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1

Thermodynamic modeling of donor splice site recognition in pre-mRNA  

NASA Astrophysics Data System (ADS)

When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5' ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our “finding with binding” method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

Garland, Jeffrey A.; Aalberts, Daniel P.

2004-04-01

2

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

3

A novel splice donor site at nt 1534 is required for long-term maintenance of HPV31 genomes  

SciTech Connect

Human papillomaviruses (HPV) are small double-stranded DNA viruses that replicate as low copy number nuclear plasmids during the persistent phase. HPV only possess nine open reading frames but extend their coding capabilities by alternative RNA splicing. We have identified in cell lines with replicating HPV31 genomes viral transcripts that connect the novel splice donor (SD) sites at nt 1426 and 1534 within the E1 replication gene to known splice acceptors at nt 3295 or 3332 within the E2/E4 region. These transcripts are polyadenylated and are present at low amounts in the non-productive and productive phase of the viral life cycle. Mutation of the novel splice sites in the context of HPV31 genomes revealed that the inactivation of SD1534 had only minor effects in short-term replication assays but displayed a low copy number phenotype in long-term cultures which might be due to the expression of alternative E1 circumflex E4 or yet unknown viral proteins. This suggests a regulatory role for minor splice sites within E1 for papillomavirus replication.

Poppelreuther, Sven; Iftner, Thomas [Sektion Experimentelle Virologie, Institut fuer Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universitaetsklinikum Tuebingen, Elfriede-Aulhorn-Str. 6, 72076 Tuebingen (Germany); Stubenrauch, Frank [Sektion Experimentelle Virologie, Institut fuer Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universitaetsklinikum Tuebingen, Elfriede-Aulhorn-Str. 6, 72076 Tuebingen (Germany)], E-mail: frank.stubenrauch@med.uni-tuebingen.de

2008-01-05

4

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. PMID:16061557

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

2005-01-01

5

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

PubMed Central

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

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

2015-01-01

6

Further investigation of the HEXA gene intron 9 donor splice site mutation frequently found in non-Jewish Tay-Sachs disease patients from the British Isles  

Microsoft Academic Search

In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have

E C Landels; P M Green; I H Ellis; A H Fensom; M M Kaback; J Lim-Steele; K Zeiger; N Levy; M Bobrow

1993-01-01

7

Escape variants of the XPR1 gammaretrovirus receptor are rare due to reliance on a splice donor site and a short hypervariable loop.  

PubMed

Entry determinants in the XPR1 receptor for the xenotropic/polytropic mouse leukemia viruses (XP-MLVs) lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor and is evolutionarily conserved in vertebrate XPR1 genes; 2 of the 3 rare replacement mutations at this site destroy this receptor determinant. The 13 residue ECL4 is hypervariable, and replacement mutations carrying an intact ECL3 site alter but do not abolish receptor activity, including replacement of the entire loop with that of a jellyfish (Cnidaria) XPR1. Because ECL4 deletions are found in all X-MLV-infected Mus subspecies, we deleted each ECL4 residue to determine if deletion-associated restriction is residue-specific or is effected by loop size. All deletions influence receptor function, although different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations severely limits the likelihood of host escape mutations. PMID:25151060

Lu, Xiaoyu; Martin, Carrie; Bouchard, Christelle; Kozak, Christine A

2014-11-01

8

Further investigation of the HEXA gene intron 9 donor splice site mutation frequently found in non-Jewish Tay-Sachs disease patients from the British Isles.  

PubMed

In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have now tested, in a blind study, 26 American TSD carriers and 28 non-carriers who have British ancestry for the intron 9 splice site mutation. Six of the carriers and none of the controls were positive for the mutation. All six had Irish ancestry, compared with nine of the 20 other (intron 9 mutation negative) TSD carriers (p < 0.05). These results confirm the previously found high frequency of the intron 9 mutation in non-Jewish TSD families of British Isles, particularly Irish, origin, and reinforce the need to screen such families for this mutation. PMID:8326491

Landels, E C; Green, P M; Ellis, I H; Fensom, A H; Kaback, M M; Lim-Steele, J; Zeiger, K; Levy, N; Bobrow, M

1993-06-01

9

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

PubMed Central

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

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

1997-01-01

10

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

11

Compensatory relationship between splice sites and exonic splicing signals depending on the length of vertebrate introns  

