wide activation of latent donor splice sites in stress and disease.  

PubMed

Sequences that conform to the 5' splice site (5'SS) consensus are highly abundant in mammalian introns. Most of these sequences are preceded by at least one in-frame stop codon; thus, their use for splicing would result in pre-maturely terminated aberrant mRNAs. In normally grown cells, such intronic 5'SSs appear not to be selected for splicing. However, under heat shock conditions aberrant splicing involving such latent 5'SSs occurred in a number of specific gene transcripts. Using a splicing-sensitive microarray, we show here that stress-induced (e.g. heat shock) activation of latent splicing is widespread across the human transcriptome, thus highlighting the possibility that latent splicing may underlie certain diseases. Consistent with this notion, our analyses of data from the Gene Expression Omnibus (GEO) revealed widespread activation of latent splicing in cells grown under hypoxia and in certain cancers such as breast cancer and gliomas. These changes were found in thousands of transcripts representing a wide variety of functional groups; among them are genes involved in cell proliferation and differentiation. The GEO analysis also revealed a set of gene transcripts in oligodendroglioma, in which the level of activation of latent splicing increased with the severity of the disease. PMID:23002147

Nevo, Yuval; Kamhi, Eyal; Jacob-Hirsch, Jasmine; Amariglio, Ninette; Rechavi, Gideon; Sperling, Joseph; Sperling, Ruth

2012-09-23

11

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada  

Microsoft Academic Search

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a G?A transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts

Peter Hechtman; Bernard Boulay; Marc De Braekeleer; Eve Andermann; Serge Melançon; Jean Larochelle; Claude Prevost; Feige Kaplan

1992-01-01

12

Information analysis of human splice site mutations  

Microsoft Academic Search

Splice site nucleotide substitutions can be analyzed by comparing the individual information contents (Ri, bits) of the normal and variant splice junction sequences (Rogan and Schneider, 1995). In the present study, we related splicing abnormalities to changes in Ri values of 111 previously reported splice site substitutions in 41 different genes. Mutant donor and acceptor sites have significantly less informa-

Peter K. Rogan; Brian M. Faux; Thomas D. Schneider

1998-01-01

13

A 6-bp deletion at the splice donor site of the first intron resulted in aberrant splicing using a cryptic splice site within exon 1 in a patient with succinyl-CoA: 3Ketoacid CoA transferase (SCOT) deficiency  

Microsoft Academic Search

Succinyl-CoA: 3-ketoacid-CoA transferase (SCOT; locus symbol OXCT, EC 2.8.3.5) deficiency is a rare genetic disorder affecting ketone body utilization in extra-hepatic tissues. A 6-bp deletion at the splice donor site of intron 1 resulted in the absence of a full-length mature SCOT mRNA with faint amounts of aberrantly spliced transcripts using a cryptic splice donor site within exon 1, which

Toshiyuki Fukao; Satomi Sakurai; Marie-Odile Rolland; Marie-Therese Zabot; Andreas Schulze; Keitaro Yamada; Naomi Kondo

2006-01-01

14

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

PubMed Central

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

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

2005-01-01

15

A novel single-base substitution (c.1124A>G) that activates a 5-base upstream cryptic splice donor site within exon 11 in the human mitochondrial acetoacetyl-CoA thiolase gene  

Microsoft Academic Search

Most mutations related to aberrant splicing occur in conserved splice acceptor and donor sites. Some exonic mutations also affect splicing. We identified and characterized a point mutation (c.1124A>G) in an Australian patient (GK43) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK43 is a homozygote of c.1124A>G, which activates a cryptic splice donor site 5 bases upstream from c.1124A>G within exon 11,

Toshiyuki Fukao; Avihu Boneh; Yusuke Aoki; Naomi Kondo

2008-01-01

16

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

SciTech Connect

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

Willing, M.; Deschenes, S. [Univ. of Iowa, Iowa City, IA (United States)

1994-09-01

17

Gonadal Mosaicism of Frasier Syndrome in 3 Chinese Siblings with Donor Splice Site Mutation of Wilms’ Tumour Gene  

Microsoft Academic Search

Frasier syndrome is a rare human developmental disorder classically affecting 46,XY females and leading to male pseudohermaphroditism and chronic renal failure. We describe a family with both 46,XX and 46,XY females affected by the syndrome due to WT1 splice site mutations. The diagnosis of Frasier syndrome in 1 of the children led to the discovery of the syndrome in 2

Wai Leung Chak; Ka Fai To; Yuk Lun Cheng; Kan Ming Tsui; Kwok Wai Lo; Hung Man Tong; Fernand Mac-Moune Lai; Francis Kin Ming Wong; Koon Shing Choi; Ka Foon Chau; Chun Sang Li

2002-01-01

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

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.

Ramirez, Jean Marie [Laboratoire Infections Retrovirales et Signalisation Cellulaire, CNRS UMR5121, UMI, IFR122, Montpellier (France); Houzet, Laurent [Laboratoire Infections Retrovirales et Signalisation Cellulaire, CNRS UMR5121, UMI, IFR122, Montpellier (France); Koller, Richard [Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD 20892-4263 (United States); Bies, Juraj [Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD 20892-4263 (United States); Centre of Molecular Medicine, Cancer Research Institute, Slovak Academy of Sciences, Bratislava (Slovakia); Wolff, Linda [Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD 20892-4263 (United States); Mougel, Marylene [Laboratoire Infections Retrovirales et Signalisation Cellulaire, CNRS UMR5121, UMI, IFR122, Montpellier (France)]. E-mail: mmougel@univ-montp1.fr

2004-12-20

19

Two novel mutations in the thyroglobulin gene as cause of congenital hypothyroidism: identification a cryptic donor splice site in the exon 19.  

PubMed

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, 52 mutations of the TG gene have been identified in humans. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report a French patient with congenital hypothyroidism, mild enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, genotyping, expression of chimeric minigenes as well as bioinformatics analysis were performed. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the paternal mutation consists of a c.3788-3789insT or c.3788dupT, whereas the maternal mutation consists of g.IVS19+3_+4delAT. Minigene analysis of the g.IVS19+3_+4delAT mutant showed that the exon 19 is skipped during pre-mRNA splicing or partially included by use of cryptic 5' splice site located to 100 nucleotides downstream of the wild type exon-intron junction. The c.3788-3789insT mutation results in a putative truncated protein of 1245 amino acids, whereas g.IVS19+3_4delAT mutation originates two putative truncated proteins of 1330 and 1349 amino acids. In conclusion, we show that the g.IVS19+3_+4delAT mutation promotes the activation of a cryptic donor splice site in the exon 19 of the TG gene. These results open up new perspectives in the knowledge of the mechanism of splicing for the TG pre-mRNA. PMID:21958696

Targovnik, Héctor M; Edouard, Thomas; Varela, Viviana; Tauber, Maithé; Citterio, Cintia E; González-Sarmiento, Rogelio; Rivolta, Carina M

2011-09-21

20

A Splice Site Mutant of Maize Activates Cryptic Splice Sites, Elicits Intron Inclusion and Exon Exclusion, and Permits Branch Point Elucidation  

Microsoft Academic Search

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5* terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One acti- vated donor site, located nine nt

Shailesh Lal; Jae-Hyuk Choi; Janine R. Shaw; L. Curtis Hannah

1999-01-01

21

Activation of a Cryptic Splice Donor in Human Immunodeficiency Virus Type1  

Microsoft Academic Search

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5? splice donor of HIV-1 was mutated in

Keith M. Borg; Justin P. Favaro; Salvatore J. Arrigo; Michael Schmidt

1999-01-01

22

Activation of a cryptic splice donor in human immunodeficiency virus type-1  

Microsoft Academic Search

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function ascis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5? splice donor of HIV-1 was mutated in the

Keith M. Borg; Justin P. Favaro; Salvatore J. Arrigo; Michael Schmidt

1999-01-01

23

Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site.  

PubMed Central

The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined. Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen-receptor-binding element. A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens. Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts. Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin. Images Fig. 3. Fig. 4.

Girotti, M; Jones, R; Emery, D C; Chia, W; Hall, L

1992-01-01

24

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

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

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

1992-02-01

25

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

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

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

1992-01-01

26

Characterization and prediction of alternative splice sites  

Microsoft Academic Search

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC

Magnus Wang; Antonio Marín

2006-01-01

27

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

SciTech Connect

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.

Wadelius, C.; Lagerkvist, A. (Univ. Hospital, Uppsala (Sweden) Uppsala Univ. (Sweden)); Molin, A.K.; Larsson, A. (Univ. Hospital, Uppsala (Sweden)); Von Doebeln, U. (Karolinska Institute, Stockholm (Sweden))

1993-08-01

28

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

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.

Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi (Kobe Univ. School of Medicine (Japan))

1994-01-01

29

Frequency of the 735G ? A mutation of the 5?-splice donor site of intron 14 of the dihydropyrimidine dehydrogenase gene ( DPYD ) in residents of novosibirsk region (Russia) as revealed with fluorescent oligonucleotides  

Microsoft Academic Search

A simple method was developed for end-point fluorescence detection of the 735G ? A mutation of the 5?-splice donor site of\\u000a intron 14 of the dihidropyrimidine dehydrogenase gene (DPYD). The method was based on allele-specific PCR with duplex Scorpion primers. The genotyping results obtained by the fluorescent\\u000a end-point PCR technique completely coincided with the results obtained by allele-specific PCR with

D. V. Mitrofanov; O. B. Chasovnikova; L. S. Koroleva; V. N. Silnikov; L. G. Zhdanova; S. P. Kovalenko

2008-01-01

30

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

PubMed Central

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

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

2000-01-01

31

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

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.

Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

1992-02-01

32

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

PubMed Central

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

Xia, Huiyu; Bi, Jianning; Li, Yanda

2006-01-01

33

Alternative 5' splice site selection induced by heat shock.  

PubMed Central

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. Images

Takechi, H; Hosokawa, N; Hirayoshi, K; Nagata, K

1994-01-01

34

A method for identifying alternative or cryptic donor splice sites within gene and mRNA sequences. Comparisons among sequences from vertebrates, echinoderms and other groups  

Microsoft Academic Search

BACKGROUND: As the amount of genome sequencing data grows, so does the problem of computational gene identification, and in particular, the splicing signals that flank exon borders. Traditional methods for identifying splicing signals have been created and optimized using sequences from model organisms, mostly vertebrate and yeast species. However, as genome sequencing extends across the animal kingdom and includes various

Katherine M Buckley; Liliana D Florea; L Courtney Smith

2009-01-01

35

Cryptic splice sites and split genes  

PubMed Central

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.

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

2011-01-01

36

Cryptic splice sites and split genes.  

PubMed

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

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

2011-04-05

37

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

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.

Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X. [Cancer Research Institute, Barcelona (Spain); Doerk, T.; Will, K. [Medizinische Hochschule Hannover (Germany); Fonknechten, N. [Institut Cochin de Genetique Moleculaire, Paris (France)

1995-03-01

38

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.  

PubMed Central

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5

Song, I; Brown, D R; Wiltshire, R N; Gantz, I; Trent, J M; Yamada, T

1993-01-01

39

Validation of Human Alternative Splice Forms Using the EASED Platform and Multiple Splice Site Discriminating Features  

Microsoft Academic Search

We have shown for a dataset of computationally predicted alternative splice sites how inherent information can be utilized\\u000a to validate the predictions by applying statistics on different features typical for splice sites. As a promising splice site\\u000a feature we investigated the frequencies of binding motifs in the context of exonic and intronic splice site flanks and between\\u000a the alternative and

Ralf Bortfeldt; Alexander Herrmann; Heike Pospisil; Stefan Schuster

40

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

SciTech Connect

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

He, Guo-Shun (Mount Siani School of Medicine, New York, NY (United States)); Grabowski, G.A. (Children's Hospital Medical Center, Cincinnati, OH (United States))

1992-10-01

41

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

Microsoft Academic Search

Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disorder caused by loss-of-function mutations in the thymidine phosphorylase gene (TYMP). We report here a patient compound heterozygous for two TYMP mutations: a novel g.4009G>A transition affecting the consensus splice donor site of intron 9, and a previously reported g.675G>C splice site mutation. The novel mutation causes exon 9 skipping but leaves the

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

2009-01-01

42

Analysis and recognition of 5? UTR intron splice sites in human pre-mRNA  

PubMed Central

Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5? untranslated regions (UTRs), and investigate correlations between this class of splice sites and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to ‘pure’ UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice site prediction, which typically relies on the change in sequence at the transition from protein coding to non-coding. By doing so, the algorithms were able to pick up subtler splicing signals that were otherwise masked by ‘coding’ noise, thus enhancing significantly the prediction of 5? UTR splice sites. For example, the non-coding splice site predicting networks pick up compositional and positional bias in the 3? ends of non-coding exons and 5? non-coding intron ends, where cytosine and guanine are over-represented. This compositional bias at the true UTR donor sites is also visible in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2–3-fold better compared with NetGene2 and GenScan in 5? UTRs. We also tested the 5? UTR trained method on protein coding regions, and discovered, surprisingly, that it works quite well (although it cannot compete with NetGene2). This indicates that the local splicing pattern in UTRs and coding regions is largely the same. The NetUTR method is made publicly available at www.cbs.dtu.dk/services/NetUTR.

Eden, E.; Brunak, S.

2004-01-01

43

Analysis and recognition of 5' UTR intron splice sites in human pre-mRNA.  

PubMed

Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice site prediction, which typically relies on the change in sequence at the transition from protein coding to non-coding. By doing so, the algorithms were able to pick up subtler splicing signals that were otherwise masked by 'coding' noise, thus enhancing significantly the prediction of 5' UTR splice sites. For example, the non-coding splice site predicting networks pick up compositional and positional bias in the 3' ends of non-coding exons and 5' non-coding intron ends, where cytosine and guanine are over-represented. This compositional bias at the true UTR donor sites is also visible in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also tested the 5' UTR trained method on protein coding regions, and discovered, surprisingly, that it works quite well (although it cannot compete with NetGene2). This indicates that the local splicing pattern in UTRs and coding regions is largely the same. The NetUTR method is made publicly available at www.cbs.dtu.dk/services/NetUTR. PMID:14960723

Eden, E; Brunak, S

2004-02-11

44

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

PubMed

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. PMID:22617876

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

2012-05-23

45

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

PubMed Central

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

Tigges, M A; Raskas, H J

1984-01-01

46

Exon Junction Sequences as Cryptic Splice Sites  

Microsoft Academic Search

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the

Terrie Sadusky; Andrew J Newman; Nicholas J Dibb

2004-01-01

47

Intrinsic differences between authentic and cryptic 5' splice sites  

Microsoft Academic Search

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5¢ splice sites (5¢ss), we systematically analyzed a set of 76 cryptic 5¢ss derived from 46 human genes. These cryptic 5¢ss have a similar frequency distribution in exons and introns, and are usually located

Xavier Roca; Ravi Sachidanandam; Adrian R. Krainer

2003-01-01

48

Prediction of Alternative Splice Sites in Human Genes  

Microsoft Academic Search

This thesis addresses the problem of predicting alternative splice sites in human genes. The most common way to identify alternative splice sites are the use of expressed sequence tags and microarray data. Since genes only produce alternative proteins under certain conditions, these methods are limited to detecting only alternative splice sites in genes whose alternative protein forms are expressed under

Douglas Simmons

2007-01-01

49

The Allele-Specific Suppressor sup-39 Alters Use of Cryptic Splice Sites in Caenorhabditis elegans  

Microsoft Academic Search

Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 59 end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes

A. Brock Roller; David C. Hoffman; Alan M. Zahler

50

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

PubMed Central

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

Bodem, J; Lochelt, M; Winkler, I; Flower, R P; Delius, H; Flugel, R M

1996-01-01

51

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

PubMed

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

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

2013-01-09

52

Processing sites involved in intron splicing of Armillaria natural product genes.  

PubMed

We analysed the structure of four genes whose transcriptional products are likely to be involved in the small molecule metabolism of the homobasidiomycete Armillaria mellea with the aim of verifying splice sites. To this end we experimentally validated in silico predicted intron/exon junctions for accuracy. Based on 78 verified junctions, a consensus for donor and acceptor sites in Armillaria is presented, along with experimental evidence for non-canonical splice sites, introns with alternative donor or acceptor junctions, and allele-selective splicing. The investigated reading frames show significant homologies to: (1) antibiotic and other small molecule efflux transporter genes; (2) phenoloxidase/laccase genes; (3) genes for dual Cys2His2/Zn(II)2Cys6 transcriptional regulators. For all of these gene categories, this is the first report on examples from the genus Armillaria. PMID:18280725

Misiek, Mathias; Hoffmeister, Dirk

2007-11-01

53

A novel splice site mutation in a Becker muscular dystrophy patient  

Microsoft Academic Search

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

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

1996-01-01

54

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.  

PubMed Central

We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain alpha-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (Mr 52,000) was absent and another protein band with an Mr of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (Mr 49,000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result of the mutation, part of the inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity. Images

Mitsubuchi, H; Nobukuni, Y; Akaboshi, I; Indo, Y; Endo, F; Matsuda, I

1991-01-01

55

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.  

PubMed

We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain alpha-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (Mr 52,000) was absent and another protein band with an Mr of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (Mr 49,000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result of the mutation, part of the inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity. PMID:2010537

Mitsubuchi, H; Nobukuni, Y; Akaboshi, I; Indo, Y; Endo, F; Matsuda, I

1991-04-01

56

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

PubMed Central

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.

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

1996-01-01

57

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

PubMed Central

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

Kohrer, K; Domdey, H

1988-01-01

58

Mechanism for Cryptic Splice Site Activation During Pre-mRNA Splicing  

Microsoft Academic Search

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby \\

Kristin K. Nelson; Michael R. Green

1990-01-01

59

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes.  

PubMed

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. PMID:20052763

Le Guédard-Méreuze, Sandie; Vaché, Christel; Baux, David; Faugère, Valérie; Larrieu, Lise; Abadie, Caroline; Janecke, Andreas; Claustres, Mireille; Roux, Anne-Françoise; Tuffery-Giraud, Sylvie

2010-03-01

60

Identification of alternative 50\\/30 splice sites based on the mechanism of splice site competition  

Microsoft Academic Search

Alternative splicing plays an important role in regu- lating gene expression. Currently, most efficient methods use expressed sequence tags or microar- ray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alterna- tive splice events with them because of their in- herent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds

Huiyu Xia; Jianning Bi; Yanda Li

2006-01-01

61

A Novel SingleBase Substitution (380C>T) That Activates a 5Base Downstream Cryptic Splice-Acceptor Site within Exon 5 in Almost All Transcripts in the Human Mitochondrial Acetoacetyl-CoA Thiolase Gene  

Microsoft Academic Search

Most mutation-related aberrant splicing occurs in the conserved splice-acceptor and -donor sites and some exonic mutations also affect splicing. We identified and characterized a point mutation (380C>T) in a Spanish patient (GK25) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK25 is a homozygote of 380C>T, which activates a cryptic splice-acceptor site 5 bases downstream from 380C>T within exon 5, causing aberrant

Kozue Nakamura; Toshiyuki Fukao; Celia Perez-Cerda; Cristobal Luque; Xiang-Qian Song; Yasuhiro Naiki; Yoshinori Kohno; Magdalena Ugarte; Naomi Kondo

2001-01-01

62

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

PubMed Central

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

Davidson, Lee; West, Steven

2013-01-01

63

Method of predicting Splice Sites based on signal interactions  

PubMed Central

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

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

2006-01-01

64

Identification of a human protein that recognizes the 3? splice site during the second step of pre-mRNA splicing  

Microsoft Academic Search

Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5? and 3? splice sites at the intron boundaries. Interactions between several splicing factors and the 5? splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3? splice site, which is cleaved during the second catalytic step, is poorly

Shaoping Wu; Michael R. Green

1997-01-01

65

A Suboptimal src 3? Splice Site Is Necessary for Efficient Replication of Rous Sarcoma Virus  

Microsoft Academic Search

Regulation of splicing of Rous sarcoma virus (RSV) RNA primary transcripts is necessary, as with other retroviruses, to allow for the accumulation of unspliced RNA and approximately equivalent amounts of spliced env and src mRNAs. Previous studies have indicated that the env 3? splice site is suboptimal because it has a nonconsensus branchpoint sequence and that this suboptimal splice site

Lei Zhang; C. Martin Stoltzfus

1995-01-01

66

Cryptic splicing involving the splice site mutation in the canine model of Duchenne muscular dystrophy  

Microsoft Academic Search

Golden retriever muscular dystrophy arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene. Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation. We are using this animal model to evaluate treatments for Duchenne muscular dystrophy, including gene repair induced by chimeric oligonucleotides. After injection of golden

S. Fletcher; T. Ly; R. M. Duff; J. McC Howell; S. D. Wilton

2001-01-01

67

Context-dependent robustness to 5' splice site polymorphisms in human populations.  

PubMed

There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5' splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5' splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5' splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only ?14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5' splice site polymorphisms, most notably a strong 3' splice site and the presence of intronic motifs downstream of the 5' splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5' splice site SNPs. In TRIM62, the upstream 3' splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5' splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5' splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5' splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells. PMID:21224255

Lu, Zhi-xiang; Jiang, Peng; Cai, James J; Xing, Yi

2010-12-28

68

Context-dependent robustness to 5? splice site polymorphisms in human populations  

PubMed Central

There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5? splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5? splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5? splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only ?14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5? splice site polymorphisms, most notably a strong 3? splice site and the presence of intronic motifs downstream of the 5? splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5? splice site SNPs. In TRIM62, the upstream 3? splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5? splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5? splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5? splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells.

Lu, Zhi-xiang; Jiang, Peng; Cai, James J.; Xing, Yi

2011-01-01

69

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

PubMed Central

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

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

2013-01-01

70

Elimination of mRNA splicing by a point mutation outside the conserved GU at 5' splice sites.  

PubMed Central

Nearly all mRNA introns begin with the dinucleotide GU. Mutations in either of these virtually invariant bases have been found to inactivate the corresponding 5' splice site. Until now single base changes in neighboring bases have not been found to completely inactivate a 5' splice site. Here we show that a single A----U transversion in the third position of the adenovirus 2 E1A 13S mRNA intron does prevent RNA splicing at the corresponding 5' splice site. Images

Montell, C; Berk, A J

1984-01-01

71

Complications at mucous membrane donor sites.  

PubMed

Full-thickness mucous membrane is an acceptable autogenous graft to replace the deficient conjunctiva resulting from intrinsic disease, surgical resection for carcinoma, or reconstruction of contracted sockets. The mouth provides an excellent source of mucous membrane graft material with few donor site complications. However, we encountered four cases of donor site complications after full-thickness mucous membrane grafting. All cases involved submucosal scarring with contracture. Because the inner aspect of the mouth is a multicontoured surface, the submucosal scarring resulted in web formation and limitation of movement of the mandible or lip. In two cases, we resected submucosal fibrotic scar tissue and designed a standard or multiple Z-plasty to release mucosal tension. This allowed a return to normal oral function. PMID:7081362

Neuhaus, R W; Baylis, H I; Shorr, N

1982-05-01

72

Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping  

Microsoft Academic Search

Mutations that affect splicing of precursor messenger RNAs play a major role in the development of hereditary diseases. Most splicing mutations have been found to eliminate GT or AG dinucleotides that define the 5' and 3' ends of introns, leading to exon skipping or cryptic splice-site activation. Although accurate description of the mis-spliced transcripts is critical for predicting phenotypic consequences

Petr Divina; Andrea Kvitkovicova; Emanuele Buratti; Igor Vorechovsky

2009-01-01

73

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.  

PubMed Central

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images

Harper, J E; Manley, J L

1991-01-01

74

Extended base pair complementarity between U1 snRNA and the 50 splice site does not inhibit splicing in higher eukaryotes, but rather increases 50 splice site recognition  

Microsoft Academic Search

Spliceosome formation is initiated by the recognition of the 50 splice site through formation of an RNA duplex between the 50 splice site and U1 snRNA. We have previously shown that RNA duplex format- ion between U1 snRNA and the 50 splice site can pro- tect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to

Marcel Freund; Martin J. Hicks; Carolin Konermann; Marianne Otte; Klemens J. Hertel; Heiner Schaal

2005-01-01

75

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

Microsoft Academic Search

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present

Shravan Kumar Mishra; Tim Ammon; Grzegorz M. Popowicz; Marcin Krajewski; Roland J. Nagel; Manuel Ares; Tad A. Holak; Stefan Jentsch

2011-01-01

76

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

PubMed Central

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5? splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M.; Krajewski, Marcin; Nagel, Roland J.; Ares, Manuel; Holak, Tad A.; Jentsch, Stefan

2013-01-01

77

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.  