PubMed Central

Background The signals that determine the specificity and efficiency of splicing are multiple and complex, and are not fully understood. Among other factors, the relative contributions of different mechanisms appear to depend on intron size inasmuch as long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode, Drosophila, and fungi. We explored the connections between the length of vertebrate introns, the strength of splice sites, exonic splicing signals, and evolution of flanking exons. Results A compensatory relationship is shown to exist between different types of signals, namely, the splice sites and the exonic splicing enhancers (ESEs). In the range of relatively short introns (approximately, < 1.5 kilobases in length), the enhancement of the splicing signals for longer introns was manifest in the increased concentration of ESEs. In contrast, for longer introns, this effect was not detectable, and instead, an increase in the strength of the donor and acceptor splice sites was observed. Conceivably, accumulation of A-rich ESE motifs beyond a certain limit is incompatible with functional constraints operating at the level of protein sequence evolution, which leads to compensation in the form of evolution of the splice sites themselves toward greater strength. In addition, however, a correlation between sequence conservation in the exon ends and intron length, particularly, in synonymous positions, was observed throughout the entire length range of introns. Thus, splicing signals other than the currently defined ESEs, i.e., potential new classes of ESEs, might exist in exon sequences, particularly, those that flank long introns. Conclusion Several weak but statistically significant correlations were observed between vertebrate intron length, splice site strength, and potential exonic splicing signals. Taken together, these findings attest to a compensatory relationship between splice sites and exonic splicing signals, depending on intron length. PMID:17156453

Dewey, Colin N; Rogozin, Igor B; Koonin, Eugene V

2006-01-01

12

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

13

Accurate splice site prediction using support vector machines  

PubMed Central

Background For splice site recognition, one has to solve two classification problems: discriminating true from decoy splice sites for both acceptor and donor sites. Gene finding systems typically rely on Markov Chains to solve these tasks. Results In this work we consider Support Vector Machines for splice site recognition. We employ the so-called weighted degree kernel which turns out well suited for this task, as we will illustrate in several experiments where we compare its prediction accuracy with that of recently proposed systems. We apply our method to the genome-wide recognition of splice sites in Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Danio rerio, and Homo sapiens. Our performance estimates indicate that splice sites can be recognized very accurately in these genomes and that our method outperforms many other methods including Markov Chains, GeneSplicer and SpliceMachine. We provide genome-wide predictions of splice sites and a stand-alone prediction tool ready to be used for incorporation in a gene finder. Availability Data, splits, additional information on the model selection, the whole genome predictions, as well as the stand-alone prediction tool are available for download at . PMID:18269701

Sonnenburg, Sören; Schweikert, Gabriele; Philips, Petra; Behr, Jonas; Rätsch, Gunnar

2007-01-01

14

Prediction of human mRNA donor and acceptor sites from the DNA sequence*1  

Microsoft Academic Search

Artificial neural networks have been applied to the prediction of splice site location in humanpre--mRNA. A joint prediction scheme where prediction of transition regions between intronsand exons regulates a cutoff level for splice site assignment was able to predict splice sitelocations with confidence levels far better than previously reported in the literature. Theproblem of predicting donor and acceptor sites in

Søren Brunak; Jacob Engelbrecht; Steen Knudsen

1991-01-01

15

An abnormal splice donor site in one allele of the thyroid peroxidase gene in FRTL5 rat thyroid cells introduces a premature stop codon: association with the absence of functional enzymatic activity.  

PubMed

Low stringency screening of an FRTL5 cDNA library with a human thyroid peroxidase (TPO) cDNA probe yielded two different types of TPO cDNA clones. One type contained the full-length structural gene, but there was an A at the first nucleotide of an intronic splice donor site that leads to alternate splicing, with the retention of part of an intron. This 54-basepair retained intron fragment contains a premature inframe stop codon that would truncate the protein at its carboxyl-terminus by 71% with the loss of enzymatic activity. The second type of TPO cDNA does not contain the retained intron fragment and premature stop codon, but it is not full-length and lacks 580-680 basepairs at its 5' end. However, Northern blot analysis reveals only full-length copies (3.2 kilobases) of TPO mRNA in FRTL5 cells, and this 5' truncation is, therefore, an artifact of library construction. The relative proportions of the two types of TPO mRNA in FRTL5 cells was determined by the polymerase chain reaction, using as template single stranded cDNA generated by reverse transcription of FRTL5 mRNA. Slightly more than half of the TPO mRNA in the FRTL5 cells represented the form with the abnormal splice donor site. The relative proportions of the two TPO mRNA forms was not influenced by TSH stimulation of the FRTL5 cells, and the proportion remained unaltered even after the FRTL5 cells were subcloned by limiting dilution. At the genomic level, we used allele-specific oligonucleotides for the mutant and normal forms of TPO, and found FRTL5 cells to have both normal and abnormal TPO alleles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2233737

Derwahl, M; Seto, P; Rapoport, B

1990-06-01

16

Improved splice site detection in Genie  

SciTech Connect

We present an improved splice site predictor for the genefinding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including sensors that integrate matches to homologous sequences from a database. One of the hardest problems in genefinding is to determine the complete gene structure correctly. The splice site sensors are the key signal sensors that address this problem. We replaced the existing splice site sensors in Genie with two novel neural networks based on dinucleotide frequencies. Using these novel sensors, Genie shows significant improvements in the sensitivity and specificity of gene structure identification. Experimental results in tests using a standard set of annotated genes showed that Genie identified 82% of coding nucleotides correctly with a specificity of 81%, versus 74% and 81% in the older system. In further splice site experiments, we also looked at correlations between splice site scores and intron and exon lengths, as well as the effect of distance to the nearest splice site on false positive rates. 27 refs., 8 figs., 3 tabs.