PubMed

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier. PMID:21614000

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M; Krajewski, Marcin; Nagel, Roland J; Ares, Manuel; Holak, Tad A; Jentsch, Stefan

2011-05-25

78

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.  

PubMed Central

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.

Romfo, C M; Wise, J A

1997-01-01

79

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

PubMed Central

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.

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

1999-01-01

80

The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA  

Microsoft Academic Search

Alternative splicing of eukaryotic messenger RNA precursors is now known to be of widespread importance in generating multiple transcripts from a single gene. This phenomenon has emphasized the problem of the way in which splice sites are selected; recent studies have discussed the role of secondary structure1 or affinity and spatial relationships2 in this selection. Splice site sequences vary widely,

L. P. Eperon; J. P. Estibeiro; I. C. Eperon

1986-01-01

81

Oriented Scanning Is the Leading Mechanism Underlying 5? Splice Site Selection in Mammals  

Microsoft Academic Search

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5? splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the

Keren Borensztajn; Marie-Laure Sobrier; Philippe Duquesnoy; Anne-Marie Fischer; Jacqueline Tapon-Bretaudière; Serge Amselem

2006-01-01

82

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

Microsoft Academic Search

BACKGROUND: The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels

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

2008-01-01

83

Detection of Splice Sites Using Support Vector Machine  

NASA Astrophysics Data System (ADS)

Automatic identification and annotation of exon and intron region of gene, from DNA sequences has been an important research area in field of computational biology. Several approaches viz. Hidden Markov Model (HMM), Artificial Intelligence (AI) based machine learning and Digital Signal Processing (DSP) techniques have extensively and independently been used by various researchers to cater this challenging task. In this work, we propose a Support Vector Machine based kernel learning approach for detection of splice sites (the exon-intron boundary) in a gene. Electron-Ion Interaction Potential (EIIP) values of nucleotides have been used for mapping character sequences to corresponding numeric sequences. Radial Basis Function (RBF) SVM kernel is trained using EIIP numeric sequences. Furthermore this was tested on test gene dataset for detection of splice site by window (of 12 residues) shifting. Optimum values of window size, various important parameters of SVM kernel have been optimized for a better accuracy. Receiver Operating Characteristic (ROC) curves have been utilized for displaying the sensitivity rate of the classifier and results showed 94.82% accuracy for splice site detection on test dataset.

Varadwaj, Pritish; Purohit, Neetesh; Arora, Bhumika

84

Intronic and exonic sequences modulate 5' splice site selection in plant nuclei.  

PubMed Central

Pre-mRNA transcripts in a variety of organisms, including plants, Drosophila and Caenorhabditis elegans, contain introns which are significantly richer in adenosine and uridine residues than their flanking exons. Previous analyses using exonic and intronic replacements between two nonequivalent 5'splice sites in the 469 nt long rbcS3A intron 1 provided the first evidence indicating that, in both tobacco and Drosophila nuclei, 5'splice site selection is strongly influenced by the position of that site relative to the AU transition point between exon and intron. To differentiate between two potential models for 5'splice site recognition, we have expressed a completely different set of intronic and exonic replacement constructs containing identical 5'splice sites upstream of beta-conglycinin intron 4 (115 nt). Mutagenesis and deletion of the upstream 5'splice site demonstrate that intronic AU-rich sequences function by promoting recognition of the most upstream 5'splice site rather than by masking the downstream 5'splice site. Sequence insertions define a role for AG-rich exonic sequences in plant pre-mRNA splicing by demonstrating that an AG-rich element is capable of promoting downstream 5'splice site recognition. We conclude that AU-rich intronic sequences, AG-rich exonic sequences and the 5'splice site itself collectively define 5'intron boundaries in dicot nuclei.

McCullough, A J; Schuler, M A

1997-01-01

85

Splice site skipping in polyomavirus late pre-mRNA processing.  

PubMed Central

Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site. Images

Luo, Y; Carmichael, G G

1991-01-01

86

A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome  

Microsoft Academic Search

Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype–phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of

Qiuming Gong; Li Zhang; Arthur J. Moss; G. Michael Vincent; Michael J. Ackerman; Jeffrey C. Robinson; Melanie A. Jones; David J. Tester; Zhengfeng Zhou

2008-01-01

87

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

Microsoft Academic Search

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause\\u000a exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion\\u000a of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series\\u000a of vectors

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

2007-01-01

88

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites  

Microsoft Academic Search

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5? splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the

Michelle L Hastings; Nicoletta Resta; Daniel Traum; Alessandro Stella; Ginevra Guanti; Adrian R Krainer

2004-01-01

89

The RNA-Binding Protein TIA-1 Is a Novel Mammalian Splicing Regulator Acting through Intron Sequences Adjacent to a 5' Splice Site  

Microsoft Academic Search

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5* splice site. We show that IAS1 can activate the use of several heterologous 5* splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of

FABIENNE DEL GATTO-KONCZAK; CYRIL F. BOURGEOIS; CAROLINE LE GUINER; LILIANE KISTER; MARIE-CLAUDE GESNEL; JAMES STEVENIN; RICHARD BREATHNACH

2000-01-01

90

Statistical analysis of DNA sequences in the neighborhood of splice sites  

Microsoft Academic Search

Prediction of gene sequences and their exon-intron structure in large eukaryotic genomic sequences is one of the central problems\\u000a of mathematical biology. Solving this problem involves, in particular, high-accuracy splice site recognition. Using statistical\\u000a analysis of a splice site-containing human gene fragment database, some characteristic features were described for nucleotide\\u000a sequences in the splicing site neighborhood, the frequencies of all

O. M. Korzinov; T. V. Astakhova; P. K. Vlasov; M. A. Roytberg

2008-01-01

91

Splice Site, Frameshift and Chimeric GFAP Mutations in Alexander Disease  

PubMed Central

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

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

2012-01-01

92

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

PubMed Central

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

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

93

A homozygous splice site mutation in TRAPPC9 causes intellectual disability and microcephaly.  

PubMed

Autosomal recessive intellectual disability is believed to be particularly prevalent in highly consanguineous populations and genetic isolates and may account for a quarter of all non-syndromic cases. Mutations in more than 50 genes have been reported to be involved in autosomal recessive intellectual disability, including TRAPPC9 (MIM 611966), mutations of which have been identified in six families from different geographical origins. We performed a clinical and molecular genetic study of a consanguineous Pakistani family segregating intellectual disability and microcephaly. SNP-array-based homozygosity mapping revealed suggestive linkage to four genomic regions including one on chromosome 8 that contained TRAPPC9. We detected a homozygous TRAPPC9 splice donor site mutation (c.1024+1G>T) that cosegregated with intellectual disability in the family and led to skipping of exon 3 and exons 3 and 4 in blood-derived patient RNA. We have thus identified a novel splice site mutation leading to exon skipping and premature termination of TRAPPC9 translation. These data further suggest that TRAPPC9 mutations -unlike mutations in the vast majority of the known intellectual disability-associated genes- constitute a more frequent cause of autosomal-recessive cognitive deficits, especially when microcephaly is also present. PMID:22989526

Kakar, Naseebullah; Goebel, Ingrid; Daud, Shakeela; Nürnberg, Gudrun; Agha, Noor; Ahmad, Adeel; Nürnberg, Peter; Kubisch, Christian; Ahmad, Jamil; Borck, Guntram

2012-08-30

94

lnteractions acmss Exons Can lnfluence Splice Site Recognition in Plant Nuclei  

Microsoft Academic Search

In vivo analyses of cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demon- strated that the 5' splice sites are selected by their position relative to AU-rich elements within plant introns and by their degree of complementarity to the U1 small nuclear RNA. To determine whether the presence of adjacent introns affects 5'splice site recognition in plant

Andrew J. McCullough; Clair E. Baynton; Mary A. Schule

95

The 3' Splice Site of Pre-Messenger RNA is Recognized by a Small Nuclear Ribonucleoprotein  

Microsoft Academic Search

A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even

Benoit Chabot; Douglas L. Black; David M. Lemaster; Joan A. Steitz

1985-01-01

96

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

PubMed

Epidermolytic palmoplantar keratoderma (EPPK) is caused by mutations in KRT9 and less often, KRT1. All known mutations in KRT9 have been found in regions of the gene encoding the conserved central ?-helix rod domain. In the present study, we investigated the molecular basis of EPPK in a patient of Ashkenazi Jewish origin. The patient was found to carry a novel missense mutation in KRT9, resulting in the substitution of a poorly conserved leucine for valine at position 11 of the amino acid sequence. Despite its unusual location, the mutation was shown to be pathogenic through activation of a cryptic donor splice site, resulting in the deletion of 162 amino acids. The present data indicate the need to screen keratin genes in their entirety, as mutations altering domains of lesser functional importance may exert their deleterious effect at the transcriptional level. PMID:23397986

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

2013-02-09

97

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

PubMed

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

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

2010-10-06

98

A G-to-T transversion at the splice acceptor site of dystrophin exon 14 shows multiple splicing outcomes that are not exemplified by transition mutations.  

PubMed

Mutations at splicing consensus sequences have been shown to induce splicing errors such as exon skipping or cryptic splice site activation. Here, we identified eight splicing products caused by a G-to-T transversion mutation at the splice acceptor site of exon 14 of the dystrophin gene (c.1603-1G>T). Unexpectedly, the most abundant product showed skipping of the two consecutive exons 14 and 15, and exon 14 skipping was observed as the second most abundant product. To examine the cause of this splicing multiplicity, minigenes containing dystrophin exons 14 and 15 with their flanking introns were constructed and subjected to in vitro splicing. Minigenes with the wild-type sequence or a G>A transition at position c.1603-1 produced only the mature mRNA. On the other hand, the minigenes with a G>T or G>C transversion mutation produced multiple splicing products. A time-course analysis of the in vitro splicing revealed that splicing of the middle intron, intron 14, was the first step in transcript maturation for all four minigene constructs. The identity of the mutant nucleotide, but not its position, is a factor leading to multiple splicing outcomes. Our results suggest that exon skipping therapy for Duchenne's muscular dystrophy should be carefully monitored for their splicing outcomes. PMID:21854195

Ota, Mitsunori; Takeshima, Yasuhiro; Nishida, Atsushi; Awano, Hiroyuki; Lee, Tomoko; Yagi, Mariko; Matsuo, Masafumi

2011-08-19

99

Positional characterisation of false positives from computational prediction of human splice sites.  

PubMed

The performance of computational tools that can predict human splice sites are reviewed using a test set of EST-confirmed splice sites. The programs (namely HMMgene, NetGene2, HSPL, NNSPLICE, SpliceView and GeneID-3) differ from one another in the degree of discriminatory information used for prediction. The results indicate that, as expected, HMMgene and NetGene2 (which use global as well as local coding information and splice signals) followed by HSPL (which uses local coding information and splice signals) performed better than the other three programs (which use only splice signals). For the former three programs, one in every three false positive splice sites was predicted in the vicinity of true splice sites while only one in every 12 was expected to occur in such a region by chance. The persistence of this observation for programs (namely FEXH, GRAIL2, MZEF, GeneID-3, HMMgene and GENSCAN) that can predict all the potential exons (including optimal and sub-optimal) was assessed. In a high proportion (>50%) of the partially correct predicted exons, the incorrect exon ends were located in the vicinity of the real splice sites. Analysis of the distribution of proximal false positives indicated that the splice signals used by the algorithms are not strong enough to discriminate particularly those false predictions that occur within +/- 25 nt around the real sites. It is therefore suggested that specialised statistics that can discriminate real splice sites from proximal false positives be incorporated in gene prediction programs. PMID:10637326

Thanaraj, T A

2000-02-01

100

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

PubMed

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

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

1995-01-16

101

Prophylactic internal fixation of the radial osteocutaneous donor site.  

PubMed

The technique of prophylactic internal fixation (PIF) of the radial osteocutaneous donor site is described and reviewed. Twenty-two donor sites were reinforced with a 3.5mm dynamic compression plate across the anterior defect. The incidence of fracture was 4.5% (1 out of 22). The single fracture was due to a technical error and was managed conservatively. Fracture at a donor site that has not been plated is more frequent and often becomes displaced, requiring secondary surgery. In contrast, the incidence of fracture, displacement and secondary surgery following prophylactic internal fixation (PIF) is relatively low. There have been no significant long-term complications with PIF. It is now the method of choice for managing the radial osteocutaneous donor site. PMID:17110005

Avery, C M E; Danford, M; Johnson, P A

2006-11-15

102

Donor Site Morbidity Post-Conchal Cartilage Grafting  

Microsoft Academic Search

.   Morbidity of the conchal cartilage donor site is evaluated in this article. Forty-two patients (from 1984–1994) with 44 donor\\u000a sites were reviewed retrospectively. In 27 cases an anterior approach was used and in 17 cases a posterior approach was used.\\u000a Four complications were observed: two cases of delayed healing in the post auricular approach, one case of flattening of

A. O. Grobbelaar; B. A. Matti; F. V. Nicolle

1997-01-01

103

Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain.  

PubMed

The yeast mitochondrial group I intron b15 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions. Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure. Folding energy provided by CBP2 drives assembly of two RNA domains that comprise the catalytic core and meditates association of an approximately 100 nt 5' domain that contains the 5' splice site. Thus, the protein assembles RNA secondary structure elements into a specific three-dimensional array while the RNA provides the catalytic center. The division of labor between RNA and protein illustrated by this simple system reveals principles applicable to complex ribonucleoprotein assemblies such as the spliceosome and ribosome. PMID:7628013

Weeks, K M; Cech, T R

1995-07-28

104

Encapsidation determinants located downstream of the major splice donor in the maedi-visna virus leader region.  

PubMed

We investigated the role of the 5'-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Delta3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Delta5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Delta5MSD mutant and a double mutant (DM) (Delta5MSD and Delta3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Delta5MSD was dispensable in FOS cells, and the DM had the same REE as the Delta3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors. PMID:16971429

Bjarnadottir, Helga; Gudmundsson, Bjarki; Gudnason, Janus; Jonsson, Jon J

2006-09-13

105

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

Microsoft Academic Search

Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local prediction of splice sites, is refined by rules based on splice site confidence values, prediction scores, coding context

Stefan M. Hebsgaard; Peter G. Korning; Niels Tolstrup; Jacob Engelbrecht; Pierre Rouzé; Søren Brunak

1996-01-01

106

The treatment of donor sites with cultured epithelial grafts.  

PubMed

In a significant number of elderly patients, the healing of split skin donor sites can be delayed. The cultured allogenic epithelial graft (CAG) has been reported to heal leg ulcers. The mechanism of action may be to improve the healing environment and thus stimulate the host skin cells. A clinical trial was undertaken to compare the healing rate of the donor sites of elderly patients using CAGs and two commercially available dressings. Compared to Jelonet, CAGs (p = 0.008) and OpSite (p = 0.013) significantly reduced the number of patients with delayed healing. There was no significant difference between CAGs and the occlusive dressing, OpSite. PMID:1993229

Blight, A; Fatah, M F; Datubo-Brown, D D; Mountford, E M; Cheshire, I M

1991-01-01

107

Evidence that RNA editing modulates splice site selection in the 5HT2C receptor gene  

Microsoft Academic Search

Adenosine to inosine editing of mRNA from the human 5-HT2C receptor gene (HTR2C) occurs at five exonic positions (A-E) in a stable stem-loop that includes the normal 5¢ splice site of intron 5 and is flanked by two alternative splice sites. Using in vitro editing, we identified a novel editing site (F) located in the intronic part of the stem-loop

Rachel Flomen; Joanne Knight; Pak Sham; Robert Kerwin; Andrew Makoff

2004-01-01

108

AG-dependent 3?-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon  

PubMed Central

In pre-mRNA splicing, a conserved AG/G at the 3?-splice site is recognized by U2AF35. A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E+1) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF35 enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF35 but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10–15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E+1. Analysis of nine other disease-causing mutations at E+1 detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3?-splice site that requires U2AF35 for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3?-splice site that does not require U2AF35 gives rise to normal splicing. The AG-dependence of the 3?-splice site that we analyzed in disease-causing mutations at E+1 potentially helps identify yet unrecognized splicing mutations at E+1.

Fu, Yuan; Masuda, Akio; Ito, Mikako; Shinmi, Jun; Ohno, Kinji

2011-01-01

109

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

PubMed Central

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

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

1994-01-01

110

Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment  

Microsoft Academic Search

Gene trap vectors developed for genome-wide muta- genesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consist- ing of a partial 3¢-terminal exon (i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3¢ splice site) were typically expressed following insertion into introns, from cellular transcripts that spliced

Anna B. Osipovich; Erica K. White-Grindley; Geoffrey G. Hicks; Michael J. Roshon; Christian Shaffer; Jason H. Moore; H. Earl Ruley

2004-01-01

111

A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures.  

PubMed

We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts. PMID:23863840

Mattioli, Chiara; Pianigiani, Giulia; Pagani, Franco

2013-07-17

112

A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures  

PubMed Central

We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3? splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3?ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3? SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

Mattioli, Chiara; Pianigiani, Giulia; Pagani, Franco

2013-01-01

113

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

PubMed

GC 5' splice sites (5'ss) are present in ?1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2? and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites. PMID:21609956

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

2011-05-23

114

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant.  

PubMed

Changes at the invariable donor splice site +1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6+1G>T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6+1G>T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6+1G>T mutation is compatible with the synthesis of functional FVII molecules (~0.01% of normal, 1pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55nM) measured in plasma from an IVS6+1G>T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition. PMID:22426302

Cavallari, Nicola; Balestra, Dario; Branchini, Alessio; Maestri, Iva; Chuamsunrit, Ampaiwan; Sasanakul, Werasak; Mariani, Guglielmo; Pagani, Franco; Bernardi, Francesco; Pinotti, Mirko

2012-03-09

115

The influenza A segment 7 mRNA 3? splice site pseudoknot/hairpin family  

PubMed Central

The 3? splice site of the influenza A segment 7 transcript is utilized to produce mRNA for the critical M2 ion-channel protein. In solution a 63 nt fragment that includes this region can adopt two conformations: a pseudoknot and a hairpin. In each conformation, the splice site, a binding site for the SF2/ASF exonic splicing enhancer and a polypyrimidine tract, each exists in a different structural context. The most dramatic difference occurs for the splice site. In the hairpin the splice site is between two residues that are involved in a 2 by 2 nucleotide internal loop. In the pseudoknot, however, these bases are canonically paired within one of the pseudoknotted helices. The conformational switching observed in this region has implications for the regulation of splicing of the segment 7 mRNA. A measure of stability of the structures also shows interesting trends with respect to host specificity: avian strains tend to be the most stable, followed by swine and then human.

Moss, Walter N.; Dela-Moss, Lumbini I.; Priore, Salvatore F.; Turner, Douglas H.

2012-01-01

116

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

PubMed

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

Kralovicova, Jana; Vorechovsky, Igor

2010-07-14

117

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

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. PMID:22785194

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

2012-07-11

118

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 Central

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 ×102 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 ?105 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.

El Chaar, Mira; El Jisr, Tamima

2012-01-01

119

The U1 snRNP Base Pairs with the 5? Splice Site within a Penta-snRNP Complex  

PubMed Central

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

Malca, Hadar; Shomron, Noam; Ast, Gil

2003-01-01

120

The strength of the HIV1 3' splice sites affects Rev function  

Microsoft Academic Search

BACKGROUND: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that

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

2006-01-01

121

Splice-Site Mutations in Atherosclerosis Candidate Genes Relating Individual Information to Phenotype  

Microsoft Academic Search

Background—Nucleotide variants in several genes for lipid and methionine metabolism influence the risk of premature atherosclerosis. Ten percent of single nucleotide substitutions in these genes involve mRNA splice sites. The effects of some of these changes on splicing and on phenotypic severity are not inherently obvious. Methods and Results—Using an information theory-based model, we measured the individual information content (R

Yskert von Kodolitsch; Reed E. Pyeritz; Peter K. Rogan

2010-01-01

122

The Cbp2 protein suppresses splice site mutations in a group I intron.  

PubMed

The Cbp2 protein facilitates the folding of a group I intron in the COB pre-mRNA of yeast mitochondria. Based on its ability to suppress mutations affecting the auto-catalytic reaction, the protein appears to play a role in the selection of splice sites. Adding Cbp2 did not overcome the effects of mutations in P1 whose primary effect was on the first step of splicing. In contrast, most mutations affecting the ligation of exons were suppressed in vitro by Cbp2. These included mutations in P1, P9.0 and P10. In fact, a mutant transcript lacking both P9.0 and P10 ligated efficiently in the presence of Cbp2. P9.0 and P10 mutations also reduced the rate of cleavage at the 5' splice junction, and this effect was only partially mitigated by adding Cbp2. A competitive secondary structure near the 3' splice junction blocked Cbp2-stimulated splicing, but this mutation could be suppressed by co-transcriptional splicing in the presence of Cbp2. Our data underscore the importance of the interaction between the 5' and 3' splice junctions in group I introns and suggest that nucleotide-nucleotide interactions that stabilize the structure of group I introns can be superceded by protein-RNA interactions. PMID:8811097

Shaw, L C; Thomas, J; Lewin, A S

1996-09-01

123

Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene  

Microsoft Academic Search

Only one of six point mutations of the sequence around one end of the larger of the introns of the rabbit beta-globin gene seriously affects the normal removal of the intron and splicing of the gene. That mutation converts a GT sequence, invariably found at the 5' end of introns, into an AT, which is no longer recognized as a

Berend Wieringa; François Meyer; Jakob Reiser; Charles Weissmann

1983-01-01

124

Interaction of the U1 snRNP with nonconserved intronic sequences affects 5? splice site selection  

PubMed Central

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5? splice site recognition when this process is impaired as a result of the presence of noncanonical 5? splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5? splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5? splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.

Puig, Oscar; Gottschalk, Alexander; Fabrizio, Patrizia; Seraphin, Bertrand

1999-01-01

125

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

PubMed Central

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

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

1985-01-01

126

Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome.  

PubMed

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5' splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERG?24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERG?24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome. PMID:21057041

Stump, Matthew R; Gong, Qiuming; Zhou, Zhengfeng

2010-11-05

127

Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3? Splice Site Choice in Vertebrate 4.1 Genes  

PubMed Central

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

Parra, Marilyn K.; Gallagher, Thomas L.; Amacher, Sharon L.; Mohandas, Narla

2012-01-01

128

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

PubMed

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

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

2013-05-13

129

Pick one, but be quick: 5? splice sites and the problems of too many choices  

PubMed Central

Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5? Splice sites (5?ss) are the critical elements at the 5? end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5?ss in the human transcriptome. Most 5?ss are recognized by base-pairing with the 5? end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5?ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5?ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5?ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5?ss might lead to selection of a single 5?ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5?ss selection.

Roca, Xavier; Krainer, Adrian R.; Eperon, Ian C.

2013-01-01

130

Pick one, but be quick: 5' splice sites and the problems of too many choices.  

PubMed

Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5' Splice sites (5'ss) are the critical elements at the 5' end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5'ss in the human transcriptome. Most 5'ss are recognized by base-pairing with the 5' end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5'ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5'ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5'ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5'ss might lead to selection of a single 5'ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5'ss selection. PMID:23348838

Roca, Xavier; Krainer, Adrian R; Eperon, Ian C

2013-01-15

131

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

SciTech Connect

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.

Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. (National Institutes of Health, Bethesda, MD (United States) Erasmus Univ. of Rotterdam (Netherlands))

1993-03-01

132

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

PubMed Central

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

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

2012-01-01

133

Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.  

PubMed Central

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary. Images

Padgett, R A; Konarska, M M; Aebi, M; Hornig, H; Weissmann, C; Sharp, P A

1985-01-01

134

A Genetic Screen for Suppressors of a Mutated 5' Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly  

Microsoft Academic Search

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the ? 1 and 123 positions and retains some residual

MaryAnn Dassah; Sophie Patzek; Valerie M. Hunt; Pedro E. Medina; Alan M. Zahler

2009-01-01

135

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

PubMed

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

Grimaud, O; Delpit, X; Hardy, P

2011-05-10

136

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

PubMed

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

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

2003-07-01

137

Transcription and splicing  

PubMed Central

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.

Brody, Yehuda

2011-01-01

138

AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster.  

PubMed Central

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco. Images

McCullough, A J; Schuler, M A

1993-01-01

139

Splicing mediates the activity of four putative cellular internal ribosome entry sites  

PubMed Central

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5? end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3? splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3? splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

Baranick, Brian T.; Lemp, Nathan A.; Nagashima, Jill; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.

2008-01-01

140

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

PubMed Central

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

Fisette, Jean-Francois; Toutant, Johanne; Dugre-Brisson, Samuel; Desgroseillers, Luc; Chabot, Benoit

2010-01-01

141

Sequence, splice site and population frequency distribution analyses of the polymorphic human tryptophan hydroxylase intron 7  

Microsoft Academic Search

A human tryptophan hydroxylase intron seven polymorphism previously associated with low CSF 5-HIAA and suicidal behavior was sequenced and characterized for its potential role in TPH pre-mRNA splicing. Two polymorphic sites were identified: A218C and A779C. The 779A allelic frequency in various populations ranged from 0.43 to 0.61 and was in strong linkage disequilibrium with the A218C site. A218C provides

David A. Nielsen; Gary L. Jenkins; Karen M. Stefanisko; Kimberly K. Jefferson; David Goldman

1997-01-01

142

Multiple transcripts of the murine immunoglobulin ? membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites  

Microsoft Academic Search

The human C? gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed ? mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary ? chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in

Shubha Anand; Facundo D. Batista; Tatiana Tkach; Dimitar G. Efremov; Oscar R. Burrone

1997-01-01

143

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

SciTech Connect

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.

Hirschhorn, R.; Yang, D.R.; Israni, A.; Huie, M.L. (New York Univ. Medical Center, NY (United States)); Ownby, D.R. (Henry Ford Hospital, Detroit, MI (United States))

1994-07-01

144

Identification ofa Specific ExonSequence ThatIsa Major Determinant intheSelection between a Natural anda Cryptic 5'Splice Site  

Microsoft Academic Search

Thefirst intron oftheearly region 3fromadenovirus type2contains a cryptic 5'splice site, Dcrl,74 nucleotides downstream fromthenatural site DI.Thecryptic site can beactivated whenthenatural site is inactivated bymutagenesis. Toinvestigate thebasis forselection between anatural andacryptic 5'splice site, we searched forcis-acting elements responsible fortheexclusive selection ofthenatural site. We showthatboth therelative intrinsic strength ofthesites andthesequencecontext affect theselection. A 120-nucleotide segment located atthe3'endofexon 1enhances splicing attheproximal site DI;initsabsence

LIONEL DOMENJOUD; HELENE GALLINARO; SYLVIE MEYER; MONIQUE JACOB

1991-01-01

145

Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression  

Microsoft Academic Search

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT–PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179bp of coding sequence and a

Scott W. Bukovac; Richard D. Bagshaw; Brigitte A. Rigat; John W. Callahan; Joe T. R. Clarke; Don J. Mahuran

2008-01-01

146

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

PubMed Central

The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects.

Gandia, Marta; del Castillo, Francisco J.; Rodriguez-Alvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderon, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

2013-01-01

147

Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect  

Microsoft Academic Search

Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha

Nikolay A Barashkov; Lilya U Dzhemileva; Sardana A Fedorova; Fedor M Teryutin; Olga L Posukh; Elvira E Fedotova; Simeon L Lobov; Elza K Khusnutdinova

2011-01-01

148

Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5? splice site domain  

Microsoft Academic Search

The yeast mitochondrial group I intron bl5 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions. Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure. Folding energy provided by CBP2 drives assembly of two RNA domains that

Thomas R Cech

1995-01-01

149

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations.  

PubMed

Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments. PMID:23088352

Zimmermann, M A; Gehrig, A; Oldenburg, J; Müller, C R; Rost, S

2012-10-23

150

Infantile systemic hyalinosis associated with a putative splice-site mutation in the ANTXR2 gene.  

PubMed

Infantile systemic hyalinosis (ISH) is a rare autosomal recessive genetic disorder characterized by dermal and subcutaneous fibromatosis, joint contractures and bone deformities. The condition usually presents at birth, resulting in death in infancy. ISH is caused by mutations in the anthrax toxin receptor 2 gene, ANTXR2, also known as CMG2. We report an Indian child with ISH in whom we identified a homozygous acceptor splice site mutation, IVS2-4G>A. In silico analysis of this sequence showed that it changed predicted cryptic splicing, leading to out-of-frame transcripts and little, if any, functional protein. Mutations in the ANTXR2 gene can also cause juvenile hyaline fibromatosis (JHF). Although there are currently no effective treatments for ISH or JHF, identification of pathogenetic mutations in the ANTXR2 gene makes DNA-based prenatal diagnosis feasible for subsequent pregnancies. PMID:22300424

Fong, K; Rama Devi, A R; Lai-Cheong, J E; Chirla, D; Panda, S K; Liu, L; Tosi, I; McGrath, J A

2012-02-02

151

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

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.

Arredondo-Vega, F.X.; Santisteban, I.; Kelly, S.; Hershfield, M.S. (Duke Univ. Medical Center, Durham, NC (United States)); Umetsu, D.T. (Stanford Univ., CA (United States)); Schlossman, C.M.

1994-05-01

152

Prediction of mutant mRNA splice isoforms by information theory-based exon definition.  

PubMed

Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon-skipping isoforms in mRNA produced by splicing mutations from the combined information contents (R(i), which measures binding-site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (R(i),total) is the sum of the R(i) values of its acceptor and donor splice sites, adjusted for the self-information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (?R(i,total)) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis (http://splice.uwo.ca) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations. PMID:23348723

Mucaki, Eliseos J; Shirley, Ben C; Rogan, Peter K

2013-02-21

153

Bupivacaine Moistened Dressing for Pain Relief on Skin Graft Donor Sites  

Microsoft Academic Search

Background: Pain at the split-thickness skin graft donor site has been a great trouble for some patients especially during the first five postoperative days. Many types of dressings have been used for split skin graft donor site especially in the last twenty years period but it could not provide the effective pain relief for prolonged period. Patients and Methods: After

Kamonwan Jenwitheesuk; Apirag Chuangsuwanich; Somsag Areewatana

154

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

PubMed Central

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

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

1984-01-01

155

Biased exon/intron distribution of cryptic and de novo 3? splice sites  

PubMed Central

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

Kralovicova, Jana; Christensen, Mikkel B.; Vorechovsky, Igor

2005-01-01

156

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

PubMed

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

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

157

Mutations in the Caenorhabditis elegans U2AF large subunit UAF-1 alter the choice of a 3' splice site in vivo.  

PubMed

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3' splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3' splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25 degrees C. These suppressors differentially affected the recognition of the cryptic 3' splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3' splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3' splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3' splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans. PMID:19893607

Ma, Long; Horvitz, H Robert

2009-11-06

158

Germline splicing mutations of CDKN2A predispose to melanoma.  

PubMed

Coding mutations of the CDKN2A gene on chromosome 9p21 cosegregate with 25-60% of familial melanoma cases, but there remains a number of 9p21-linked kindreds that lack germline coding mutations of CDKN2A. We sequenced CDKN2A exons 1alpha, 2, 3, and the adjacent intronic regions in 167 melanoma-prone families (at least two affected first-degree relatives), and detected four splice site variations, three of which cosegregate with the disease. RT-PCR experiments verified that these three variants, including an AGgt to ATgt mutation that demonstrates a founder effect, do affect splicing. While an exon 1alpha splice donor site mutation incompletely abolishes splicing, the correctly spliced mRNA yields a protein (Q50P) that cannot effectively interact with CDK4. We also performed RT-PCR on mRNA from 16 melanoma-prone kindreds to search for cryptic splice sites deep within introns, but identified no splice variants. Meanwhile, we screened 139 affected families using allele-specific PCR for the recently discovered IVS2-105A>G mutation, but found only one family that possesses this alteration. We conclude that splice site mutations do predispose to disease in a subset of melanoma-prone kindreds. Characterization of additional splice site variants and other noncoding alterations of CDKN2A should allow us to detect a wider range of mutations in at-risk patients. PMID:14508519

Loo, Joanne C Y; Liu, Ling; Hao, AiHua; Gao, LuZhuang; Agatep, Ron; Shennan, Michael; Summers, Anne; Goldstein, Alisa M; Tucker, Margaret A; Deters, Carolyn; Fusaro, Ramon; Blazer, Kathleen; Weitzel, Jeffrey; Lassam, Norman; Lynch, Henry; Hogg, David

2003-09-25

159

Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms.  

PubMed

The molecular mechanisms that direct splice-site selection and assure orderly exon juxtaposition have not been fully clarified. The extraordinary nature of the dystrophin gene points to several hurdles in the processing of transcripts. In this study, dystrophin statistical and thermodynamic splicing parameters have been evaluated providing the first comprehensive description for a single human gene. We show that concomitant use of consensus values (CV) and DeltaDG degrees 37 values for U1snRNA annealing better discriminates between real donor sites and donor-like sequences. Evidence is also provided that, on average, out-of-frame dystrophin exons have significantly stronger CVs and more favorable DeltaDG degrees 37 values; this feature has never been reported and might reflect evolutionary-driven minimization of out-of-frame exon misplicing. Dystrophin splicing mutations have been reported to determine either Duchenne or Becker Muscular Dystrophy, but no comprehensive genotypic/phenotypic correlation has ever been investigated. We have analyzed splicing affecting single base-pair substitutions in the dystrophin gene with respect to their effect on splicing parameters; functional and clinical consequences are also reported. We have found 5'-splice-site mutation occurrence to be statistically related to mutability quotients and propose the use of DeltaDG degrees 37 values as a more effective tool than CV alone to describe donor site mutation consequences. Our analysis also indicates a nearly 100% correlation between clinical phenotype and the reading-frame rule determined at the RNA level. We consider that elucidation of the relative importance of splicing determinants might help to clarify the molecular mechanisms that direct correct splicing in complex genes and might be useful in the validation of predictive models. PMID:11479738

Sironi, M; Pozzoli, U; Cagliani, R; Comi, G P; Bardoni, A; Bresolin, N

2001-07-01

160

A hidden donor site for resurfacing finger tip defects: the 'ring graft'.  

PubMed

Skin defects on the finger tip, are commonly treated with skin grafts or flaps. Hidden areas are usually preferred as donor sites. In this study, skin over the dorsal aspect of the base of the injured finger or another finger in the same area is used as a donor site. Since the donor area can be hidden beneath a ring, it was named as the 'ring graft'. The skin elasticity over this area, allows a fairly large graft. This method was performed in defects of 27 patients involving 32 fingers. Donor sites were closed primarily without any tension. Results on follow-ups of the patients were satisfactory concerning colour and texture match of the graft. We recommend the 'ring graft' as a hidden alternative donor site for resurfacing of a finger in selected cases. PMID:14615255

Sarifakioglu, Nedim; Terzioglu, Ahmet; Bingul, Ferruh; Aslan, Gurcan

2003-12-01

161

EGCG corrects aberrant splicing of IKAP mRNA in cells from patients with familial dysautonomia  

Microsoft Academic Search

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disorder. The most prevalent causative mutation is a T?C transition in a donor splice site of the IKBKAP transcript, resulting in aberrant splicing and a truncated protein. The mutation’s position and leaky nature suggested that its impact might be moderated by altering the level of splice-regulating proteins. The reported ability of (?)-epigallocatechin

Sylvia L. Anderson; Jinsong Qiu; Berish Y. Rubin

2003-01-01

162

Multiple transcription start sites and alternative splicing in the methylenetetrahydrofolate reductase gene result in two enzyme isoforms.  

PubMed

Methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major carbon donor in the remethylation of homocysteine to methionine. Mild MTHFR deficiency, due to a common variant at nucleotide 677, has been reported to alter risk for several disorders including cardiovascular disease, neural tube defects, pregnancy complications, and certain cancers. Little is known about MTHFR regulation, since the complete cDNA and gene sequences have not been determined. In earlier work, we isolated and expressed a 2.2-kb human cDNA comprised of 11 coding exons, and we demonstrated that it encoded an active 70-kDa isoform. However, transcript sizes of approximately 7.5 kb and 9.5 kb and the presence of a second isoform of 77 kDa on Western blots suggested that cDNA sequences were incomplete. In this report, we characterized the complete cDNA and gene structure in human and mouse. Variable 5? and 3? UTR regions were identified, resulting in transcript heterogeneity. The 5? and 3? termini of the MTHFR cDNA were found to overlap with the 5? terminus of a chloride ion channel gene (CLCN-6) and the 3? terminus of an unidentified gene, respectively; this finding has resulted in finer mapping of MTHFR on Chromosome (Chr) 1p36.3. Ribonuclease protection assays identified clusters of transcriptional start sites, suggesting the existence of multiple promoters. MTHFR has several polyadenylation sites creating 3?UTR lengths of 0.2 kb-5.0 kb or 0.6 kb-4.0 kb in human and mouse, respectively. In both species, the previously reported exon 1 was redefined to approximately 3.0 kb in length and shown to be alternatively spliced. An important splice variant contains novel coding sequences; this cDNA was expressed and shown to encode the isozyme of 77 kDa. Our results, which suggest intricate regulation of MTHFR, will facilitate additional regulatory and functional studies of the different isoforms. PMID:12370778

Tran, Pamela; Leclerc, Daniel; Chan, Manuel; Pai, Aditya; Hiou-Tim, Francois; Wu, Qing; Goyette, Philippe; Artigas, Carmen; Milos, Renate; Rozen, Rima

2002-09-01

163

Splice Site Mutations in the P-Cadherin Gene Underlie Hypotrichosis with Juvenile Macular Dystrophy  

PubMed Central

Background Hypotrichosis with juvenile macular dystrophy (HJMD; OMIM 601553) is a rare autosomal recessive disorder characterized by hypotrichosis with short scalp hair and progressive macular dystrophy leading to blindness between the second and the fourth decades of life. HJMD is caused by mutations in the P-cadherin gene (CDH3), a member of the family of classical cadherins. Methods We analyzed the DNA from members of 2 consanguineous Pakistani families with HJMD for mutations in the P-cadherin gene through direct sequencing. Results We identified 2 splice site mutations in the P-cadherin gene in these families. One was a novel mutation, Ivs12-2A?G and the other a recurrent mutation, Ivs10-1G?T. A screening assay for the novel mutation ruled out the possibility of a polymorphism. Using haplotype analysis, we determined that the mutation, Ivs10-1G?T, is a founder mutation in the Pakistani population. Conclusion We identified 2 splice site mutations in the CDH3 gene leading to HJMD, further enriching our understanding of HJMD versus ectodermal dysplasia, ectrodactyly and macular dystrophy syndrome.

Shimomura, Y.; Wajid, M.; Kurban, M.; Christiano, A.M.

2010-01-01

164

First report of glucose transporter 1 deficiency syndrome in Korea with a novel splice site mutation.  

PubMed

Glucose transporter type 1 deficiency syndrome (Glut-1DS) is caused by autosomal dominant haplodeficiency or autosomal recessive with homozygous mutation of the glucose transporter 1 (SLC2A1) gene and is characterized by severe seizures, developmental delay, ataxia and acquired microcephaly. We describe the first known Korean patient with glucose transporter 1 deficiency syndrome, who had a novel mutation in the splice site. The patient began having intractable seizures at 4 days of age that initially presented as eye blinking and apnea, evolving into generalized tonic seizures. A lumbar puncture revealed low glucose concentration in the cerebrospinal fluid (CSF) in the setting of normoglycemia (blood glucose, 106 mg/dl; CSF glucose 21 mg/dl, and CSF to blood glucose ratio 0.20). The results of a 3-O-methylglucose uptake study in erythrocytes (RBC) revealed that glucose uptake reduced to 48% of his parents in the patient. The patient responded to a ketogenic diet that was initiated at 4 months of age and currently is on the modified Atkins diet (MAD) without seizures. He does not require antiepileptic medication. We diagnosed the first Glut-1 patient in Korea with a novel splice site mutation on the basis of clinical features, deficient glucose uptake and a mutation in the SLC2A1 gene. PMID:22814174

Woo, Sat Byul; Lee, Kon-Hee; Kang, Hoon-Chul; Yang, Hong; De Vivo, Darryl C; Kim, Sung Koo

2012-07-17

165

Anterior talar dome as an alternative donor site for osteochondral transplantation for medial talar dome lesions.  

PubMed

An alternative donor site for osteochondral transplantation for medial osteochondral defects of the talus eliminates the need for a second surgical site and decreases operative time. Additionally, the donor osteochondral grafts topographical shape and contour more closely resembles the medical talar shoulder than grafts harvested from the lateral femoral condyle or the superolateral margin of the intra-articular notch. This approach has been used with good preliminary results and no evidence of donor site complications. Long-term follow-up will be needed to adequately evaluate the validity of this procedure. Further investigation and clinical evaluation is warranted. PMID:11499180

Lee, M S

2001-07-01

166

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3?-splice site recognition  

PubMed Central

Recognition of the 3?-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3?-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.

Zhang, Yun; Madl, Tobias; Bagdiul, Ivona; Kern, Thomas; Kang, Hyun-Seo; Zou, Peijian; Mausbacher, Nina; Sieber, Stephan A.; Kramer, Angela; Sattler, Michael

2013-01-01

167

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

PubMed

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

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

2012-03-25

168

A novel intronic mutation in SHOX causes short stature by disrupting a splice acceptor site: direct demonstration of aberrant splicing by expression of a minigene in HEK-293T cells.  

PubMed

SHOX, the short stature homeobox-containing gene, encodes a critical regulatory protein controlling long bone growth. We examined patients in one family, identified an intronic mutation, and expressed SHOX minigenes in HEK293T cells to characterize the effect on gene splicing. We identified a novel mutation at position -3 (c.-432-3C>A;g.6120C>A) of the intron 1 splice acceptor site; three short (height Z-score -2.4 to -1.7) children were heterozygous and the father (height Z-score -3.4) was homozygous. A wild-type minigene produced alternative transcripts; one utilized the normal splice site between intron 1 and exon 2, the other a cryptic splice site in exon 2. Mutant SHOX minigene generated only the smaller transcript. The exon 2 acceptor splice site is weak; an alternative transcript is normally produced using a downstream cryptic splice site. The c.-432-3C>A mutation causes further weakening, and the cryptic splice site is preferentially utilized, resulting in SHOX deficiency and short stature. PMID:23426818

Danzig, Jennifer; Levine, Michael A

2012-01-01

169

Structure Function and Splice Site Analysis of the Synaptogenic Activity of the Neurexin-1? LNS Domain  

PubMed Central

Recent findings suggest that the neurexin-neuroligin link promotes both GABAergic and glutamatergic synaptogenesis, but the mechanism by which neurexins influence the clustering of appropriate neuroligins and postsynaptic differentiation remains unclear. Previous studies suggested that the presence or absence of alternatively spliced residues at splice site 4 (S4) in the neurexin LNS domain may regulate neurexin function. We demonstrate that addition of the S4 insert selectively reduces the ability of neurexin-1? to cluster neuroligin-1/3/4 and glutamatergic postsynaptic proteins, although clustering of neuroligin-2 and GABAergic postsynaptic proteins remain strong. Furthermore, addition of the S4 insert decreases the binding affinity of neurexin-1? to neuroligins-1 and -4 but has little effect on binding to neuroligins-2 and -3. Additional structure-function studies reveal the neurexin binding interface mediating synaptogenic activity to be composed primarily of residues in the ?2?3, ?6?7, and ?10?11 loops on one rim of the LNS domain ? sandwich. Mutation of two predicted Ca2+-binding residues disrupts postsynaptic protein clustering and binding to neuroligins, consistent with previous findings that neurexin-neuroligin binding is Ca2+ dependent. Glutamatergic postsynaptic clustering was more readily disrupted by the mutagenesis than GABAergic postsynaptic protein clustering. Perhaps neurexins-neuroligins, or neurexin-1? at least, is most important for GABA synapse formation or controlling the balance of GABA and glutamate synapses. These results suggest that differential neurexin-neuroligin binding affinities and splice variations may play an instructive role in postsynaptic differentiation.

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

2010-01-01

170

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

PubMed

Selection of any free flap donor site must not only meet the requirements of the recipient site but also minimize untoward sequela at the donor site itself. Although the anterolateral thigh (ALT) perforator flap is an ideal soft tissue donor site, a major drawback can be its nonesthetic appearance if a skin graft was needed. This detriment can be ameliorated by using traditional tissue expansion techniques. In a retrospective review over the past decade, 14 patients had ALT free flap donor site tissue expansion. These were subcategorized as pretransfer, concurrent, or posttransfer tissue expansion. In this group, mean ALT flap width was 12.2 ± 4.2 cm, which precluded direct donor site closure. Rectangular expanders were generally recommended. Multiple expanders are suggested for larger defects. The duration of expansion averaged 291.4 ± 163.9 days. The mean instilled volume ratio exceeded 2.43 ± 0.9 times the maximum vendor recommendation. Small skin graft residua were still left in four patients. Tissue expansion proved to be an important modality to consider for minimizing the stigmata of the skin grafted ALT free flap donor site. However, this process is time consuming and requires an additional surgical procedure. As such, this option must be reserved for the most motivated and compliant patients. PMID:23784789

Hallock, Geoffrey G

2013-06-19

171

Reconstruction of the radial forearm free flap donor site using integra artificial dermis.  

PubMed

Autologous skin grafting to the donor site in patients who undergo radial forearm free flap reconstruction (RFFF) is associated with cosmetic and functional morbidity. Integra artificial dermis (Integra Lifesciences, Plainsboro, NJ) is a bovine collagen based dermal substitute that can be used as an alternative to primary autologous skin transplantation of the donor site. We describe a staged reconstruction using Integra followed by ultrathin skin grafting that results in highly aesthetic and functional outcomes for these defects. A retrospective review of 29 patients undergoing extirpative head and neck oncologic resection were examined. Integra graft placement was performed at the time of RFFF harvest followed by autologous split thickness skin grafting at 1 to 5 weeks postoperatively. Healing fully occurred within 4-6 weeks with negligible donor site complications, excellent cosmesis, and minimal scar contracture. Composite reconstruction with Integra artificial dermis offers advantages over traditional methods of coverage for select cases of radial forearm free flap donor site closures. PMID:20939003

Murray, Ryan C; Gordin, Eli A; Saigal, Kapil; Leventhal, Douglas; Krein, Howard; Heffelfinger, Ryan N

2010-10-11

172

Clinical comparative study of aquacel and paraffin gauze dressing for split-skin donor site treatment.  

PubMed

The management of split-thickness skin graft donor sites is targeted towards promoting the healing process, while minimizing adverse effects and complications. The aim of this study was to compare donor site treatment outcome between Aquacel, a carboxymethylcellulose-based hydrofiber dressing, and the standard mesh paraffin gauze dressing. The study included 23 adult patients. Half of the skin graft donor site in the proximal thigh was dressed with paraffin gauze and the rest with Aquacel. The results indicated that patients treated with Aquacel experienced significantly less pain and a more rapid rate of epithelialization compared with patients treated with mesh paraffin gauze dressing. Final scarring (ie, after the 1-year follow-up) was significantly better with the Aquacel dressing. We conclude that Aquacel dressing is superior to the standard mesh paraffin gauze dressing for split-thickness donor site area in pain relief, ease of treatment, promotion of epithelialization, and the quality of scarring. PMID:15269581

Barnea, Yoav; Amir, Aharon; Leshem, David; Zaretski, Arik; Weiss, Jerry; Shafir, Raphael; Gur, Eyal

2004-08-01

173

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites  

SciTech Connect

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.

Bodaghi, Sohrab; Jia Rong [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Zheng Zhiming [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)], E-mail: zhengt@exchange.nih.gov

2009-03-30

174

Identification of activated cryptic 5' splice sites using structure profiles and odds measure.  

PubMed

The activation of cryptic 5' splice sites (5' SSs) is often related to human hereditary diseases. The DNA-based mutation screening strategies are commonly used to recognize the cryptic 5' SSs, because features of the local DNA sequence can influence the choice of cryptic 5' SSs. To improve the identification of the cryptic 5' SSs, we developed a structure-based method, named SPO (structure profiles and odds measure), which combines two parameters, the structural feature derived from hydroxyl radical cleavage pattern and odds measure, to assess the likelihood of a cryptic 5' SS activation in competing with its paired authentic 5' SS. Compared to the current tools for identifying activated cryptic 5' SSs, the SPO algorithm achieves higher prediction accuracy than the other methods, including MaxEnt, MDD, Markov model, weight matrix model, Shapiro and Senapathy matrix, R(i) and ?G. In addition, the predicted ?SPO scores from the SPO algorithm exhibited a greater degree of correlation with the strength of cryptic 5' SS activation than that measured from the other seven methods. In conclusion, the SPO algorithm provides an optimal identification of cryptic 5' SSs, can be applied in designing mutagenesis experiments for various splicing events and may be helpful to investigate the relationship between structural variants and human hereditary diseases. PMID:22323516

Tsai, Kun-Nan; Wang, Daryi

2012-02-09

175

Myopathy in a woman and her daughter associated with a novel splice site MTM1 mutation.  

PubMed

We have investigated a woman and her daughter with an early onset, slowly progressive myopathy. Muscle biopsy showed in both cases severe atrophy with marked fatty replacement. Frequent fibers with internalized nuclei were present but no typical features of centronuclear myopathy. There were also many fibers with deep invaginations of the plasma membrane. The presence of necklace fibers provided clue to correct genetic diagnosis. Both patients had a novel heterozygous splice site mutation in the myotubularin gene, MTM1 (c.867+1G>T). Analysis of MTM1 cDNA revealed that the mutation resulted in aberrant splicing with variable exon skipping. The expression of normal transcripts was markedly reduced and there was reduced expression of myotubularin protein. Although the expression of the allele without the mutation was reduced we did not obtain evidence of skewed X-chromosome inactivation. Other factors than skewed X-inactivation may cause allele inactivation and manifestation of severe myopathy in heterozygous carriers of pathogenic MTM1 mutations. PMID:22101172

Hedberg, Carola; Lindberg, Christopher; Máthé, Gyöngyvér; Moslemi, Ali-Reza; Oldfors, Anders

2011-11-18

176

Identification of activated cryptic 5? splice sites using structure profiles and odds measure  

PubMed Central

The activation of cryptic 5? splice sites (5? SSs) is often related to human hereditary diseases. The DNA-based mutation screening strategies are commonly used to recognize the cryptic 5? SSs, because features of the local DNA sequence can influence the choice of cryptic 5? SSs. To improve the identification of the cryptic 5? SSs, we developed a structure-based method, named SPO (structure profiles and odds measure), which combines two parameters, the structural feature derived from hydroxyl radical cleavage pattern and odds measure, to assess the likelihood of a cryptic 5? SS activation in competing with its paired authentic 5? SS. Compared to the current tools for identifying activated cryptic 5? SSs, the SPO algorithm achieves higher prediction accuracy than the other methods, including MaxEnt, MDD, Markov model, weight matrix model, Shapiro and Senapathy matrix, Ri and ?G. In addition, the predicted ?SPO scores from the SPO algorithm exhibited a greater degree of correlation with the strength of cryptic 5? SS activation than that measured from the other seven methods. In conclusion, the SPO algorithm provides an optimal identification of cryptic 5? SSs, can be applied in designing mutagenesis experiments for various splicing events and may be helpful to investigate the relationship between structural variants and human hereditary diseases.

Tsai, Kun-Nan; Wang, Daryi

2012-01-01

177

Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.  

PubMed

Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis. PMID:23239986

Wappenschmidt, Barbara; Becker, Alexandra A; Hauke, Jan; Weber, Ute; Engert, Stefanie; Köhler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K

2012-12-11

178

Analysis of 30 Putative BRCA1 Splicing Mutations in Hereditary Breast and Ovarian Cancer Families Identifies Exonic Splice Site Mutations That Escape In Silico Prediction  

PubMed Central

Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

Hauke, Jan; Weber, Ute; Engert, Stefanie; Kohler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K.

2012-01-01

179

A Comparison of Full and Split Thickness Skin Grafts in Radial Forearm Donor Sites  

Microsoft Academic Search

To formally evaluate the functional and aesthetic outcomes between full versus split thickness skin graft coverage of radial\\u000a forearm free flap donor sites. A retrospective chart review of 47 patients who underwent pedicled or free radial forearm free\\u000a flap reconstruction from May 1997 to August 2004 was performed. Comparisons were made between patients who had donor site\\u000a coverage with split

Wellington J. Davis; Cindy Wu; David Sieber; Darl K. Vandevender

2011-01-01

180

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

PubMed Central

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

de Witt, Christien A.; Verdonck-de Leeuw, Irma M.; Quak, Jasper J.; Leemans, C. Rene

2007-01-01

181

A controlled subatmospheric pressure dressing increases the rate of skin graft donor site reepithelialization.  

PubMed

The ability to increase the rate of skin graft donor site reepithelialization significantly in a cost-effective manner has important implications for the patient undergoing major reconstructive procedures. In this study the effect of externally applied reduced pressure (the V.A.C.) on the rate of healing of donor site wounds was initially investigated using a porcine model (N = 4), then repeated on humans (N = 10). Split-thickness skin grafts were harvested from the backs of pigs using standard technique. Half of the donor sites were treated with subatmospheric pressure (125 mmHg) and half were treated with an OpSite dressing. Biopsies taken every 48 hours demonstrated that sites exposed to reduced pressure healed at a much faster rate than sites treated with a standard occlusive dressing. Similarly, donor sites in humans reepithelialized faster in 7 of 10 patients, the rate was the same in 2 of 10 patients, and OpSite was faster in 1 of 10 patients. We believe this technology has the potential to be a relatively simple and cost-efficient method for increasing the rate of donor site healing. PMID:9523602

Genecov, D G; Schneider, A M; Morykwas, M J; Parker, D; White, W L; Argenta, L C

1998-03-01

182

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

PubMed Central

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

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

1996-01-01

183

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

PubMed Central

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

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

2012-01-01

184

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

PubMed

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

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

2009-11-01

185

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

PubMed

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

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

2010-06-30

186

Activation of a Cryptic Splice Site of PTEN and Loss of Heterozygosity in Benign Skin Lesions in Cowden Disease  

Microsoft Academic Search

Cowden disease is an autosomal dominant syndrome characterized by facial trichilemmomas, acral keratoses, papillomatous papules, mucosal lesions, and an increased risk for breast and nonmedullary thyroid cancer. Here, we describe a novel PTEN splicing site mutation in a family with classical Cowden disease and we studied benign skin lesions typical for Cowden disease for loss of heterozygosity. We found a

Joerg Trojan; Guido Plotz; Angela Brieger; Jochen Raedle; Stephen J. Meltzer; Manfred Wolter; Stefan Zeuzem

2001-01-01

187

Unusual patterns of exon skipping in Bruton tyrosine kinase are associated with mutations involving the intron 17 3' splice site.  

PubMed

Seven individuals with the diagnosis of X-linked agammaglobulinemia were analyzed for mutations in Bruton tyrosine kinase (Btk) gene at both the cDNA transcript and genomic DNA levels. In addition, maternal carrier status was determined in six of the seven families by examining X chromosome-inactivation patterns for B cells in comparison with other types of blood cells. Three categories of mutations were identified: (1) three patients have missense mutations in either the pleckstrin or SH2 domains of Btk; (2) three patients exhibit mutations at or near intron/exon splice sites, two of which represent inherited mutations within the kinase domain; and (3) one patient has inherited a 2.5-kb deletion with the loss of a DNA segment encoding three exons of the kinase domain. Variation in the lengths of Btk transcripts was evident in two patients with splice-site mutations and in the patient with the DNA deletion. Sequences of the different cDNA transcripts from the patients with 3' splice-site mutations reveal complex patterns of exon skipping involving from one to four exons of the kinase domain. These findings implicate 3' splice sites of the penultimate exon in the recognition or processing of upstream exons. PMID:9106525

Haire, R N; Ohta, Y; Strong, S J; Litman, R T; Liu, Y; Prchal, J T; Cooper, M D; Litman, G W

1997-04-01

188

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

PubMed Central

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

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

2009-01-01

189

Identification of Proteins That Interact with Exon Sequences, Splice Sites, and the Branchpoint Sequence during Each Stage of Spliceosome Assembly  

Microsoft Academic Search

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5*and 3*splice sites, the branchpoint sequence (BPS), or the exons with 32 P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal

MARIA DOLORES CHIARA; MARIA BENNETT; PATRICK CHAMPION-ARNAUD; LEON PALANDJIAN; ANDROBIN REED

1996-01-01

190

Anteromedial thigh perforator-assisted closure of the anterolateral thigh free flap donor site.  

PubMed

Primary closure of the anterolateral thigh free flap donor site is advisable as skin grafting can be associated with higher morbidity. However, this is not possible when anterolateral thigh free flap width is over 8-9 cm with a corresponding flap width-to-thigh circumference ratio over 16%. The authors report their experience and technique with the anteromedial thigh perforator dissection during anterolateral thigh free flap donor-site closure that, on demand, can be used to design a local perforator flap to achieve primary closure of the donor site. Between July and December 2012, 20 consecutive patients underwent elective anterolateral thigh free flap reconstruction for head and neck oncologic surgery. Attempts to close directly the anterolateral thigh free flap donor site failed in two patients with large flaps and V-Y anteromedial thigh perforator flaps were advanced to close the defect. Flaps healed uneventfully. Except two patients, at least one>1-mm perforator was found in all the remaining thighs. Further investigation is needed to establish the maximum anterolateral thigh free flap donor-site width that can be served by this reconstruction. This represents an ideal model for residents to start training on perforator dissection. PMID:23523166

Visconti, Giuseppe; Salgarello, Marzia

2013-03-20

191

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

PubMed Central

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-?2 gene (LAMA2). Laminin-?2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8–15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-?2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-?2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

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

2012-01-01

192

Comparison of Donor-Site Morbidity and Satisfaction between Anterolateral Thigh and Parascapular Free Flaps in the Same Patient.  

PubMed

The purpose of this study was to compare donor-site morbidity after anterolateral thigh (ALT) and parascapular (PS) free flap harvest in the same patient. A total of 13 patients were included in this study. Because of initial flap loss, each patient received ALT as well as PS free flap transplantation. A total of 10 patients were available for follow-up. The average follow-up time was 44.5 months. Besides physical examination, range of motion and scar dimensions were assessed to objectify donor-site deficiencies. The subjective donor-site morbidity was assessed by the patients using a self-report questionnaire. In addition, patients were requested to state their donor-site preference. ALT donor site revealed more sensitivity deficiencies compared with the PS harvest site (8 vs. 4). The latter provoked less functional impairments (1 vs. 2). Scar dimensions were larger at PS harvest site (25.8 × 4.3 cm vs. 23.3 × 3.6 cm). Patients' satisfaction was in favor of the PS donor site (1.9 vs. 2.7). Among the 10 patients, 7 patients preferred the PS and 3 patients preferred the ALT donor site. Comparison of donor-site morbidities in the same patient reveals a valuable tool to diminish individual bias. Despite the low number of cases, we were able to demonstrate the superiority of PS compared with ALT donor sites.The Level of Evidence of the study is III. PMID:23982858

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

2013-08-27

193

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

PubMed Central

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

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

2008-01-01

194

Comparative Clinical Study of Bactigras and Telfa AMD for Skin Graft Donor-Site Dressing  

PubMed Central

The Bactigras® paraffin tulle coated with chlorhexidine is normally used for the treatment of donor-site wounds in burn patients who received split-thickness skin grafts in several centers. It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD®, a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras® or the Telfa AMD® wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (?90%) was significantly shorter in patients treated with the Telfa AMD® compared to the Bactigras® group (14.00 ± 3.05 vs. 9.25 ± 1.88 days for Bactigras® and Telfa AMD® groups, respectively, p < 0.001). The average pain score was also significantly lower in the Telfa AMD® group (1.57 ± 0.55 vs. 4.70 ± 1.16, p < 0.001). There was no difference in the cost of treatment between the groups (4.64 ± 1.97 vs. 5.72 ± 2.54 USD, p = 0.19). This study indicated that the Telfa AMD® was an effective dressing for the treatment of donor-site wounds.

Muangman, Pornprom; Nitimonton, Sooksan; Aramwit, Pornanong

2011-01-01

195

Processing of fish Ig heavy chain transcripts: diverse splicing patterns and unusual nonsense mediated decay.  

PubMed

While the diversification of the antigen-binding sites is realized by genomic VDJ rearrangements during B cell differentiation, different forms of immunoglobulin (Ig) heavy (H) chains can be produced through multiple splicing pathways. In most vertebrates, the secreted (S) and membrane (Mb) forms of IgM chain are created by alternative splicing through usage of a cryptic splice site in C?4 allowing the junction to the TM exon. The processing pattern for Ig? is different in teleosts, which generally use the C?3 donor site instead. In ancient fish lineages, multiple unusual splicing patterns were found for Ig H chain, involving donor sites that do not always follow the classical consensus. The production of IgD versus IgM H chains seems to be generally realized by alternative splicing in all vertebrates, but typical teleost IgD H chains are chimeric and contains a C?1 domain. Together, these observations raise questions on how different fish regulate RNA splicing and if their splicing machinery is especially complex. A preliminary scan of the zebrafish and stickleback genomes provides evidence that gene orthologs to the mammalian main splice factors are highly conserved as single copy genes, while the snRNPs U repertoire may be different and may explain other particular features of RNA processing in fish. PMID:21168434

Quiniou, Sylvie M A; Wilson, Melanie; Boudinot, Pierre

2010-12-17

196

A method for identifying splice sites and translational start sites in eukaryotic mRNA  

Microsoft Academic Search

This paper describes a new method for determining theconsensus sequences that signal the start of translationand the boundaries between exons and introns (donorand acceptor sites) in eukaryotic mRNA. The methodtakes into account the dependencies between adjacentbases, in contrast to the usual technique of consideringeach position independently. When coupled with a dynamicprogram to compute the most likely sequence, newconsensus sequences emerge.

Steven L. Salzberg

1997-01-01

197

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.  

PubMed Central

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Shih, S R; Nemeroff, M E; Krug, R M

1995-01-01

198

Tn7 Transposition Creates a Hotspot for Homologous Recombination at the Transposon Donor Site  

PubMed Central

Homologous recombination at the bacterial transposon Tn7 donor site is stimulated 10-fold when Tn7 is activated to transpose at high frequency in RecD(-) Escherichia coli, where recombination is focused near the ends of double-chain breaks. This is observed as an increase in recombination between two lacZ heteroalleles when one copy of lacZ carries within it a Tn7 that is transposing at high frequency. This stimulation of recombination is dependent upon the presence of homology with the donor site, is independent of SOS induction, and is not due to a global stimulation of recombination. When stimulated by Tn7 transposition, the conversion events giving rise to Lac(+) recombinants occur preferentially at the site of Tn7, suggesting that transposition is stimulating gene conversion at the donor site. These results support the model that Tn7 transposition occurs by a ``cut and paste'' mechanism, leaving a double-chain break at the donor site that is repaired by the host homologous recombination machinery; normally, repair would use homology in a sister chromosome to regenerate a copy of the transposon. This proposed series of events allows transposition that is nonreplicative, per se, to be effectively replicative.

Hagemann, A. T.; Craig, N. L.

1993-01-01

199

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

PubMed Central

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

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

1992-01-01

200

XLA Patients with BTK Splice-Site Mutations Produce Low Levels of Wild-Type BTK Transcripts  

Microsoft Academic Search

X-linked agammaglobulinemia is caused by mutations in the BTK gene, which result in a precursor B-cell differentiation arrest in the bone marrow and the absence of or strongly reduced B lymphocytes in blood. We identified a patient with a mild clinical phenotype, low numbers of B lymphocytes, and a splice-site mutation in the BTK gene. The precursor B-cell compartment in

Jeroen G. Noordzij; Sandra de Bruin-Versteeg; Nico G. Hartwig; Corry M. R. Weemaes; Egbert J. A. Gerritsen; Eva Bernatowska; Stefaan van Lierde; Ronald de Groot; Jacques J. M. van Dongen

2002-01-01

201

Activation of a cryptic splice site in the tax gene of HTLV-I by a single nucleotide change  

Microsoft Academic Search

We identified a T-to-C mutation 2 nucleotides (nt) upstream from the AG in a GT-AG intron between exons 2 and 3 in the human T-cell leukemia virus type I (HTLV-I) tax mRNA. This mutation resulted in the preferential usage of an alternative splice site, causing a 75-nt elongation of tax mRNA and reduced production of viral antigens. When the clone

Takeo Ohsugi

2006-01-01

202

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

PubMed

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

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

2013-03-08

203

Reduction of donor site morbidity of free radial forearm flaps: what level of evidence is available?  

PubMed

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

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

2012-02-03

204

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

PubMed

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

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

2013-09-09

205

Three-Dimensional Visualization of Transcription Sites and Their Association with Splicing Factor-Rich Nuclear Speckles  

PubMed Central

Transcription sites are detected by labeling nascent transcripts with BrUTP in permeabilized 3T3 mouse fibroblasts followed by laser scanning confocal microscopy. Inhibition and enzyme digestion studies confirm that the labeled sites are from RNA transcripts and that RNA polymerase I (RP I) and II (RP II) are responsible for nucleolar and extranucleolar transcription, respectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and three-dimensional networklike arrays. The number of transcription sites, their three-dimensional organization and arrangement into functional zones (Wei et al. 1998) is strikingly maintained after extraction for nuclear matrix. Significant levels of total RP II mediated transcription sites (45%) were associated with splicing factor–rich nuclear speckles even though the speckles occupied <10% of the total extranucleolar space. Moreover, the vast majority of nuclear speckles (>90%) had moderate to high levels of associated transcription activity. Transcription sites were found along the periphery as well as inside the speckles themselves. These spatial relations were confirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speckles and their surrounding regions are major sites of RP II-mediated transcription in the cell nucleus, and support the view that both speckle- and nonspeckle-associated regions of the nucleus contain sites for the coordination of transcription and splicing processes.

Wei, Xiangyun; Somanathan, Suryanarayan; Samarabandu, Jagath; Berezney, Ronald

1999-01-01

206

The Pivotal Roles of TIA Proteins in 5? Splice-Site Selection of Alu Exons and Across Evolution  

PubMed Central

More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5? splice site (5?ss), even in the presence of a stronger 5?ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5?ss. This enhancing effect depended on the strength of the downstream 5?ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5?ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within ?20 nt downstream of the 5?ss. For most metazoans, the strength of the 5?ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

Ram, Oren; Eyras, Eduardo; Ast, Gil

2009-01-01

207

Regulation of Alternative Splicing by Histone Modifications  

Microsoft Academic Search

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing

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

2010-01-01

208

Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection.  

PubMed

TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 3' untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA(1). This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA(1) by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 3' end processing to effect autoregulation of TDP-43. PMID:22855830

Avendaño-Vázquez, S Eréndira; Dhir, Ashish; Bembich, Sara; Buratti, Emanuele; Proudfoot, Nicholas; Baralle, Francisco E

2012-08-01

209

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

PubMed

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

Catania, Francesco; Lynch, Michael

2013-04-09

210

Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA.  

PubMed

Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo. Here we describe a strategy to map accessible sites on sickle beta-globin (beta(s)-globin) transcripts in vitro and in vivo and to use this information to enhance RNA repair efficiency. Two sites upstream of the sickle mutation were identified as accessible in some fraction of the beta-globin RNA by mapping with a ribozyme library and the accessibility of those sites was assessed by in vitro cleavage analyses. Ribozymes targeting either site could only convert a certain fraction of the beta(s)-globin RNA to product but not drive the reaction to completion. However, cleavage and splicing reactions were driven further toward completion when the two ribozymes were both added to the reactions, suggesting that the substrate RNA is present in multiple conformations in vitro. These two ribozymes were each able to repair beta(s)-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Moreover, the relative accessibility of the targeted sites in vivo is as predicted by mapping and in vitro analyses. These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA. PMID:10985955

Lan, N; Rooney, B L; Lee, S W; Howrey, R P; Smith, C A; Sullenger, B A

2000-09-01

211

Lysyl oxidase-like 4 is alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities.  

PubMed

Lysyl oxidase-like enzymes (LOXL) are expressed in various cancers. We analyzed the expression of LOXL2, LOXL3, and LOXL4 in cancers involving the serosal cavities-breast carcinoma, ovarian carcinoma, and malignant mesothelioma using reverse-transcriptase polymerase chain reaction. We discovered two new alternative splice variants of LOXL4. The spliced segments were exon 9 (splice variant 1) or both exons 8 and 9 (splice variant 2). In ovarian carcinoma, splice variant 1 was significantly elevated in effusions compared to solid lesions (p < 0.001). Splice variant 2 appeared only in effusions. In breast carcinoma, LOXL4 was expressed only in the effusion samples. In malignant mesothelioma, LOXL4 and its splice variants were expressed at all sites. Breast carcinoma effusions showed significantly higher LOXL2 (p = 0.003) and lower LOXL3 (p < 0.001) expression compared to primary carcinomas. Our data show differences in LOXL messenger RNA expression as a function of anatomic site and tumor type in cancers affecting the serosal cavities. PMID:19015874

Sebban, Shulamit; Davidson, Ben; Reich, Reuven

2008-11-18

212

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

PubMed Central

Background Donor site wounds after split-skin grafting are rather 'standard' wounds. At present, lots of dressings and topical agents for donor site wounds are commercially available. This causes large variation in the local care of these wounds, while the optimum 'standard' dressing for local wound care is unclear. This protocol describes a trial in which we investigate the effectiveness of various treatment options for these donor site wounds. Methods A 14-center, six-armed randomized clinical trial is being carried out in the Netherlands. An a-priori power analysis and an anticipated dropout rate of 15% indicates that 50 patients per group are necessary, totaling 300 patients, to be able to detect a 25% quicker mean time to complete wound healing. Randomization has been computerized to ensure allocation concealment. Adult patients who need a split-skin grafting operation for any reason, leaving a donor site wound of at least 10 cm2 are included and receive one of the following dressings: hydrocolloid, alginate, film, hydrofiber, silicone dressing, or paraffin gauze. No combinations of products from other intervention groups in this trial are allowed. Optimum application and changes of these dressings are pursued according to the protocol as supplied by the dressing manufacturers. Primary outcomes are days to complete wound healing and pain (using a Visual Analogue Scale). Secondary outcomes are adverse effects, scarring, patient satisfaction, and costs. Outcome assessors unaware of the treatment allocation will assess whether or not an outcome has occurred. Results will be analyzed according to the intention to treat principle. The first patient was randomized October 1, 2009. Discussion This study will provide comprehensive data on the effectiveness of different treatment options for donor site wounds. The dressing(s) that will prevail in effectiveness, satisfaction and costs will be promoted among clinicians dealing with such patients. Thus, we aim to contribute a well-designed trial, relevant to all clinicians involved in the care for donor site wounds, which will help enhance uniformity and quality of care for these patients. Trial registration http://www.trialregister.nl, NTR1849. Date registered: June 9, 2009

2011-01-01

213

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing  

PubMed Central

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site.

Penn, Andrew C.; Balik, Ales; Greger, Ingo H.

2013-01-01

214

A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa.  

PubMed

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

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

2010-05-06

215

Spliced leader RNA trans-splicing in dinoflagellates.  

PubMed

Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), to the 5' end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 50-60 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter. The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF 512889, DQ 864761-DQ 864971, DQ 867053-DQ 867070, DQ 884413-DQ 884451, EF 133854-EF 133905, EF 133961-EF 134003, EF 134083-EF 134402, EF 141835, and EF 143070-EF 143105). PMID:17360573

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

2007-03-02

216

Spliced leader RNA trans-splicing in dinoflagellates  

PubMed Central

Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), to the 5? end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 50–60 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter.

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

2007-01-01

217

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

Microsoft Academic Search

Mutations in the gene encoding the enzyme iduronate-2-sulfatase (IDS) were reported as the cause of the X-linked recessive lysosomal disease, mucopolysaccharidosis II (MPS II). Amongst the different mutations, it emerges that nearly 10% are nucleotide substitutions causing splicing mutations. We now report the molecular characterisation of three MPS II patients with multiple aberrant transcripts due to three different point mutations.

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

2006-01-01

218

Exon 10 skipping caused by intron 10 splice donor site mutation in cholesteryl ester transfer protein gene results in abnormal downstream splice site selection  

Microsoft Academic Search

Cholesteryl ester transfer protein (CETP) defi- ciency is the most common cause of hyperalphalipoprote- inemia in Japan. However, the genetic basis of this disorder has not been fully characterized. We have studied a 49-year- old Japanese male presenting with total cholesterol, HDL- cholesterol, and apolipoprotein A-I levels of 300, 236, and 233 mg\\/dl, respectively, and total absence of CETP activity

Naohiko Sakai; Silvia Santamarina-Fojo; Shizuya Yamashita; Yuji Matsuzawa

219

An alternative coverage for split thickness skin graft donor site wounds.  

PubMed

Diabetic foot and ankle soft tissue reconstruction poses a difficult challenge to the treating surgeon, especially in cases associated with previous infection or amputation. Maintenance of a functional, plantigrade limb is important with regard to prevention of persistent or recurrent cutaneous compromise following diabetic limb salvage. Wound coverage by means of application of a split thickness skin graft (STSG) is a useful technique; however, donor site wounds require care during the early postoperative period, and can pose a challenge to wound healing in and of themselves. In this article, we describe a technique of management of STSG donor site wounds that we have found to be useful and well tolerated by our patients. PMID:21420327

Sagray, Bryan A; Lalani, Samir; Mehan, Vineet

2011-03-21

220

Cryptic splice site activation during RNA processing of MLL\\/ AF4 chimeric transcripts in infants with t(4;11) positive ALL  

Microsoft Academic Search

Co-expression of multiple variants of the MLL\\/AF4 fusion transcript is a common phenomenon in patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). Different transcriptional and post-transcriptional mechanisms were found to contribute to the heterogeneity of the chimeric transcripts. Multiple splice variants are generated by utilizing alternative splice sites that result in the joining of different MLL-exons within the breakpoint cluster

Vladimir Divoky; Jan M. Trka; Franz Watzinger; Thomas Lion

2000-01-01

221

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression  

PubMed Central

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5? splice sites (5? ss) and three 3? splice sites (3? ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5? ss (nt 221 5? ss and nt 191 5? ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5? ss and nt 409 3? ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3? ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3? ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

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

2012-01-01

222

A Dual Reporter Splicing Assay Using HaloTag-containing Proteins  

PubMed Central

To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag–luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5’-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.

Oshima, Koichi; Nagase, Takahiro; Imai, Kohsuke; Nonoyama, Shigeaki; Obara, Megumi; Mizukami, Tomoyuki; Nunoi, Hiroyuki; Kanegane, Hirokazu; Kuribayashi, Futoshi; Amemiya, Shin; Ohara, Osamu

2012-01-01

223

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

Microsoft Academic Search

The objective of this study was the evaluation of donor site morbidity in head and neck cancer patients after reconstruction\\u000a using a free vascularized radial forearm flap with emphasis on subjective complaints. Fifty patients who underwent at least\\u000a 6 months before a reconstruction using a free vascularized radial forearm flap were asked to fill out two questionnaires regarding\\u000a cosmetics and sensibility

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

2007-01-01

224

Solid phase electron donors control denitrification in groundwater at agricultural sites  

NASA Astrophysics Data System (ADS)

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

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

2011-12-01

225

Evidence for a direct role of the disease modifier SCNM1 in splicing.  

PubMed

We originally isolated Scnm1 as a disease modifier gene that is required for efficient in vivo splicing of a mutant splice donor site in the sodium channel Scn8a. It was previously unclear whether the modifier effect on splicing was direct or indirect. We now report evidence that sodium channel modifier 1 (SCNM1) has a direct role in splicing. SCNM1 protein interacts with the spliceosome protein U1-70K in the yeast two-hybrid system, and is co-localized with U1-70K in nuclear speckles in mammalian cells. SCNM1 is also co-immunoprecipitated with the spliceosomal core Smith (Sm) proteins and demonstrates functional activity in a minigene splicing assay. In a yeast two-hybrid screen, SCNM1 interacted with LUC7L2, a mammalian homolog of a yeast protein involved in recognition of non-consensus splice donor sites. This interaction requires the acidic C-terminal domain of SCNM1 which is truncated by the disease susceptibility variant Scnm1(R187X) in mouse strain C57BL/6J. Luc7L2 transcripts are widely distributed in mammalian tissues, and undergo alternative splicing and polyadenylation. LUC7L2 is also co-localized with U1-70K and may function with SCNM1 in recognition of weak splice donor sites. In summary, Scnm1 is the first example of a modifier gene which influences disease severity through a trans-effect on splicing of the disease gene transcript. PMID:17656373

Howell, Viive M; Jones, Julie M; Bergren, Sarah K; Li, Li; Billi, Allison C; Avenarius, Matthew R; Meisler, Miriam H

2007-07-26

226

Directing alternative splicing: cast and scenarios  

Microsoft Academic Search

Recent progress in the study of alternative RNA splicing indicates that the interaction of RNA-binding proteins with specific target elements modulates splice site recognition and spliceosome assembly. The identity of splicing signals, the presence of modulating elements and differences in the distribution of RNA-binding proteins are key determinants involved in the tissue-specific regulation of splice site selection.

Benoit Chabot

1996-01-01

227

Iatrogenic implantation of giant cell tumor at bone graft donor site and clinical recommendations to prevent "a rare avoidable complication".  

PubMed

The treatment of giant cell tumor of bone is directed toward local control without sacrificing joint function. This is achieved by intralesional curettage. When autograft is used for the reconstruction of the curetted cavity, there is always a theoretical risk of contamination of graft donor site. We report a case of iatrogenic implantation of giant cell tumor at the bone graft donor site after intralesional curettage and bone grafting of giant cell tumor of distal femur. Patient was treated with repeat intralesional curettage and excision of implantation lesion at bone graft donor site. We recommend precautionary measures to prevent this avoidable complication. PMID:23412188

Gulia, Ashish; Puri, Ajay; Salunke, Abhijeet; Desai, Subhash; Jambhekar, N A

2012-08-08

228

[Donor site morbidity after free gracilis muscle flap. Report of 32 cases.  

PubMed

BACKGROUND: Coverage of tissue defects of the lower limbs is a complex problem. Free gracilis muscle flap is a reliable surgical technique and the morbidity of its donor site is considered as minimal. Our retrospective study involved all patients who underwent a free gracilis muscle flap in a reconstructive surgery of the lower limb. To the best of our knowledge, this is the first study to assess comprehensively the aesthetic and functional morbidity of the free gracilis flap donor site. PATIENTS AND METHODS: Thirty-two patients underwent a gracilis muscle free flap in our plastic surgery department, between January 2009 and April 2012, as part of a reconstructive surgery of the lower limb. All medical datas were carried out using computerized records. Aesthetic and functional assessments of the donor site were done by the patient using questionnaires and by a plastic surgeon and a physiotherapist using a clinical evaluation, 6months after surgery. A comparative study between both limbs including the thigh perimeter analysis, an isokinetic study of the knee, a study of the range of motion of hip and knee, and an assessment of the strength of adduction of the hip were conducted. RESULTS: Concerning the aesthetic outcomes, the clinical and subjective scores were satisfactory with a Vancouver score under 1. Five patients had a decrease in the volume of the thigh after surgery. Concerning the functional outcomes, no motor or sensory defects were reported. No statistically significant difference was demonstrated for the range of motion of the hip and knee between both limbs. The strength of hip adduction was not altered by the removal of the gracilis muscle. CONCLUSION: This study confirms the low aesthetic and functional donor site morbidity of the free gracilis muscle flap. The aftermath of the donor area of the flap are very well accepted by patients, which is a sign of good acceptance of the whole reconstruction. Because of these findings and the suitability of the flap at the recipient site, the gracilis muscle free flap should be part of the armamentarium of any reconstructive surgeon. PMID:23707083

Besset, M; Penaud, A; Quignon, R; Bahe, L; Brilhault, J; Fouquet, B

2013-05-23

229

Novel mutations in 21 patients with tuberous sclerosis complex and variation of tandem splice-acceptor sites in TSC1 exon 14.  

PubMed

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by epilepsy, mental retardation, skin lesions, and tumors in various organs. However, TSC is sometimes difficult to diagnose because of its broad phenotypic spectrum. In such cases, it is essential to find a mutation in the disease-causing genes, TSC1 and TSC2. In this study, we analyzed 21 TSC patients from 16 families using a combination method of DHPLC and nucleotide sequencing. We identified 16 novel mutations in the 16 families: nine mutations in TSC1 (1 insertion, 7 deletion and 1 nonsense mutations) and seven mutations in TSC2 (2 insertion, 2 deletion, 1 missense mutations and 2 splicing abnormalities). We also tested the possibility of very short alternative splicing due to a variation of the tandem splice-acceptor sites of TSC1 exon 14 in a patient. RT-PCR and sequencing analysis indicated that no alternative splicing occurred in the patient. In conclusion, we confirmed the diagnosis of all patients using mutation analysis and clarified that variation of the tandem splice-acceptor sites in TSC1 exon 14 does not cause a splicing abnormality. PMID:18772611

Sasongko, Teguh Haryo; Wataya-Kaneda, Mari; Koterazawa, Keiko; Gunadi; Yusoff, Surini; Harahap, Indra Sari Kusuma; Lee, Myeong Jin; Matsuo, Masafumi; Nishio, Hisahide

2008-05-23

230

Secondary alveolar bone grafting: the dilemma of donor site selection and morbidity.  

PubMed

Fresh autogenous cancellous bone is ideal for secondary alveolar cleft bone grafting because it supplies living, immunocompatible bony cells that integrate fully with the maxilla and are essential for osteogenesis. Recent animal studies have shown that the dynamics of cancellous inlay bone grafts are different from those of cortical onlay bone grafts, and they refute the assumption that membranous bone grafts are superior to endochondral bone grafts because of their embryological origin. These studies prove that inlay endochondral cancellous specimens have a higher percentage increase in actual bony volume than cortical membranous and cortical endochondral inlay bone grafts. There are various donor sites for secondary alveolar cleft bone grafts. Currently the main sites for autogenous cancellous bone are iliac crest, calvarium, mandibular symphysis, and tibia. Some authors have suggested that the iliac crest donor site causes an unacceptably high degree of postoperative morbidity, but it is still the first choice for secondary alveolar cleft bone grafts and should not be rejected solely because of such concerns. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is now an attractive bony substitute that promotes the differentiation of pluripotential cells into bone-forming cells that lay down new host bone in the site of the defect. Much more research and development are necessary to find a suitable carrier for rhBMP-2, and to study the properties of newly formed bone that it has induced before it can be a substitute for autogenous bone. PMID:18760515

Rawashdeh, Ma'amon A; Telfah, Hani

2008-08-29

231

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

SciTech Connect

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

Brown, W.R.; Kacskovics, I.; Amendt, B.A. [Univ. of Iowa, Iowa City, IA (United States)] [and others

1995-04-15

232

Functional Recognition of the 5? Splice Site by U4\\/U6.U5 tri-snRNP Defines a Novel ATP-Dependent Step in Early Spliceosome Assembly  

Microsoft Academic Search

A sensitive assay based on competition between cis- and trans- splicing suggested that factors in addition to U1 snRNP were important for early 5? splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4\\/U6.U5 tri-snRNP and the 5? splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point.

Patricia A. Maroney; Charles M. Romfo; Timothy W. Nilsen

2000-01-01

233

Analysis of LDLR mRNA in patients with familial hypercholesterolemia revealed a novel mutation in intron 14, which activates a cryptic splice site.  

PubMed

Familial hypercholesterolemia (FH) is caused by a defective low-density lipoprotein receptor (LDLR), and >1000 mutations in LDLR have been identified. However, in some patients with clinically defined FH, no mutation can be detected within the exons and adjacent intronic segments of the LDLR. We have analyzed RNA extracted from blood samples of patients with clinically defined FH and identified an aberrantly spliced mRNA containing an 81-bp insert from intron 14. The aberrant splicing was caused by a novel intronic mutation, c.2140+86C>G, which activated a cryptic splice site. Although the cryptic splice site does not completely surpass the normal splice site, the mutation was found to cosegregate with high cholesterol levels in a family, which supports the notion that c.2140+86C>G causes FH. The insertion of 81? bp in LDLR mRNA encodes an in-frame insertion of 27 amino acids in the LDLR. However, the insertion was found to hamper LDLR activity by preventing the receptor from leaving the endoplasmic reticulum, probably because of misfolding of the protein. In patients with clinically defined hypercholesterolemia, despite normal results from sequencing of exonic regions of the LDLR gene, characterization of the LDLR mRNA might identify the underlying genetic defect. PMID:20703241

Kulseth, Mari Ann; Berge, Knut Erik; Bogsrud, Martin Prøven; Leren, Trond P

2010-08-12

234

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

PubMed

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

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

2013-06-05

235

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

PubMed Central

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

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

2013-01-01

236

Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles.  

PubMed

In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations. PMID:22607811

Vigentini, Ileana; De Lorenzis, Gabriella; Picozzi, Claudia; Imazio, Serena; Merico, Annamaria; Galafassi, Silvia; Piškur, Jure; Foschino, Roberto

2012-03-03

237

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

SciTech Connect

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.

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

2008-11-07

238

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

Microsoft Academic Search

Introns are among the hallmarks of eukaryotic genes. Splicing of introns is directed by three main splicing signals: the 5 splice site (5ss), the branch site (BS), and the polypyrimdine tract\\/3splice site (PPT-3ss). To study the evolution of these splicing signals, we have conducted a systematic comparative analysis of these signals in over 1.2 million introns from 22 eukaryotes. Our

Schraga H. Schwartz; João Silva; David Burstein; Tal Pupko; Eduardo Eyras; Gil Ast

2007-01-01

239

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

PubMed

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

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

2012-11-08

240

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

SciTech Connect

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

Schiavi, A.; Bartlett, R. [Univ. of Miami, FL (United States); Brown, M. [Emory Univ., Atlanta, GA (United States)] [and others

1994-09-01

241

A novel 3' splice-site mutation and a novel gross deletion in leukocyte adhesion deficiency (LAD)-1.  

PubMed

A patient was diagnosed with leukocyte adhesion deficiency-1. She was born in 1996 and her parents are not known to be related. Her leukocytes expressed less than 2% of the CD18 antigens relative to normal individuals. Molecular analysis revealed that she is a compound heterozygote. She inherited a 27,703bp deletion from her father (g.43201_PTTG1IP:10890del27703), spanning from intron 11 of the gene for the ?2 integrin (ITGB2, CD18, NG_007270.2) to intron 2 of the gene for the Pituitary Tumor-Transforming Gene 1 Interacting Protein (PTTG1IP, NC_000021.8). The maternal allele has a g.23457C>A mutation at position -10 in intron 2 of the ITGB2 gene, resulting in the activation of a cryptic 3' splice site in intron 2 to include 43 intronic nucleotides (r.[59-43_59-1ins;59-10C>A]). PMID:21195692

Bernard Cher, T H; Chan, Hwee Sing; Klein, Georg F; Jabkowski, Jörg; Schadenböck-Kranzl, Gabriela; Zach, Otto; Roca, Xavier; Law, S K Alex

2010-12-31

242

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

PubMed Central

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

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

1989-01-01

243

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

PubMed Central

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

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

1999-01-01

244

A glucose-6-phosphate dehydrogenase (G6PD) splice site consensus sequence mutation associated with G6PD enzyme deficiency.  

PubMed

A glucose-6-phosphate dehydrogenase (G6PD) deficient strain of mouse (GPDX) which was developed using the ethylating agent ethylnitrosourea (ENU) has been used to study clonality in epithelial tissues. While the biochemical defect has been quantified, the genetic basis of the deficiency is unknown. The G6PD gene is composed of 13 exons. Exon 1 is not translated, and the ATG start site is near the 5' end of exon 2. Direct sequencing of the exonic regions of the gene from GPDX, C3H, 101, C57BL/6 and BALB/c mice was carried out. The coding region, in which (with a single exception) all mutations found to cause G6PD deficiency in man are situated, showed identical sequences in three of the four strains studied (101 coding region sequence was not examined). However, the G6PD gene in the GPDX mouse showed a single base difference from the other four strains and from the published mouse G6PD sequence (BALB/c) in the 5' splice site consensus sequence at the 3' end of exon 1, part of the untranslated region. The difference was confirmed in four different GPDX mice. This mutation was of the type (A to T transversion) that is known to be induced by ENU; its effect is likely to be exerted through a defect in transcription, splicing or translation, leading to a reduction in protein levels. By Western blot we have found a marked decrease in the G6PD protein levels in the GPDX mouse, with the C3H X GPDX heterozygote showing a lesser decrease. Recently, an increasing number of mutations in the untranslated regions of genes have been found which have effects on protein levels. We believe that the reduced enzyme activity in the GPDX mouse is due to the mutation in the 5' untranslated region (UTR), and that similar mutations may be relevant in other inherited conditions. PMID:9067418

Sanders, S; Smith, D P; Thomas, G A; Williams, E D

1997-03-01

245

A Girl with a Novel Splice Site Mutation in VDR Supports the Role of a Ligand-Independent VDR Function on Hair Cycling  

Microsoft Academic Search

Mutations in vitamin D receptor (VDR) cause hereditary vitamin D resistant rickets (HVDRR). We reported a Thai girl with HVDRR, presenting with an early onset of rickets and partial alopecia. She was a product of a consanguineous couple. Mutation analysis showed that she was homozygous for a novel splice site mutation of the VDR gene, 462 + 1 G ?

Paravee Katavetin; Pisut Katavetin; Suttipong Wacharasindhu; Vorasuk Shotelersuk

2006-01-01

246

The Pathobiology of Splicing  

PubMed Central

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

Ward, Amanda J.; Cooper, Thomas A.

2010-01-01

247

Activation of cryptic splice sites is a frequent splicing defect mechanism caused by mutations in exon and intron sequences of the OPA1 gene  

Microsoft Academic Search

Mutations in OPA1 are the most frequent cause underlying autosomal dominant optic atrophy (adOA). Until now only few putative splicing mutations\\u000a in the OPA1 gene have been investigated at the mRNA level and all these result in exon skipping. Here, we report the identification and\\u000a cDNA analysis of four intronic and three exonic OPA1 gene mutations that cause a variety

Simone Schimpf; Simone Schaich; Bernd Wissinger

2006-01-01

248

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

PubMed Central

Background Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC?+?K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. Results Healing time was reduced from 13.9?±?0.5 days (mean?±?SEM) in the control group to 7.2?±?0.2 days in the PC group (P?

2013-01-01

249

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

PubMed Central

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

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

2012-01-01

250

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.  

PubMed Central

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.

Reyes, J L; Kois, P; Konforti, B B; Konarska, M M

1996-01-01

251

Regulation of Alternative Splicing by Histone Modifications  

PubMed Central

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

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

2010-01-01

252

Regulation of alternative splicing by histone modifications.  

PubMed

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

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

2010-02-04

253

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

SciTech Connect

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.

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.; (Sydney)

2009-09-02

254

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

Microsoft Academic Search

Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder

Francois-Olivier Desmet; Dalil Hamroun; Marine Lalande; Gwenaelle Collod-Beroud; Mireille Claustres; Christophe Beroud

2009-01-01

255

A Spontaneous Fatp4/Scl27a4 Splice Site Mutation in a New Murine Model for Congenital Ichthyosis  

PubMed Central

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3?-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.

Tao, Jianning; Koster, Maranke I.; Harrison, Wilbur; Moran, Jennifer L.; Beier, David R.; Roop, Dennis R.; Overbeek, Paul A.

2012-01-01

256

Xanthogranulomatous inflammation involving latissimus dorsi donor site and implant breast reconstruction: case report and literature review  

PubMed Central

Xanthogranulomatous inflammation is a rare clinico-pathological condition involving many organ systems. Breast involvement with this rare condition reported from a few cases of mastitis has been limited to only microscopic involvement on histology. We would like to report an unusual presentation of this inflammatory process presenting as a solid lump mimicking malignancy in latissimus dorsi donor site scar and implant-based breast reconstruction as a result of a ruptured silicone gel implant. To our knowledge there have been no previous reports on similar presentation published in the literature. This case highlights a rare complication of a leaked silicone gel implant triggering a xanthomatous response in the absence of the usual infective or obstructing etiologies. This condition is of benign nature with complete clearance on surgical excision and excellent clinical prognosis reported from other organ involvement.

2012-01-01

257

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

PubMed Central

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

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

2012-01-01

258

The U11-48K Protein Contacts the 5' Splice Site of U12Type Introns and the U11-59K Protein  

Microsoft Academic Search

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5 splice sites (5ss). This interaction did not require branch point recognition and was sensitive to 5ss mutations, suggesting that 48K interacts with the 5ss

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

2008-01-01

259

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing*  

PubMed Central

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5? splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Guan, Wuxiang; Huang, Qinfeng; Cheng, Fang; Qiu, Jianming

2011-01-01

260

Splice-mediated insertion of an Alu sequence inactivates ornithine. delta. -aminotransferase: A role for Alu elements in human mutation  

SciTech Connect

In studies of mutations causing deficiency of ornithine {delta}-aminotransferase the authors found an allele whose mature mRNA has a 142-nucleotide insertion at the junction of sequences from exons 3 and 4. The insert derives from an Alu element in ornithine {delta}-aminotransferase intron 3 oriented in the direction opposite to transcription (an antisense Alu). A guanine {r arrow} cytosine transversion creates a donor splice site in this Alu, activating a cryptic acceptor splice site at its 5{prime} end and causing splice-mediated insertion of an Alu fragment into the mature ornithine-{delta}-aminotransferase mRNA. The authors note that the complement of the Alu consensus sequence has at least two cryptic acceptor sites and several potential donor sequences and predict that similar mutations will be found in other genes.

Mitchell, G.A.; Labuda, D.; Fontaine, G. (Hopital Ste-Justine, Montreal, Ontario (Canada)); Saudubray, J.M.; Bonnefont, J.P.; Lyonnet, S. (Inst. National de la Sante et de la Recherche Medicale, Paris (France)); Brody, L.C.; Steel, G.; Obie, C.; Valle, D. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States))

1991-02-01

261

Splice site selection in the proteolipid protein (PLP) gene transcript and primary structure of the DM-20 protein of central nervous system myelin.  

PubMed Central

Proteolipid protein (PLP) is the major myelin membrane protein of the central nervous system. We have isolated a copy of an alternatively spliced PLP gene transcript from a mouse brain cDNA library that was screened for PLP-related sequences. The encoded 241-amino acid protein differs from PLP by an internal deletion of 35-amino acid residues (116-150) from the major hydrophilic domain. This PLP variant is identical with the DM-20 protein of myelin, previously described as a brain-specific myelin component and known to be related to PLP. We determined the corresponding nucleotide sequence of the rat PLP gene and found that DM-20 mRNA results when a second 5' splice site, located 105 nucleotides within the third exon of the primary PLP transcript, is utilized in precursor mRNA (pre-mRNA) splicing. This demonstrates that alternative 5' splice site selection can determine the protein product of a cellular gene. DM-20 mRNA is expressed in rat brain with approximately 50% abundance relative to PLP mRNA and appears to be developmentally coregulated. Images

Nave, K A; Lai, C; Bloom, F E; Milner, R J

1987-01-01

262

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

PubMed

In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed sequence tag database represents sequences derived from rotifers. In summary, the description of SL-mediated trans-splicing in Rotifera extends its representation to at least five metazoan phyla, making it increasingly probable that this is a phylogenetically widespread and therefore ancient phenomenon. PMID:15788744

Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

2005-03-23

263

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

SciTech Connect

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.

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

1988-04-01

264

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

Microsoft Academic Search

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

C. Wadelius; A. Lagerkvist; A. K. Molin; A. Larsson; U. Von Doebeln

1993-01-01

265

[Baropodometric analysis of the functional donor-site morbidity after gastrocnemius or soleus muscle-flap procedure].  

PubMed

The triceps surae muscle is a major donor-site for muscle-flap to cover soft-tissue defects of the leg. There are very limited datas on the functional donor-site morbidity in the literature. From a retrospective study on 14 patients, we realized a baropodometric analysis comparing the operated lower limb with the healthy non operated side and a functional evaluation by a questionary. The modified functional score of Kitatoka was good (87/100). Ninety percent of the patients were able to resume a professional activity and 2/3 to resume the sport. The baropodometric analysis did not show statistically significant difference of propulsion and absorption between the healthy side and the operated side, but a modification of the programming of the step. The absence of important functional donor-site morbidity is probably bound to a compensation of the remaining triceps surae muscles and/or to mechanisms of adaptation. Our study confirms the little functional donor-site morbidity of the partial triceps surae muscle-flap procedure. These flaps remain a good solution for the coverage of the soft-tissue defects of the leg. PMID:21440973

Lasserre, G; Cornu, J-Y; Vidal, C; Laveaux, C; Lepage, D; Obert, L; Pauchot, J; Tropet, Y

2011-03-26

266

Comparison of the ionic silver-containing hydrofiber and paraffin gauze dressing on split-thickness skin graft donor sites.  

PubMed

The split-thickness skin graft (STSG) donor site dressing has been an inconclusive topic. Each of the Hydrofiber (Aquacel, ConvaTec A Bristol-Myers Squibb Company, Deeside, UK) and silver dressings have applied in many types of wound care with favorable outcomes. Our study compared the ionic silver-containing Hydrofiber dressing and paraffin gauze dressing. The subjects were randomized into group A: ionic silver-containing Hydrofiber and group B: paraffin gauze. From February 2006 to 2007, 20 donor sites were recorded. The mean donor site surface area was 145.5 cm2 (group A) and 135.8 cm2 (group B). The completed re-epithelization day was 7.90 and 11.20 days, respectively (P = 0.031). The average pain score at rest were 0.74 and 0.80, respectively (P = 0.894). The average pain score on dressing removal were 3.12 and 4.70, respectively (P = 0.027). There was no infection or seroma in both groups. In conclusion, ionic silver-containing Hydrofiber dressing can reduce STSG donor site pain and promote re-epithelization compared to paraffin gauze dressing. PMID:19325350

Lohsiriwat, Visnu; Chuangsuwanich, Apirag

2009-04-01

267

A novel T->G splice site mutation of CRYBA1/A3 associated with autosomal dominant nuclear cataracts in a Chinese family  

PubMed Central

Purpose The purpose of this study was to identify the disease-causing mutation and the molecular phenotype that are responsible for the presence of an autosomal dominant congenital nuclear cataract disease in a Chinese family. Methods The family history and clinical data were recorded. The patients were given a physical examination and their blood samples were collected for DNA extraction. Direct sequencing was used to detect the mutation. Transcription analysis of the mutant crystallin, beta A1 (CRYBA1/A3) gene was performed to verify whether the defective mutation had influenced the splice of the mature mRNA. Results The phenotype of the congenital cataract in the family was identified as a nuclear cataract type, by using slit-lamp photography. Direct sequencing revealed a novel mutation IVS3+2 T?G in CRYBA1/A3. This mutation co-segregated with all affected individuals in the family, but was not found in unaffected family members nor in the 100 unrelated controls. Transcription analysis of the mutant CRYBA1/A3 gene indicated that this mutation had influenced the splice of the mature mRNA. Conclusions Our study identified a novel splice site mutation in CRYBA1/A3. This mutation was responsible for aberrant splicing of the mature mRNA and had caused the congenital nuclear cataracts in the family. This is the first report relating an IVS3+2 T?G mutation of CRYBA1/A3 to congenital cataracts.

Yang, Zhenfei; Su, Dongmei; Li, Qian; Yang, Fan; Ma, Zicheng; Ma, Xu

2012-01-01

268

Molecular and cellular characterization of a new ?-thalassemia mutation (HBA2:c.94A>C) generating an alternative splice site and a premature stop codon.  

PubMed

The identification of ?-thalassemia (?-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the ?2-globin gene, causing a mild ?-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of ?-thal. PMID:22524210

Qadah, Talal; Finlayson, Jill; Newbound, Christopher; Pell, Nicole; Pascoe, Michelle; Greenwood, Laura; Holmes, Paula; Grey, Dianne; Beilby, John; Ghassemifar, Reza

2012-04-23

269

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

PubMed Central

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

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

2004-01-01

270

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

PubMed Central

Sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins required for the lysosomal degradation of various sphingolipids with a short oligosaccharide chain by their exohydrolases. Four of the five known activator proteins (sap-A-sap-D), also called "saposins," are derived from a common precursor by proteolytic processing. sap-B stimulates hydrolysis of sulfatides by arylsulfatase A in vivo. Its recessively inherited deficiency results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is caused by a defect of arylsulfatase A. Here we report on a patient with sap-B deficiency. Reverse-transcription-PCR studies on the patient's mRNA revealed the occurrence of two distinct mutant species: one with an in-frame deletion of the first 21 bases of exon 6, the other with a complete in-frame deletion of this exon. The patient was homozygous for the underlying mutation, which was found to be a G-->T transversion within the acceptor splice site between intron e and exon 6, abolishing normal RNA splicing. Allele-specific oligonucleotide hybridization revealed that the parents and both grandfathers of the patient were carriers of this mutation. In order to analyze the fate of the mutant precursor proteins, both abnormal cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments showed that the deletion of 21 bp had no effect on the transport and the maturation of the encoded precursor. All sap forms except sap-B were detectable by immunochemical methods. The cDNA bearing a complete deletion of exon 6 encoded a shortened precursor of only 60 kD, and no mature SAPs were detectable. The carbohydrate chains of this polypeptide were of the high-mannose and hybrid type, indicating no transport of the mutant precursor beyond early Golgi apparatus. An endoplasmic-reticulum localization of this polypeptide was supported by indirect immunofluorescence analysis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 5b Figure 6 Figure 7 Figure 7b

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

1996-01-01

271

RNA Splicing in a New Rhabdovirus from Culex Mosquitoes?†  

PubMed Central

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.

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

2011-01-01

272

RNA splicing in a new rhabdovirus from Culex mosquitoes.  

PubMed

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

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

2011-04-20

273

Expression of the Volvox gene encoding nitrate reductase: Mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA  

Microsoft Academic Search

Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153–81 resides in a G-to-A transition of the first nucleotide in the 5' splice site of nitA intron 2. This mutation resulted in at

Heribert Gruber; Stefan H. Kirzinger; Rüdiger Schmitt

1996-01-01

274

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

PubMed Central

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

Burd, C G; Dreyfuss, G

1994-01-01

275

Analgesic infiltration at the site of bone marrow harvest significantly reduces donor morbidity  

Microsoft Academic Search

Little information has been published concerning the severity of pain experienced by bone marrow donors or the use of local analgesia following bone marrow harvesting procedures. The aims of this study were to assess duration and severity of pain experienced by bone marrow donors and the effectiveness of bupivacaine as a local analgesic agent following bone marrow harvest. During a

B Chern; N McCarthy; C Hutchins; STS Durrant

1999-01-01

276

Accelerating autograft maturation in instrumented posterolateral lumbar spinal fusions without donor site morbidity.  

PubMed

Properly harvested iliac crest bone autograft applied to a meticulously prepared fusion bed produces a consistently high rate of fusion with a low incidence of donor site morbidity. Some reports advocate substituting bone morphogenic protein (BMP) for iliac crest bone autograft, but in posterolateral lumbar spinal fusion, BMP appears better suited to facilitate iliac crest bone autograft maturation than to substitute for it. In this single-center, nonrandomized, prospective study (minimum 2-year follow-up), cancellous-only iliac crest bone autograft was harvested for use in posterolateral lumbar spinal fusion. Reviewers blinded to graft condition and age assigned fusion scores to the random radiographs of 31 consecutive patients who underwent 1- to 3-level posterolateral lumbar spinal fusion using iliac crest bone autograft supplemented with either an implanted spinal fusion stimulator or BMP. There was no significant immediate or remote iliac crest bone autograft harvest morbidity, and there was a significant reduction in pain scores postoperatively (P<.001). At 12 months, BMP radiographs were more likely than spinal fusion stimulator radiographs to be rated as fused (P<.019). All BMP patients were deemed fused at 12 months and all spinal fusion stimulator patients at 24 months. In this study, iliac crest bone autograft supplemented with either BMP or spinal fusion stimulator resulted in a solid contiguous fusion without significant iliac crest bone autograft harvest-related morbidity. Bone morphogenic protein-supplemented iliac crest bone autograft fused at a faster rate, producing the more mature-appearing, trabeculated, robust fusion. PMID:19902899

Rogozinski, Abraham; Rogozinski, Chaim; Cloud, Gregory

2009-11-01

277

Therapeutic Potential of Splice-Switching Oligonucleotides  

PubMed Central

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

Bauman, John; Jearawiriyapaisarn, Natee

2009-01-01

278

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

NASA Astrophysics Data System (ADS)

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

Pollastro, Pasquale; Rampone, Salvatore

279

Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation  

PubMed Central

Background Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups. Case presentation In this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475?+?1G?>?C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa. Conclusion This report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling.

2013-01-01

280

Effect of subcutaneous epinephrine/saline/local anesthetic versus saline-only injection on split-thickness skin graft donor site perfusion, healing, and pain.  

PubMed

The technique for split-thickness skin graft harvest varies among surgeons. Even though there is scientific evidence that the subcutaneous injection of modified tumescent solution reduces blood loss during burn surgery, the technique has not been unanimously adapted because of, in part, fear of healing retardation. This study prospectively examines the effect of tumescent injection on donor site perfusion, healing, and pain. Ten burn patients in need of grafting with a need for two distinctly different donor sites were included. During the grafting procedure, the two donor areas were randomly assigned to receive either modified tumescent solution or warm sterile saline solution subcutaneously before skin graft harvest with a dermatome. Perfusion, pain, pruritus, and donor site healing were measured, and a follow-up evaluation on scar quality was performed. Baseline perfusion on day 1 was significantly less in the donor site injected with modified tumescent solution (62.26 vs 95.71 perfusion units; P = .031), whereas the response to heat was similar in both sites. The physiologic response to injury (hyperemia) on days 2 and 3 was not suppressed in the modified tumescent group. Pain reported on day 1 was 2.38/10 in the tumescent site and 3.38/10 in the saline site (P = .21). On all other days, measurements showed no difference between the two sites. Donor sites healed in an average of 16.1 days with modified tumescent solution and in 16.4 days with saline. Late follow-up showed no difference in scar quality. The subcutaneous injection of modified tumescent solution before split-thickness donor site harvest has no adverse effect on donor site perfusion past day 1 or donor site healing. The addition of a local anesthetic may decrease pain for 24 hours postoperatively, but the difference in this study group was not significant. This technique should be universally recommended. PMID:23237817

Blome-Eberwein, Sigrid; Abboud, Michael; Lozano, Daniel D; Sharma, Rohit; Eid, Sherrine; Gogal, Christina

281

Painless split skin donor sites: a controlled double-blind trial of Opsite, scarlet red and bupivacaine.  

PubMed

A prospective randomized double-blind trial comparing Opsite alone, Opsite after application of bupivacaine, scarlet ointment dressing alone and scarlet ointment after bupivacaine was done to assess the effect of these four dressing regimens on split skin donor site pain and healing. Significantly less pain was reported by those dressed with Opsite and this was thought to be due to the immobility of an Opsite dressing. Many of the patients dressed with scarlet ointment felt no pain. It was concluded that movement of dressings is the main factor in pain production and that bupivacaine appeared to have no effect. There was no difference in healing rates between those treated with Opsite and those treated with scarlet ointment. It is concluded that using Opsite is a convenient way of preventing donor site pain, but that to gain maximum advantage from this it should not be applied under tension. PMID:2202283

Morris, W T; Lamb, A M

1990-08-01

282

A Pyrimidine-Rich Exonic Splicing Suppressor Binds Multiple RNA Splicing Factors and Inhibits Spliceosome Assembly  

Microsoft Academic Search

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately down-stream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind

Zhi-Ming Zheng; Martijn Huynen; Carl C. Baker

1998-01-01

283

Role of the C. elegans U2 snRNP protein MOG-2 in sex determination, meiosis, and splice site selection.  

PubMed

In Caenorhabditis elegans, germ cells develop as spermatids in the larva and as oocytes in the adult. Such fundamentally different gametes are produced through a fine-tuned balance between feminizing and masculinizing genes. For example, the switch to oogenesis requires repression of the fem-3 mRNA through the mog genes. Here we report on the cloning and characterization of the sex determination gene mog-2. MOG-2 is the worm homolog of spliceosomal protein U2A'. We found that MOG-2 is expressed in most nuclei of somatic and germ cells. In addition to its role in sex determination, mog-2 is required for meiosis. Moreover, MOG-2 binds to U2B?/RNP-3 in the absence of RNA. We also show that MOG-2 associates with the U2 snRNA in the absence of RNP-3. Therefore, we propose that MOG-2 is a bona fide component of the U2 snRNP. Albeit not being required for general pre-mRNA splicing, MOG-2 increases the splicing efficiency to a cryptic splice site that is located at the 5' end of the exon. PMID:21504747

Zanetti, Simone; Meola, Marco; Bochud, Arlette; Puoti, Alessandro

2011-04-12

284

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

PubMed

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. PMID:21347446

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

2011-02-08

285

Discovery and mass spectrometric analysis of novel splice-junction peptides using RNA-Seq.  

PubMed

Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Eighty million paired-end Illumina reads and ?500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing. PMID:23629695

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

2013-04-29

286

A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria  

PubMed Central

The Pet54p protein is an archetypical example of a dual functioning (‘moonlighting’) protein: it is required for translational activation of the COX3 mRNA and splicing of the aI5? group I intron in the COX1 pre-mRNA in Saccharomyces cerevisiae mitochondria (mt). Genetic and biochemical analyses in yeast are consistent with Pet54p forming a complex with other translational activators that, in an unknown way, associates with the 5? untranslated leader (UTL) of COX3 mRNA. Likewise, genetic analysis suggests that Pet54p along with another distinct set of proteins facilitate splicing of the aI5? intron, but the function of Pet54 is, also, obscure. In particular, it remains unknown whether Pet54p is a primary RNA-binding protein that specifically recognizes the 5? UTL and intron RNAs or whether its functional specificity is governed in other ways. Using recombinant protein, we show that Pet54p binds with high specificity and affinity to the aI5? intron and facilitates exon ligation in vitro. In addition, Pet54p binds with similar affinity to the COX3 5? UTL RNA. Competition experiments show that the COX3 5?UTL and aI5? intron RNAs bind to the same or overlapping surface on Pet54p. Delineation of the Pet54p-binding sites by RNA deletions and RNase footprinting show that Pet54p binds across a similar length sequence in both RNAs. Alignment of the sequences shows significant (56%) similarity and overlap between the binding sites. Given that its role in splicing is likely an acquired function, these data support a model in which Pet54p's splicing function may have resulted from a fortuitous association with the aI5? intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5? splicing and provided a possible means to coregulate COX1 and COX3 expression.

Kaspar, Benjamin J.; Bifano, Abby L.; Caprara, Mark G.

2008-01-01

287

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

PubMed

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

Alkindy, Adila; Chuzhanova, Nadia; Kini, Usha; Cooper, David N; Upadhyaya, Meena

2012-08-13

288

Measurements of T2 of Electron Spins at Bound Donor Sites in Si:P.  

National Technical Information Service (NTIS)

The goal of the project was to establish the limitations on the transverse spin relaxation of bound donors in dilute semiconductors, in particular in Si:P. The experiments were undertaken in two steps, first using the existing pulsed EPR spectrometer in P...

W. G. Clark K. Holczer S. Brown E. Yablonovitch

2005-01-01

289

Solution structure of domain 6 from a self-splicing group II intron ribozyme: a Mg(2+) binding site is located close to the stacked branch adenosine.  

PubMed

Group II intron self-splicing is essential for the correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism that is apparently analogous to that of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5gamma. The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix, being partially stacked between two flanking GU wobble pairs. We identified a novel prominent Mg(2+) binding site in the major groove of the branch site. Importantly, Mg(2+) addition does not impair the stacking of the branch adenosine, rather it strengthens the interaction with the flanking uridines, as shown by NMR and fluorescence studies. This means that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation. PMID:17200997

Erat, Michèle C; Zerbe, Oliver; Fox, Thomas; Sigel, Roland K O

2007-02-12

290

SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.  

PubMed

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

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

2012-08-31

291

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

PubMed

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

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

2013-09-01

292

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

PubMed

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

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

2011-11-04

293

Donor-Site Morbidity After Pedicled TRAM Breast Reconstruction: A Comparison of Two Different Types of Mesh.  

PubMed

Many different approaches have been used to minimize the risk of bulge or hernia formation when using autologous abdominal tissue for breast reconstruction. Studies have shown that further reinforcement of the abdominal wall using a mesh may decrease the complication rate.The current study included 40 consecutive patients having unilateral breast reconstruction with the pedicled transverse rectus abdominus musculocutaneous flap. The defect in the abdominal fascia was closed primarily and further reinforced using a Prolene mesh (Ethicon), n = 20, or using a self-fixating Parietex ProGrip mesh (Covidien), n = 20. The patients were examined at an outpatient consultation, with a minimum follow-up of 1 year and questioned about donor-site symptoms using a standardized questionnaire.Of the 20 patients in the Prolene group, 2 (10%) developed abdominal wall bulging, and of the 20 patients in the ProGrip group, 11 (55%) developed abdominal wall bulging (P = 0.006). In both the Prolene and the ProGrip group, most patients reported having continued donor-site symptoms at the time of the follow-up (70% and 80%, respectively); 15% and 30%, respectively, reported having symptoms that influenced their daily or physical activities (not a significant difference). All but 1 patient in our study reported being very happy with the reconstruction and would have done it again, had they known what they did at the time of the follow-up.We conclude that the self-gripping properties of the Parietex ProGrip mesh are not sufficient in withstanding the abdominal wall tension at the donor site after transverse rectus abdominus musculocutaneous-flap harvest and do not recommend using the Parietex ProGrip mesh without fixating sutures for this procedure. PMID:23392261

Sværdborg, Mille; Damsgaard, Tine Engberg

2013-11-01

294

A G->T splice site mutation of CRYBA1/A3 associated with autosomal dominant suture cataracts in a Chinese family  

PubMed Central

Purpose To identify the genetic defect in a five-generation Chinese family with congenital Y-suture cataracts. Methods A five-generation Chinese family with inherited Y-suture cataract phenotype was recruited. Detailed family history and clinical data of the family were recorded. Candidate genes sequencing was performed to screen out the disease-causing mutation. Results The congenital cataract phenotype of the family was identified as Y-suture cataract type by using slit-lamp photography. Direct sequencing revealed a G?T splice site mutation in crystallin, beta A1 (CRYBA1/A3).This mutation co-segregated with all affected individuals in the family and was not found in unaffected family members or 100 unrelated controls. Conclusions Our study identified a novel type of a splice site mutation in CRYBA1/A3 .The mutation was responsible for the congenital Y-suture cataracts in the family. This is the first report relating a G?T mutation of CRYBA1/A3 to congenital Y-suture cataract.

Yang, Zhenfei; Li, Qian; Ma, Zicheng; Guo, Yuanyuan; Ma, Xu

2011-01-01

295

Complete thyroxine-binding globulin (TBG) deficiency produced by a mutation in acceptor splice site causing frameshift and early termination of translation (TBG-Kankakee).  

PubMed

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids. PMID:9768672

Carvalho, G A; Weiss, R E; Refetoff, S

1998-10-01

296

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects  

PubMed Central

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5?ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5?ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5?ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

Fernandez Alanis, Eugenio; Pinotti, Mirko; Dal Mas, Andrea; Balestra, Dario; Cavallari, Nicola; Rogalska, Malgorzata E.; Bernardi, Francesco; Pagani, Franco

2012-01-01

297

Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model.  

PubMed

Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes. PMID:12915465

Yang, Yongping; Swaminathan, Srividya; Martin, Betty K; Sharan, Shyam K

2003-07-08

298

Identification of beta1C-2, a novel variant of the integrin beta1 subunit generated by utilization of an alternative splice acceptor site in exon C.  

PubMed Central

A new splice variant of the human integrin subunit beta1 has been identified and designated beta1C-2. It differs from the previously reported beta1C (in this report designated beta1C-1) by 18 nucleotides, and is generated by splicing from exon 6 to an alternative splice acceptor site within exon C, causing an in-frame deletion of six amino acids of the cytoplasmic region of beta1C-1. The beta1C-2 mRNA is present in several human cell lines and tissues at low levels, similarly to beta1C-1. In peripheral T-lymphocytes, beta1C-2 is the selectively expressed isoform. Neither beta1C-1 nor beta1C-2 mRNA could be detected in mouse tissues, and Southern hybridization of a mouse genomic beta1 clone with a human exon-C-specific probe failed to identify a corresponding mouse exon. The antisense orientation of exon C is highly homologous to an Alu element. Since Alu elements are restricted to primates, the beta1C-1 and beta1C-2 variants of the integrin subunit beta1 are specific for these species. The protein coded for by the beta1C-2 cDNA can be expressed and localized to the surface of beta1 deficient mouse cells. However, while stable transformed clones expressing high levels of the beta1A were commonly found, the beta1C-1 and beta1C-2 expressing clones expressed barely detectable amounts of the beta1 protein. Hence, high levels of beta1C-2 may be incompatible with cell proliferation, as previously suggested for beta1C-1.

Svineng, G; Fassler, R; Johansson, S

1998-01-01

299

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

PubMed Central

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

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

1993-01-01

300

Lysyl oxidase-like 4 is alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities  

Microsoft Academic Search

Lysyl oxidase-like enzymes (LOXL) are expressed in various cancers. We analyzed the expression of LOXL2, LOXL3, and LOXL4\\u000a in cancers involving the serosal cavities—breast carcinoma, ovarian carcinoma, and malignant mesothelioma using reverse-transcriptase\\u000a polymerase chain reaction. We discovered two new alternative splice variants of LOXL4. The spliced segments were exon 9 (splice\\u000a variant 1) or both exons 8 and 9 (splice

Shulamit Sebban; Ben Davidson; Reuven Reich

2009-01-01

301

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei  

Microsoft Academic Search

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5 splice sites, branch points, pyrimidine-rich regions (poly(Y) tracts), 3 splice sites (3SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in

T. Nicolai Siegel; Kevin S. W. Tan; George A. M. Cross

2005-01-01

302

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

PubMed Central

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

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

2012-01-01

303

A splice-site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas.  

PubMed

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173?+ 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation. PMID:20872591

Yang, Liu; Hui, Wing Sum; Chan, Wilson C W; Ng, Vivian C W; Yam, Teresa H Y; Leung, Helen C M; Huang, Jian-Dong; Shum, Daisy K Y; Jie, Qiang; Cheung, Kenneth M C; Cheah, Kathryn S E; Luo, Zhoujing; Chan, Danny

2010-11-01

304

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron.  

PubMed Central

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions. Images

Green, R; Szostak, J W; Benner, S A; Rich, A; Usman, N

1991-01-01

305

A new VCAN/versican splice acceptor site mutation in a French Wagner family associated with vascular and inflammatory ocular features  

PubMed Central

Purpose To detail the highly variable ocular phenotypes of a French family affected with an autosomal dominantly inherited vitreoretinopathy and to identify the disease gene. Methods Sixteen family members with ten affected individuals underwent detailed ophthalmic evaluation. Genetic linkage analysis and gene screening were undertaken for genes known to be involved in degenerative and exudative vitreoretinopathies. Qualitative reverse transcriptase-PCR analysis of the versiscan (VCAN) transcripts was performed after mutation detection in the VCAN gene. Results The first index patient of this French family was referred to us because of a chronic uveitis since infancy; this uveitis was associated with exudative retinal detachment in the context of a severe uncharacterized familial vitreoretinopathy. Genetic linkage was obtained to the VCAN locus, and we further identified a new pathogenic mutation at the highly conserved splice acceptor site in intron 7 of the VCAN gene (c.4004–2A>T), which produced aberrantly spliced VCAN transcripts. Conclusions Extensive molecular investigation allowed us to classify this familial vitreoretinopathy as Wagner syndrome. This study illustrates the need to confirm clinical diagnosis by molecular genetic testing and adds new ocular phenotypes to the Wagner syndrome, such as vascular and inflammatory features.

Brezin, Antoine P.; Nedelec, Brigitte; Barjol, Amandine; Rothschild, Pierre-Raphael; Delpech, Marc

2011-01-01

306

A novel splice site mutation of the MEN1 gene identified in a patient with primary hyperparathyroidism.  

PubMed

Heterozygous germline mutation of the tumor suppressor gene MEN1 is responsible for multiple endocrine neoplasia type 1 (MEN1), a familial cancer syndrome characterized by pituitary, parathyroid and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and its incomplete forms often manifested as familial isolated hyperparathyroidism and apparently sporadic parathyroid tumor. Mutation analysis of the MEN1 gene is a powerful tool for the early diagnosis of MEN1; however, the clinical significance of the identified mutations is not always obvious. In this study, a previously unreported missense MEN1 mutation, c.824G>T was identified in a patient with primary hyperparathyroidism and evaluated for its pathogenicity. This mutation was predicted to generate a putative missense menin protein, R275M. A stability test of the menin protein demonstrated that the stability of R275M mutant was reduced only slightly as compared with wild type menin, and therefore could not preclude the possibility that it was a rare benign polymorphism. However, further analysis of leukocyte mRNA and minigene experiments indicated that the mutant c.824G>T allele gives rise to abnormally spliced menin mRNA, and thereby confirmed that c.824G>T mutation is causative for MEN1. Thus, leukocyte mRNA analysis has been demonstrated useful to identify a splicing mutation of the MEN1 gene. PMID:22447146

Nagamura, Yuko; Yamazaki, Masanori; Shimazu, Satoko; Sano, Kenji; Tsukada, Toshihiko; Sakurai, Akihiro

2012-03-23

307

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

PubMed Central

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

Di Bari, Roberto; Coronelli, Roberto

2013-01-01

308

A splice site variant in the bovine RNF11 gene compromises growth and regulation of the inflammatory response.  

PubMed

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

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

2012-03-15

309

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

PubMed Central

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

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

2012-01-01

310

Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect.  

PubMed

Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha Republic (Yakutia) located in Eastern Siberia (Russian Federation). The Yakut population exhibits high frequency of some Mendelian disorders, which are rare in other populations worldwide. Mutational analysis of GJB2 gene in 86 unrelated Yakut patients with congenital HI without other clinical features has been performed. In this study, we registered a large cohort of Yakut patients homozygous for the IVS1+1G>A mutation (70 unrelated deaf subjects in total). Detailed audiological analysis of 40 deaf subjects with genotype IVS1+1G>A/IVS1+1G>A revealed significant association of this genotype with mostly symmetrical bilateral severe to profound HI (85% severe-to-profound HI versus 15% mild-to-moderate HI, P<0.05). The highest among six investigated Eastern Siberian populations carrier frequency of the IVS1+1G>A mutation (11.7%) has been found in Yakut population. Reconstruction of 140 haplotypes with IVS1+1G>A mutation demonstrates the common origin of all mutant chromosomes found in Yakuts. The age of mutation was estimated to be approximately 800 years. These findings characterize Eastern Siberia as the region with the most extensive accumulation of the IVS1+1G>A mutation in the world as a result of founder effect. PMID:21776002

Barashkov, Nikolay A; Dzhemileva, Lilya U; Fedorova, Sardana A; Teryutin, Fedor M; Posukh, Olga L; Fedotova, Elvira E; Lobov, Simeon L; Khusnutdinova, Elza K

2011-07-21

311

cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene.  

PubMed

The human ryanodine receptor type 3 (RYR3) was cloned from a fetal brain cDNA library and its complete sequence was determined (EMBL accession number AJ001515). The sequenced cDNA spanned 15,564 bp and contained an open reading frame of 14,613 bp. The corresponding protein consisted of 4870 amino acids with a calculated molecular mass of 552 kDa. Amino acid sequence identities to the RYR3 proteins from rabbit, mink, and chicken were 96%, 95%, and 83% respectively. A previously unidentified alternative splice site was detected generating a transcript that lacked bases 11,569-11,650 and encoded a truncated protein. PMID:9515741

Leeb, T; Brenig, B

1998-02-27

312

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation.  

PubMed

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer. PMID:22815844

Hersmus, Remko; van der Zwan, Yvonne G; Stoop, Hans; Bernard, Pascal; Sreenivasan, Rajini; Oosterhuis, J Wolter; Brüggenwirth, Hennie T; de Boer, Suzan; White, Stefan; Wolffenbuttel, Katja P; Alders, Marielle; McElreavy, Kenneth; Drop, Stenvert L S; Harley, Vincent R; Looijenga, Leendert H J

2012-07-18

313

Effects of splicing mutations on NF2-transcripts: transcript analysis and information theoretic predictions.  

PubMed

This study examined the effects of 22 putative splicing mutations in the NF2 gene by means of transcript analysis and information theory based prediction. Fourteen mutations were within the dinucleotide acceptor and donor regions, often referred to as (AG/GT) sequences. Six were outside these dinucleotide regions but within the more broadly defined splicing regions used in the information theory based model. Two others were in introns and outside the broadly defined regions. Transcript analysis revealed exon skipping or activation of one or more cryptic splicing sites for 17 mutations. No alterations were found for the two intronic mutations and for three mutations in the broadly defined splicing regions. Concordance and partial concordance between the calculated predictions and the results of transcript analysis were found for 14 and 6 mutations, respectively. For two mutations, the predicted alteration was not found in the transcripts. Our results demonstrate that the effects of splicing mutations in NF2 are often complex and that information theory based analysis is helpful in elucidating the consequences of these mutations. PMID:21563229

Ellis, James R; Heinrich, Bianca; Mautner, Victor-F; Kluwe, Lan

2011-05-11

314

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

PubMed Central

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.

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

315

Genetic therapies for RNA mis-splicing diseases.  

PubMed

RNA mis-splicing diseases account for up to 15% of all inherited diseases, ranging from neurological to myogenic and metabolic disorders. With greatly increased genomic sequencing being performed for individual patients, the number of known mutations affecting splicing has risen to 50-60% of all disease-causing mutations. During the past 10years, genetic therapy directed toward correction of RNA mis-splicing in disease has progressed from theoretical work in cultured cells to promising clinical trials. In this review, we discuss the use of antisense oligonucleotides to modify splicing as well as the principles and latest work in bifunctional RNA, trans-splicing and modification of U1 and U7 snRNA to target splice sites. The success of clinical trials for modifying splicing to treat Duchenne muscular dystrophy opens the door for the use of splicing modification for most of the mis-splicing diseases. PMID:21497936

Hammond, Suzan M; Wood, Matthew J A

2011-04-15

316

Gene Expression: The Close Coupling of Transcription and Splicing  

Microsoft Academic Search

Increasing evidence indicates that the transcriptional machinery can influence the efficiency of splicing as well as splice-site selection. Surprisingly, it has now been demonstrated that splicing components influence the efficiency of transcription. This mutual stimulation has important implications for the regulation of gene expression.

Emanuel Rosonina; Benjamin J Blencowe

2002-01-01

317

A synonymous codon variant in two patients with autosomal recessive bestrophinopathy alters in vitro splicing of BEST1  

PubMed Central

Purpose Autosomal recessive bestrophinopathy (ARB) is a newly defined retinal dystrophy caused by biallelic mutations in bestrophin-1 (BEST1) and is hypothesized to represent the null bestrophin-1 phenotype in humans. The aim was to determine whether a synonymous BEST1 variant, c.102C>T, identified in two unrelated ARB patients, alters pre-mRNA splicing of the gene. Additionally a detailed phenotypic characterization of this distinctive condition is presented for both patients. Methods BEST1 was analyzed by direct sequencing. Patients underwent standard ophthalmic assessment. In silico and in vitro analysis using a minigene system was performed to assess whether a synonymous variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing of BEST1. Results Both ARB patients harbored either proven (patient 1; c.102C>T p.Gly34Gly and c.572T>C p.Leu191Pro) or presumed (patient 2; c.102C>T p.Gly34Gly and c.1470_1471delCA, p.His490GlnfsX24) biallelic mutations in BEST1 and were found to have phenotypes consistent with ARB. In vitro analysis of the synonymous variant, c.102C>T p.Gly34Gly, demonstrated it to introduce a cryptic splice donor site 52 nucleotides upstream of the actual splice donor site. Conclusions The novel BEST1 variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing in vitro and is potentially pathogenic. In vivo this splicing variant is predicted to lead to the production of an mRNA transcript with a premature termination codon (p.Glu35TrpfsX11) that is predicted to be degraded by NMD.

Davidson, Alice E.; Sergouniotis, Panagiotis I.; Burgess-Mullan, Rosemary; Hart-Holden, Nichola; Low, Sancy; Foster, Paul J.; Manson, Forbes D.C.; Black, Graeme C.M.

2010-01-01

318

Deciphering the splicing code  

Microsoft Academic Search

Alternative splicing has a crucial role in the generation of biological complexity, and its misregulation is often involved in human disease. Here we describe the assembly of a `splicing code', which uses combinations of hundreds of RNA features to predict tissue-dependent changes in alternative splicing for thousands of exons. The code determines new classes of splicing patterns, identifies distinct regulatory

Yoseph Barash; John A. Calarco; Weijun Gao; Qun Pan; Xinchen Wang; Ofer Shai; Benjamin J. Blencowe; Brendan J. Frey

2010-01-01

319

Design, synthesis, and molecular modeling studies of 5?-deoxy-5?-ureidoadenosine: 5?-ureido group as multiple hydrogen bonding donor in the active site of S-adenosylhomocysteine hydrolase  

Microsoft Academic Search

5?-Deoxy-5?-ureidoadenosine was designed and synthesized as a potent inhibitor of S-adenosylhomocysteine hydrolase (SAH), in which 5?-ureido group acted as multiple hydrogen bonding donor in binding with active site residues of SAH in the molecular modeling study.

Ting Wang; Hyun Joo Lee; Dilip K. Tosh; Hea Ok Kim; Shantanu Pal; Sun Choi; Yoonji Lee; Hyung Ryong Moon; Long Xuan Zhao; Kang Man Lee; Lak Shin Jeong

2007-01-01

320

Genome-Wide Landscape of Alternative Splicing Events in Brachypodium distachyon  

PubMed Central

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

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

2013-01-01

321

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

PubMed Central

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

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

1999-01-01

322

Rbm20 regulates titin alternative splicing as a splicing repressor  

PubMed Central

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3? splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.

Li, Shijun; Guo, Wei; Dewey, Colin N.; Greaser, Marion L.

2013-01-01

323

Splicing mutations in DMD/BMD detected by RT-PCR/PTT: detection of a 19AA insertion in the cysteine rich domain of dystrophin compatible with BMD.  

PubMed Central

We have used an RNA based mutation detection method to screen the total coding region of the dystrophin gene of a Duchenne and a Becker muscular dystrophy patient in whom DNA based mutation detection methods have so far failed to detect mutations. By RT-PCR and the protein truncation test (PTT) we could identify point mutations in both cases. DMD patient DL184.3 has a T-->A mutation in intron 59 at position -9, creating a novel splice acceptor site for exon 60. As a result seven intronic bases are spliced into the mRNA, causing a frameshift and premature translation termination 20 codons downstream. Since this patient had died and only fibroblasts were available, we applied MyoD induced myodifferentiation of stored fibroblasts to enhance muscle specific gene expression. With the results of this mutation analysis, prenatal diagnosis could subsequently be performed in this family. BMD patient BL207.1 carries a G-->C mutation at position +5 of intron 64, disrupting the splice donor consensus sequence and activating a cryptic splice donor site 57bp downstream. The inclusion of these 57 intronic bases in the mRNA leaves the reading frame open and results in the insertion of 19 amino acids into the cysteine rich domain of dystrophin. Interestingly, this insertion in a part of the dystrophin considered to interact with the dystrophin binding complex of the sarcolemma is apparently compatible with mild BMD-like clinical features. Both mutations reported are missed by analysis of multiplex PCR products designed for deletion screening of the coding region. Extrapolation from existing point mutation detection efficiencies by DNA and RNA based methods emphasises that RNA based methods are more sensitive and that most of the remaining undetected mutations may affect splice or branch sites or create cryptic splice sites. Images

Roest, P A; Bout, M; van der Tuijn, A C; Ginjaar, I B; Bakker, E; Hogervorst, F B; van Ommen, G J; den Dunnen, J T

1996-01-01

324

Biomechanical study of prophylactic internal fixation of the radial osteocutaneous donor site using the sheep tibia model.  

PubMed

This study investigated the strengthening effect of different types of plate and position after osteotomy of the sheep tibia, which is a model for the radial osteocutaneous donor site. Fifty matched pairs of adult sheep tibias were tested in torsion and four-point bending. Firstly, the weakening effect of an osteotomy was compared with the intact bone. Then pairs of bones with an osteotomy were compared with and without reinforcement with different types of 3.5mm plate. The plate was placed in either the anterior (over the defect) or posterior (on the intact cortex) position. In torsion the mean strength of the intact bone was 45% greater than after osteotomy (P=0.02). The reinforced bone was on average 61% stronger than the unreinforced bone (P<0.001). In bending the mean strength of the intact bone was 188% greater than after osteotomy (P=0.02). The reinforced bone was on average 184% stronger then the unreinforced bone (P<0.001). The tibia was able to withstand much greater loads in bending. The dynamic compression plate was the strongest reinforcement in both torsion and bending. The position of the plate did not alter the strengthening effect in torsion but the posterior position resisted greater bending loads (P=0.01). This may not be relevant in clinical practice as the radius is likely to fracture first as a result of lower torsional forces. PMID:17188407

Avery, C M E; Best, A; Patterson, P; Rolton, J; Ponter, A R S

2006-12-22

325

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

PubMed

Transplants from the anterior iliac crest are used for most reconstructive procedures in cranio-maxillofacial surgery. The advantages are easy accessibility, the ability to work in two teams and the amount of corticocancellous bone available; disadvantages are postoperative pain and gait disturbances. To reduce donor-site pain, the effect of a single dose of bupivacaine (10 cc of 2.5mg/cc with 1:80.000 epinephrine) was studied. 200 consecutive patients, who underwent anterior iliac crest bone harvesting for reconstructive procedures, were randomly divided into those receiving bupivacaine and those not. They completed a standardized questionnaire. Patients scored the intensity of the pain and difficulties walking at different times with a visual analogue scale. They recorded analgesics used. 98 questionnaires were eligible for analysis. No differences between the bupivacaine and the control group were detected for postoperative pain and gait disturbance. There is no support for administration of a single dose of bupivacaine to reduce pain in the first postoperative days. The surface area of the removed bone had a significant influence on pain and walking; pain is related to the local osseous damage or periosteal stripping rather than to the length of incision or the operation time. PMID:19959335

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

2009-12-02

326

[Mechanism of inhibition by local anesthetics of electron transport at the donor site of the photosystem II].  

PubMed

The mechanism of inhibition by local anaesthetics of the procaine group of electron transport at the donor site of photosystem II (PS II) from pea chloroplasts was investigated. It was found that besides the inactivation of the O2 release system the anaesthetics used at one order of magnitude lesser concentration exert an uncoupling effect. With a rise in pH the inhibiting activity increases; however, this process is not coupled with the protonophore effect but is due to the generation of a neutral form of the amine. The increment of the inhibiting activity of the anaesthetics in the course of deprotonation seems to be regulated by changes in the coefficient of distribution between the membrane and the aqueous phase. The rate of inactivation of the H2O-dissociating complex increases considerably upon illumination. Electron transport through PS II in anaesthetic-treated chloroplasts in restored by diphenylcarbaside, but not by hydroxylamine. It is concluded that the anaesthetics induce the inhibition by interacting with the electron carrier. The role of the Ca2+--calmodulin-like protein in the functioning of the electron transport chain of PS II is discussed. PMID:3311175

Semin, B K; Chudinovskikh, M N; Ivanov, I I

1987-08-01

327

Do Functional Keratin Dressings Accelerate Epithelialization in Human Partial Thickness Wounds? A Randomized Controlled Trial on Skin Graft Donor Sites  

PubMed Central

Objective: To determine if the experimental (keratin-based) dressing accelerates epithelialization rates during healing of partial-thickness wounds, relative to a Standard Care dressing. Method: A randomized control trial was conducted using a Standard Care dressing side by side with the experimental dressing on a sample (n=26) of partial-thickness donor site wounds. The proximal/distal placement of the control and treatment was randomized. Percentage epithelialization after approximately 7 days was estimated from which time to fully epithelialize can be inferred. Patients were grouped into “young” (?50 y/o) and “old” (>50 y/o). Results: For the “old” patients (n=15), the median epithelialization percentage at 7 days is 5% and was significantly (P=.023) greater for the experimental dressing. For the “young” patients (n=11), the median epithelialization percentage at 7 days was 80% and there is no significant difference between the experimental and Standard Care control dressings. Conclusions: The experimental dressing significantly increases the rate of epithelialization of acute, traumatic partial-thickness wounds in older patients. We suggest that the dressing may be clinically useful in similar situations where epithelialization may be delayed because of patient or wound characteristics.

Davidson, Andrew; Jina, N. Hamesh; Marsh, Clive; Than, Martin; Simcock, Jeremy W.

2013-01-01

328

TassDB2 - A comprehensive database of subtle alternative splicing events  

Microsoft Academic Search

BACKGROUND: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus,

Rileen Sinha; Thorsten Lenser; Niels Jahn; Ulrike Gausmann; Swetlana Friedel; Karol Szafranski; Klaus Huse; Philip Rosenstiel; Jochen Hampe; Stefan Schuster; Michael Hiller; Rolf Backofen; Matthias Platzer

2010-01-01

329

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

PubMed Central

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

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

2011-01-01

330

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

PubMed Central

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

Hebeisen, Michael; Drysdale, John; Roy, Richard

2008-01-01

331

Novel splice-site mutations and a large intragenic deletion in PLA2G6 associated with a severe and rapidly progressive form of infantile neuroaxonal dystrophy.  

PubMed

Infantile neuroaxonal dystrophy, INAD, is a severe progressive psychomotor disorder with infantile onset and characterized by the presence of axonal spheroids throughout the central and peripheral nervous systems. A subset of INAD patients shows also brain iron accumulation which represents instead the distinctive feature of the idiopathic neurodegeneration with brain iron accumulation, NBIA. These diseases share the same causative gene, PLA2G6, encoding iPLA2-VIA, a calcium-independent phospholipase. Mutations that lead to a complete absence of protein are associated with a severe INAD profile, while compound heterozygous mutations with possibly a residual protein activity are instead associated with the less severe NBIA phenotype. Here we describe two INAD patients both with an unusually rapid disease progression and a peculiar neuroradiological presentation in one of them. Compound heterozygosity for a large intragenic deletion and a nonsense mutation was found in one of them while the other is carrying two novel splice-site mutations. Breakpoint-sequence analysis suggests a non-allelic-homologous-recombination (NAHR) event, probably underlying the rearrangement. These findings, while supporting the genotype-phenotype correlation already observed in INAD patients, provide the first sequence characterization of a genomic rearrangement in PLA2G6 gene, thus orienting the search for missing mutant alleles in PLA2G6 related diseases. PMID:20584031

Tonelli, A; Romaniello, R; Grasso, R; Cavallini, A; Righini, A; Bresolin, N; Borgatti, R; Bassi, M T

2010-11-01

332

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

PubMed Central

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

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

2009-01-01

333

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties  

Microsoft Academic Search

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US

H. Frances J. Bligh; Patrick D. Larkin; Paul S. Roach; Christopher A. Jones; Hongyong Fu; William D. Park

1998-01-01

334

The Arabic allele: A single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia  

Microsoft Academic Search

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base-pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron

Said M. Shawar; Mohammad A. Al-Drees; Ahmad R. Ramadan; Najat H. Ali; Suad M. AlFadhli

335

Synthetic Intron Improves Transduction Efficiency of Trans-Splicing Adeno-Associated Viral Vectors  

PubMed Central

Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many gene therapy applications. We have shown that rational selection of the gene-splitting site in a therapeutic target gene can lead to extremely efficient trans-splicing vectors [Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S., and Duan, D. (2005). Nat. Biotechnol. 23, 1435–1439]. Our original strategy requires the screening of endogenous introns that are capable of overcoming the mRNA accumulation barrier. To further develop trans-splicing vectors, we have tested whether the use of a generic synthetic intron can bypass the labor-intensive intron-screening process. Two previously characterized exon/intron/exon junctions (60/60/61 and 63/63/64, respectively) in the 6 kb minidystrophin gene were used as templates to represent highly efficient (60/60/61) and relatively poor (63/63/64) gene-splitting sites. We compared RNA production from the reconstituted viral genome and transduction efficiency of the trans-splicing vectors in dystrophin-null mdx mouse skeletal muscle. Our results suggest that a synthetic intron can successfully overcome the mRNA accumulation barrier at the exon 63/64 junction. Furthermore, when the gene was split at the exon 63/64 junction, the synthetic intron-based vectors performed better than the endogenous intron-based vectors. When the gene was split at the exon 60/61 junction, we observed only nominal improvement in mRNA production. Nevertheless, vectors based on the exon 60/61 junction remain the best set in transduction efficiency. Taken together, our results suggest that optimizing intron sequence may boost the transduction efficiency of trans-splicing AAV vectors. OVERVIEW SUMMARY Successful transduction of trans-splicing AAV vectors depends on the efficient uptake of both donor and acceptor vectors, unidirectional head-to-tail viral genome recombination, and productive transcription and splicing of the reconstituted genome. In this study, we explored the strategy of using synthetic intron to overcome the transcription/splicing barrier. We hypothesized that substituting a less than optimal endogenous intron with a highly conserved synthetic intron will lead to enhanced mRNA production. On the basis of our previous studies of minidystrophin gene trans-splicing AAV vectors, we replaced endogenous introns with synthetic introns in artificially reconstituted viral genomes and in trans-splicing vectors, respectively. Consistent with our initial hypothesis, we observed significant improvement in mRNA production from constructs based on a poor gene-splitting site, namely, the exon 63/64 junction. Synthetic intron replacement also increased transduction efficiency in vectors based on this junction. However, it never reached the level of our best trans-splicing vectors, namely, those based on the exon 60/61 junction. In summary, we have developed a convenient method of using synthetic intron to overcome the mRNA accumulation barrier in trans-splicing vectors. Additional investigation of factors other than mRNA accumulation will likely help to develop better trans-splicing vectors.

LAI, YI; YUE, YONGPING; LIU, MINGJU; DUAN, DONGSHENG

2007-01-01

336

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3? Splice Site In Vivo  

Microsoft Academic Search

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes

Long Ma; H. Robert Horvitz

2009-01-01

337

Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site  

Microsoft Academic Search

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG trans- lation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This

KOEN VERHOEF; PATRICIA S. BILODEAU; Wamel van J. L. B; JØRGEN KJEMS; C. MARTIN STOLTZFUS; BEN BERKHOUT

2001-01-01

338

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in Flaveria linearis.  

PubMed

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO(2)-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis. PMID:20882321

Slater, S M H; Micallef, M C; Zhang, J; Micallef, B J

2010-10-01

339

HPV-18 E2^E4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation.  

PubMed

The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1^E4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2^E4 transcripts resulting from 2 splice donor sites in the 5' part of E2, while the splice acceptor site is the one used for E1^E4. Both E2^E4 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, E2^E4-S and E2^E4-L. Whereas we could not differentiate E2^E4-S from E1^E4 in vivo, E2^E4-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 E2^E4 products. PMID:22541938

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

2012-04-27

340

A splice variant of the human CCA-adding enzyme with modified activity.  

PubMed

The human CCA-adding enzyme (tRNA nucleotidyltransferase) is an essential enzyme that catalyzes the addition of the CCA terminus to the 3' end of tRNA precursors, a reaction which is a fundamental prerequisite for mature tRNAs to become aminoacylated and to participate in protein biosynthesis. To date only one form of this enzyme has been identified in humans. Here, we describe the sequence and activity of a splice variant of the human CCA-adding enzyme identified in public cDNA databases. The in silico analyses performed on this splice variant indicate that there is conservation of the alternative splice donor site among species and indicate that it seems to be used in vivo. Moreover, the recombinantly expressed protein is active in vitro and accepts tRNA transcripts as substrates incorporating the dinucleotide sequence CC to their 3' end, in contrast to the activity of the full length enzyme. These findings strongly suggest that the splice variant of the human CCA-adding enzyme is expressed in the cell although the in vivo function remains unclear. PMID:17204286

Lizano, Esther; Schuster, Jens; Müller, Martin; Kelso, Janet; Mörl, Mario

2006-12-12

341

CYP17A1 Intron Mutation Causing Cryptic Splicing in 17?-Hydroxylase Deficiency  

PubMed Central

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

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

2011-01-01

342

Alternative splicing of the unique "PLUS" domain of chicken PG-M/versican is developmentally regulated.  

PubMed

We investigated the occurrence of alternatively spliced forms (V0, V1, V2, and V3) of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retinas, using the reverse transcription-polymerase chain reaction. We characterized the PLUS domain, which is apparently unique to the chicken molecule and is regulated by alternative splicing. PG-M in chicken retinas consisted of four forms with (V0, V1, V2, and V3) and two forms without (V1 and V3) the PLUS domain (PG-M+ and PG-M-, respectively). The four forms of PG-M+ were found in all samples examined, but the occurrence of the two PG-M- forms was regulated developmentally. Genomic analysis has revealed that the PLUS and CS-alpha domains are encoded by a single exon, and this exon has an internal alternative 5'-splice donor site, allowing alternative spliced forms that do not include the 3'-end of the exon. Sequences corresponding to the chicken PLUS domain (plus) were not found in mouse and human and may have disappeared during evolution. Sequence similarity suggests that the PLUS domain corresponds to the keratan sulfate attachment domain of aggrecan and that it has a distinct function in the chicken eye. PMID:9083069

Zako, M; Shinomura, T; Kimata, K

1997-04-01

343

Progress toward therapy with antisense-mediated splicing modulation  

PubMed Central

Antisense oligonucleotides (AO) or antisense RNA can complementarily bind to a target site in pre-mRNA and regulate gene splicing, either to restore gene function by reprogramming gene splicing or to inhibit gene expression by disrupting splicing. These two applications represent novel therapeutic strategies for several types of diseases such as genetic disorders, cancers and infectious diseases. In this review, the recent developments and applications of antisense-mediated splicing modulation in molecular therapy are discussed, with emphasis on advances in antisense-mediated splice targeting, applications in diseases and systematic delivery.

2009-01-01

344

CASE REPORT Persistent Seromas in Abdominal Free Flap Donor Sites After Postmastectomy Breast Reconstruction Surgery: Case Reports and Literature Review  

PubMed Central

Objectives: Donor site seroma formation is a common occurrence following abdominal free flap breast reconstructions. Although such seromas usually resolve spontaneously after a few weeks or months, we recently encountered 3 patients with abdominal seromas persisting for up to 2 years postoperatively. We therefore investigated possible predisposing factors in our patient group. Methods: Patients with persistent abdominal seromas, arbitrarily defined as present after 3 months following abdominal free flap harvest were identified. Their demographic characteristics, comorbidities, reconstruction details, frequency, and volume of abdominal aspirations were documented. Results: Three obese patients (Mean body mass index = 35) with an average age of 49 years bilaterally reconstructed with superior inferior epigastric artery or deep inferior epigastric artery flaps fitted the aforementioned criteria. Seroma aspirations commenced at 3 weeks and continued for a maximum of 26 months postoperatively. The average number of aspirations was 11 with a mean volume of 338 mL (range: 100-864 mL) per visit. The patients were aspirated either weekly or fortnightly depending on the speed of seroma reaccumulation and symptoms. All the 3 patients needed excision of the seroma sac to achieve permanent resolution. Discussion and Conclusion: In addition to their nuisance value (notably frequent aspirations and outpatient clinic visits), persistent seromas can cause significant morbidity and eventually require surgical excision. Possible predisposing factors in our patients included obesity, bilateral reconstructions, and superior inferior epigastric artery flap harvest. Such “high risk” patients should be warned about the likelihood of persistent seromas needing repeated aspirations and possible surgical interventions for ultimate resolution.

Sadeghi, Abtin; Malata, Charles

2013-01-01

345

Alternative splice acceptor utilization during human immunodeficiency virus type 1 infection of cultured cells.  

PubMed Central

The utilization of alternative splice acceptors for excision of the 5' major intron of human immunodeficiency virus type 1 RNA was observed after infection in vitro. Specific splice events were monitored by a cDNA-polymerase chain reaction. These splice events shared a common splice donor but utilized several alternative splice acceptors. In addition to identifying the previously documented splice acceptors for tat and nef (S. K. Arya, C. Guo, S. F. Josephs, and F. Wong-Staal, Science 229:69-73, 1985), nucleotide sequence analysis of cDNA-polymerase chain reaction fragments also revealed the following: (i) two splice acceptors 15 and 9 nucleotides upstream from the rev start codon, which are utilized to create transcripts suitable for specific rev expression; and (ii) use of the splice acceptor previously attributed to nef to generate a singly spliced, env-encoding transcript. Hybridization signals representing the nef/env, tat, and rev splice events increased in intensity between 6 and 12 h after infection of CEM cells with the LAV-1BRU strain of human immunodeficiency virus type 1. In contrast, the signal for utilization of the nef/env splice acceptor for the singly spliced env transcript appeared first at 12 h and increased to maximum intensity by 24 h. The nef/env splice acceptor was dominant at all time points examined. We propose that this dominance ensures efficient downstream splicing proximal to the env initiation codon in singly spliced transcripts. However, early after infection, the dominance of the nef/env splice acceptor appears to divert primary transcripts away from tat- and rev-specific processing paths. The relative proportions of hybridization signals representing these alternative splice events remained constant throughout the viral replicative cycle. This result suggests that trans-acting factors that might influence splice choices are not induced during infection, but rather that cis-acting, sequence-specific splice preferences determine the relative efficiency of alternative acceptor utilization. Images

Guatelli, J C; Gingeras, T R; Richman, D D

1990-01-01

346

Seroma and Quilting Suture at the Donor Site of the TRAM Flap in Breast Reconstruction: A Prospective Randomized Double-Blind Clinical Trial.  

PubMed

ABSTRACT: Seroma formation at the donor site of the transverse rectus abdominis myocutaneous flap was evaluated in 48 patients who underwent breast reconstruction with either quilting sutures and suction drains (QS+DN group) or quilting sutures alone (QS group) or suction drains alone (DN group). Clinical and ultrasound examinations were performed to assess seroma formation in 5 regions of the abdominal wall on postoperative days 7 and 14. The incidence of seroma detected by ultrasound examination was significantly higher in the DN group (P = 0.008) than that in the other 2 groups. No difference in seroma volume (puncture) was found between the QS+DN and QS groups (P = 1.00). Seroma formation was observed in the iliac region in the DN group but not in the QS+DN and QS groups (P = 0.028). Quilting sutures at the transverse rectus abdominis myocutaneous flap donor site were efficient in reducing seroma formation. PMID:23407260

Rossetto, Luis Antonio; Garcia, Elvio Bueno; Abla, Luiz Eduardo Felipe; Ferreira, Lydia Masako

2013-02-12

347

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

PubMed

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

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

2011-07-19

348

mRNA 5?-leader trans-splicing in the chordates  

PubMed Central

We report the discovery of mRNA 5?-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5?-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SL trans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SL trans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.

Vandenberghe, Amanda E.; Meedel, Thomas H.; Hastings, Kenneth E.M.

2001-01-01

349

The percutaneous trephine technique for harvesting iliac crest bone graft in alveolar clefts and its effect on donor site morbidity and quality of bone graft  

Microsoft Academic Search

The percutaneous trephine technique uses a bone marrow biopsy needle to harvest cancellous bone graft from the anterior iliac\\u000a crest. The subjects of this study were 41 patients with 47 alveolar clefts who underwent secondary bone grafting over a period\\u000a of 5 years, using the above technique. The donor site morbidity was evaluated retrospectively by means of a postal questionnaire

S. Jain; F. A. Mackay; T. M. Milward

1998-01-01

350

Indium-labeled white blood cells apheresed from donors receiving G-CSF localize to sites of inflammation when infused into allogeneic bone marrow transplant recipients  

Microsoft Academic Search

G-CSF administration to normal donors results in granulocyte apheresis yields generally greater than those observed with other neutrophil mobilizing agents. In vitro, neutrophils cultured with G-CSF exhibit prolonged survival; however, the random migration of neutrophils exposed to this agent is inhibited. Although transfused neutrophils mobilized with agents other than G-CSF migrate to sites of inflammation or infection in vivo, this

D Adkins; H Goodgold; L Hendershott; M Johnston; D Cravens; G Spitzer

1997-01-01

351

Potential therapeutic applications of antisense morpholino oligonucleotides in modulation of splicing in primary immunodeficiency diseases  

PubMed Central

Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate splicing process, such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs. This review discusses AMO-mediated splicing modulation approaches and their potential applications in PIDs.

Du, Liutao; Gatti, Richard A.

2011-01-01

352

Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila  

PubMed Central

To gain insight into splicing regulation, we developed a microarray to assay all annotated alternative splicing events in Drosophila melanogaster and identified the alternative splice events controlled by four splicing regulators: dASF/SF2, B52/SRp55, hrp48, and PSI. The number of events controlled by each of these factors was found to be highly variable: dASF/SF2 strongly affects >300 splicing events, whereas PSI strongly affects only 43 events. Pairwise analysis also revealed many instances of splice site usage affected by multiple factors and provides the framework to understand the network controlling the alternatively spliced mRNA isoforms that compose the Drosophila transcriptome.

Blanchette, Marco; Green, Richard E.; Brenner, Steven E.; Rio, Donald C.

2005-01-01

353

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

PubMed

Tongue reconstruction was performed using a deep inferior epigastric perforator (DIEP) free flap in a 6-year-old girl with undifferentiated sarcoma of the tongue. After hemi-glossectomy with upper neck dissection, a 3-lobed DIEP free flap was used for the reconstruction. Donor site was closed primarily with suturing umbilicus in proper position. No flap loss, leakage, or infection occurred. Postoperatively, the patient was able to consume a normal diet without difficulty or aspiration and displayed good speech function. No donor site morbidity, e.g., herniation or bulging, was observed, and the patient was able to perform their normal daily activities. DIEP flaps provide a pliable skin paddle, an adequate amount of adipose tissue, and reduced donor site morbidity, even in children. We did not have any difficulty harvesting the DIEP flap or with the microvascular anastomosis. We consider DIEP free flaps to be the ideal option for pediatric tongue reconstruction. © 2013 Wiley Periodicals, Inc. Microsurgery 33:487-490, 2013. PMID:23836433

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

2013-07-09

354

Use of a cryptic splice donor site in the chloramphenicol acetyltransferase (CAT)SV40 small-t antigen cassette generates alternative transcripts in transgenic rats  

Microsoft Academic Search

The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9kb), we identified an abundant 1.5kb

Z. D. Burke; T. Wells; D. A. Carter; R. Baler

2000-01-01

355

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  

Microsoft Academic Search

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 man-

INNOCENZO SAMMARCO; PAOLA GRIMALDI; PELLEGRINO ROSSI; MARCO CAPPA; COSTANZO MORETTI; GAETANO FRAJESE; RAFFAELE GEREMIA

2010-01-01

356

Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3' splice site.  

PubMed Central

A 12-yr-old hypothyroid girl was diagnosed at birth as athyreotic because her thyroid gland could not be visualized by isotope scanning. Goiter development due to incomplete thyrotropin suppression, a thyroidal radioiodide uptake of < 1%, and a low saliva to plasma ratio of 2.5 suggested iodide (I-) transport defect. mRNA isolated from her thyroid gland and injected into Xenopus oocytes failed to increase I- transport. Sequencing of the entire Na+/I- symporter (NIS) cDNA revealed a C to G transversion of nucleotide (nt) 1146 in exon 6, resulting in a Gln 267 (CAG) to Glu (GAG) substitution. This missense mutation produces an NIS with undetectable I- transport activity when expressed in COS-7 cells. Although only this missense mutation was identified in thyroid and lymphocyte cDNA, genotyping revealed that the proposita and her unaffected brother and father were heterozygous for this mutation. However, amplification of cDNA with a primer specific for the wild-type nt 1146 yielded a sequence lacking 67 nt. Genomic DNA showed a C to G transversion of nt 1940, producing a stop codon as well as a new downstream cryptic 3' splice acceptor site in exon 13, responsible for the 67 nt deletion, frameshift, and premature stop predicting an NIS lacking 129 carboxy-terminal amino acids. This mutation was inherited from the mother and present in the unaffected sister. Thus, although the proposita is a compound heterozygote, because of the very low expression (< 2.5%) of one mutant allele, she is functionally hemizygous for an NIS without detectable bioactivity.

Pohlenz, J; Rosenthal, I M; Weiss, R E; Jhiang, S M; Burant, C; Refetoff, S

1998-01-01

357

Diagnostics of pathogenic splicing mutations: does bioinformatics cover all bases?  

PubMed

Pathogenic splicing alterations caused by point mutations in both splice sites and auxiliary cis-regulatory elements are increasingly recognized as an important mechanism through which gene mutations cause human disease. Unfortunately, in routine genetic diagnostic settings, splicing mutations may escape identification, due to the lack of RNA samples. Since most patients are genotyped only, any computational prediction of mutation effects on splicing can be beneficial for the human geneticist. Here, we review common techniques to identify human point mutations and delineate the molecular basis for splice site recognition. Moreover, this article provides basic insights into web-tools predicting splice sites and cis-regulatory elements and discusses their benefits for judgment of clinically identified sequence variants of disease-specific genes. PMID:18508431

Hartmann, Linda; Theiss, Stephan; Niederacher, Dieter; Schaal, Heiner

2008-05-01

358

Alternative splicing in ascomycetes.  

PubMed

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation. PMID:23515838

Kempken, Frank

2013-03-21

359

Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei  

PubMed Central

Trans-splicing of leader sequences onto the 5?ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5?splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5? splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

Nilsson, Daniel; Gunasekera, Kapila; Mani, Jan; Osteras, Magne; Farinelli, Laurent; Baerlocher, Loic; Roditi, Isabel; Ochsenreiter, Torsten

2010-01-01

360

Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations.  

PubMed

UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5' and 3' of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype. PMID:22189268

Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P

2011-12-21

361

Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations  

PubMed Central

UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5? and 3? of each exon, typically 30?bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype.

Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P

2012-01-01

362

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

PubMed

3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha (MCCA) and smaller beta (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G-->A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G-->A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G-->A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G-->A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon. PMID:19706617

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

2009-08-24

363

Integrated Study of Ice-Rafted Debris, Temperaturess, and Stable Isotopes on a Spliced Record (piston cores and ODP Site 177-1090) From the South Atlantic  

NASA Astrophysics Data System (ADS)

We have conducted an integrated study of ice-rafted debris (IRD) and stable isotopes on a spliced record (TN057-6-PC4/ODP Site 177-1090, about 43° S, 9° E) raised on the Agulhas Ridge, in the South Atlantic. The site is just north of the northern boundary of the present-day Polar-Front Zone (PFZ), and is in a very sensitive location to record both ice-rafting and stable-isotopic-ratio changes. Our combined record reveals a pattern of ice-rafting episodes that may be characteristic for the subantarctic South Atlantic, at least for locations N of the PFZ. Ice rafting occurs during the waxing stages of each glaciation, and ends at, or before, the peak of each glaciation. IRD peaks are also associated with strong stadials during "cold" interglacials, e.g. MIS 7. A little IRD shows up during the entire interval studied here, from the Holocene to mid-MIS 14. We suggest that the IRD record at this site is essentially a temperature record on glacial-interglacial timescales. If the temperature is low enough, enough icebergs survive to melt at this location. If the temperature is too warm, only an occasional iceberg survives to deliver debris. A peculiar aspect of the combined record is the fact that during Ice-rafting events (IREs), the planktic oxygen-isotopic ratios are higher at the end of an IRE compared to the beginning. Further, by comparing our records with the Summer Sea Surface Temperature record of Becquey and Gersonde (2002) for a nearby (respectedly the same) site (PS2489-2/ODP177-1090), we see that the temperature is generally very similar at the beginning and the end of an IRE. The same age model provided by Venz and Hodell (2002) was used for both sites, allowing such direct comparisons of the data. Assuming as a working hypothesis that the IRD record is a pure temperature record, and ignoring the salinity effect for the present, then this difference in oxygen-isotopic ratios must be the ice volume effect. For MIS 12, the difference in planktic oxygen-isotopic ratios at the beginning and the end of the IRE is about 1.3 permil, which translates to 130 m sea level equivalent. The present-day temperature at the site is about 10° C (Levitus and Boyer, 1998). To attain a temperature of about 4° C (presently located at about 47° S in this area), as indicated by the SSST record of Becquey and Gersonde (2002) for the IRE during MIS 12, the Polar Front Zone (the zone of major iceberg melting) had to move north by about 4° latitude (about 240 nautical miles), a not unreasonable assumption.

Warnke, D. A.; Teitler, L.; Becquey, S.; Gersonde, R.; Venz, K.; Hodell, D. A.

2003-12-01

364

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

PubMed Central

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

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

2011-01-01

365

Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript  

PubMed Central

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Standiford, D. M.; Davis, M. B.; Sun, W.; Emerson-Jr., C. P.

1997-01-01

366

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

PubMed Central

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

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

2010-01-01

367

The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing  

Microsoft Academic Search

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.

Yehuda Brody; Noa Neufeld; Nicole Bieberstein; Sebastien Z. Causse; Eva-Maria Böhnlein; Karla M. Neugebauer; Xavier Darzacq; Yaron Shav-Tal

2011-01-01

368

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

PubMed Central

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

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

2008-01-01

369

Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 50 end  

Microsoft Academic Search

Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to .30 mRNAs. Productive infection depends on inef- ficient and regulated splicing and appears to pro- ceed in a tight 50 to 30 order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted

Jens Bohne; Harald Wodrich; Hans-Georg Krausslich

370

An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5? splice site of tau exon 10, whose misregulation causes frontotemporal dementia  

PubMed Central

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer’s disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.

Wang, Yan; Wang, Junning; Gao, Lei; Stamm, Stefan; Andreadis, Athena

2011-01-01

371

Topical phenytoin in the treatment of split-thickness skin autograft donor sites: a comparative study with polyurethane membrane drape and conventional dressing.  

PubMed

The effectiveness of topical phenytoin as a wound healing agent was compared with that of OpSite (Smith & Nephew) and a conventional topical antibiotic dressing (Soframycin, Roussel) in a controlled study of 60 patients with partial-thickness skin autograft donor sites on the lower extremities. Mean time to complete healing (complete epithelialization) was 6.2 +/- 1.6 days in the phenytoin-treated group (30 patients), compared to 8.6 +/- 2.2 days with OpSite (15 patients), and 12.6 +/- 3.4 days in the 15 Soframycin-treated patients. The differences between the treatment groups were significant at P < 0.001. Mean pain scores were also lower in the phenytoin-treated group, 0.40 +/- 0.55 vs. 0.66 +/- 0.60 with OpSite (P < 0.05) and 1.4 +/- 0.50 with the conventional dressing (P < 0.001). Both phenytoin and OpSite were superior to the Soframycin dressing with respect to bacterial contamination and wound infection as measured by Gram stains of wound smears, swab and aspirate (OpSite) cultures, and clinical assessments (P < 0.001) carried out on the fifth day of treatment. No local or systemic adverse effects of the three agents used were noted. Phenytoin appears to be an effective, low-cost and safe method for the treatment of partial-thickness skin graft donor sites, comparing very favourably with, and in some aspects superior to, occlusive dressings. Further clinical use and evaluation of topical phenytoin are merited. PMID:8357478

Yadav, J K; Singhvi, A M; Kumar, N; Garg, S

1993-08-01

372

Endoplasmic Reticulum Stress and Apoptosis Contribute to the Pathogenesis of Dominantly Inherited Isolated GH Deficiency Due to GH1 Gene Splice Site Mutations.  

PubMed

Dominantly inherited isolated GH deficiency is mainly caused by a heterozygous donor site mutation of intron 3 in the GH1 gene. An exon 3 deletion in GH (del32-71 GH) is produced from a mutant allele, whereas wild-type GH is produced from the other allele. Several studies have demonstrated a dominant negative effect of del32-71 GH on wild-type GH secretion, but the precise molecular mechanisms remain unclear. We hypothesized that unfolded del32-71 GH accumulates in the endoplasmic reticulum (ER) and causes ER stress and apoptosis in somatotrophs, promoting GH deficiency. To evaluate del32-71 GH-mediated ER stress, we established GH4C1 cell lines with doxycycline (dox)-controlled del32-71 GH expression. In 20 of 23 dox-controlled cell lines, the concentration of wild-type GH in the culture medium significantly decreased with del32-71 GH induction, demonstrating the dominant negative effect of this mutant. Cell viability, mRNA abundance of ER stress-response genes, caspase activation, and DNA fragmentation were evaluated in 5 dox-controlled cell lines selected as cellular models. In 4 of the 5 cell lines, del32-71 GH induction decreased cell viability, increased expression of 3 major ER stress response pathways (PRKR-like endoplasmic reticulum kinase [PERK], activating transcription factor-6 [ATF6], and inositol requirement 1 [IRE1]), and induced caspase-3 and caspase-7 activation. In 1 of the 4 cell lines, DNA fragmentation was demonstrated. Finally, overexpression of XBP1(S), a nuclear transcription factor downstream of IRE1, completely reversed the observed caspase activation. These data suggested that del32-71 GH-mediated ER stress and apoptosis contributed to the decrease in wild-type GH secretion observed in GH deficiency due to the GH1 gene slice-site mutations. PMID:23736291

Ariyasu, Daisuke; Yoshida, Hiderou; Yamada, Makoto; Hasegawa, Yukihiro

2013-06-04

373

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

SciTech Connect

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

Williams, C.J.; Ganguly, A.; Considine, E. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others

1996-06-14

374

Genomic features defining exonic variants that modulate splicing  

PubMed Central

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

2010-01-01

375

Human Immunodeficiency Virus Type 1 hnRNP A/B-Dependent Exonic Splicing Silencer ESSV Antagonizes Binding of U2AF65 to Viral Polypyrimidine Tracts  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3? splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3? splice sites. We further show evidence suggesting that U1 snRNP binds the 5? splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5? splice site interactions during virus replication are discussed.

Domsic, Jeffrey K.; Wang, Yibin; Mayeda, Akila; Krainer, Adrian R.; Stoltzfus, C. Martin

2003-01-01

376

Processing of carnitine octanoyltransferase pre-mRNAs by cis and trans-splicing.  

PubMed

Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur. PMID:10709632

Caudevilla, C; Serra, D; Miliar, A; Codony, C; Asins, G; Bach, M; Hegardt, F G

1999-01-01

377

Proximity-dependent and proximity-independent trans-splicing in mammalian cells.  

PubMed

Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not. PMID:18441053

Viles, Kristi D; Sullenger, Bruce A

2008-04-25

378

Proximity-dependent and proximity-independent trans-splicing in mammalian cells  

PubMed Central

Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not.

Viles, Kristi D.; Sullenger, Bruce A.

2008-01-01

379

Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis  

Microsoft Academic Search

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position 12 (relative to the 5 splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function

Avraham Hury; Hanoch Goldshmidt; Itai Dov Tkacz; Shulamit Michaeli

2009-01-01

380

A randomized comparison study of Aquacel Ag and Glucan II as donor site dressings with regard to healing time, cosmesis, infection rate, and patient's perceived pain: a pilot study.  

PubMed

This study was undertaken to compare pain, healing time, infection rate, and cosmetic outcome between Aquacel Ag (Convatec) and Glucan II (Brennan Medical) as donor site dressings. The authors performed a prospective, randomized, patient-controlled study. Eligible patients had two donor sites harvested. One site was dressed with Aquacel Ag and the other site with Glucan II. Patients were followed at set time points for 6 months to determine the rate of epithelialization, patient's perceived pain, infection rate, and the cosmetic outcome. A total of 20 patients were enrolled in the study. All patient data were collected through reepithelialization. The average time to wound healing for Aquacel Ag was 12.5 ± 2.07 days compared with Glucan II 12.7 ± 1.99 days. Perceived pain scores for each donor site were recorded. On postoperative day 5, patients reported significantly less pain with the Aquacel Ag site (Aquacel Ag 1.75 vs Glucan II 2.5, P = .02). Three donor sites showed clinical signs of infection (two Glucan II and one Aquacel Ag) prompting culture and dressing removal. There was no statistically significant difference in cosmetic outcomes of the donor sites at any time point. When comparing Aquacel Ag and Glucan II, our study has determined that there is no significant difference with regard to healing time, infection rates, and cosmetic outcomes. Both dressings are comparable with regard to ease of application and postoperative care. PMID:21844815

Bailey, Suzanne; Carmean, Melissa; Cinat, Marianne; Burton, Kimberly; Lane, Christopher; Malinoski, Darren