Reese, M.G.; Eeckman, F.H. [Lawrence Berkeley National Lab., CA (United States); Kulp, D. [Univ. of California, Santa Cruz, CA (United States)] [and others

1997-12-01

17

Unusual splice site mutations disrupt FANCA exon 8 definition.  

PubMed

The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position -3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition. PMID:24704046

Mattioli, Chiara; Pianigiani, Giulia; De Rocco, Daniela; Bianco, Anna Monica Rosaria; Cappelli, Enrico; Savoia, Anna; Pagani, Franco

2014-07-01

18

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype  

SciTech Connect

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

19

Two novel mutations in splice donor sites of CYP11B1 in congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency.  

PubMed

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Genetic analysis showed two new base substitutions of CYP11B1, a conservative transition at the last base of exon 5, and a IVS8+4A-->G transition in intron 8. Difficulties with suppressive therapy resulted in severe hypertension. A laparoscopic adrenalectomy was decided which lead to normalization of blood pressure. In vitro, steroidogenesis by adrenal cells showed no measurable 11beta-hydroxylase activity. Analysis of CYP11B1 mRNA by RT-PCR and sequencing showed expression of a mRNA which lacked exon 8, presumably resulting from the intron 8 mutation. In addition a highly truncated mRNA was detected corresponding to exons 1, 2, 8, 9, with the loss of exons 3-7, presumably related to the exon 5 mutation. Western blot analysis showed a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus adrenalectomy in this patient allowed effective treatment of severe hypertension and helped to understand the mechanisms of two novel mutations responsible for aberrant splicing of CYP11B1. PMID:11196457

Chabre, O; Portrat-Doyen, S; Vivier, J; Morel, Y; Defaye, G

2000-11-01

20

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

21

Improved splice site detection in Genie  

Microsoft Academic Search

We present an improved splice site predictor for the genehnding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including

Martin G. Reese; Frank H. Eeckman; David Kulp; David Haussler

1997-01-01

22

iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition  

PubMed Central

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

Feng, Peng-Mian; Chou, Kuo-Chen

2014-01-01

23

SpliceAid-F: a database of human splicing factors and their RNA-binding sites.  

PubMed

A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called 'SpliceAid-F' (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA-SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA-SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 'no-binding' sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements. PMID:23118479

Giulietti, Matteo; Piva, Francesco; D'Antonio, Mattia; D'Onorio De Meo, Paolo; Paoletti, Daniele; Castrignanò, Tiziana; D'Erchia, Anna Maria; Picardi, Ernesto; Zambelli, Federico; Principato, Giovanni; Pavesi, Giulio; Pesole, Graziano

2013-01-01

24

Splice site strength–dependent activity and genetic buffering by poly-G runs  

E-print Network

Pre-mRNA splicing is regulated through the combinatorial activity of RNA motifs, including splice sites and splicing regulatory elements. Here we show that the activity of the G-run (polyguanine sequence) class of splicing ...

Xiao, Xinshu

25

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. PMID:8878424

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

1996-01-01

26

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.  

PubMed

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

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

2014-01-01

27

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

PubMed Central

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

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

2014-01-01

28

Splicing proofreading at 5? splice sites by ATPase Prp28p  

PubMed Central

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5? splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5?SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5?SS. Prp28p is thought to facilitate the disruption of 5?SS–U1 snRNA pairing to allow for 5?SS–U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5?SS proofreading by Prp28p is dependent on competition with the stability of the 5?SS:U6 duplex, but not the 5?SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5?SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5?SS. PMID:23462954

Yang, Fei; Wang, Xiu-Ye; Zhang, Zhi-Min; Pu, Jia; Fan, Yu-Jie; Zhou, Jiahai; Query, Charles C.; Xu, Yong-Zhen

2013-01-01

29

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. PMID:23716637

Davidson, Lee; West, Steven

2013-01-01

30

Analysis and prediction of gene splice sites in four Aspergillus genomes.  

PubMed

Several Aspergillus fungal genomic sequences have been published, with many more in progress. Obviously, it is essential to have high-quality, consistently annotated sets of proteins from each of the genomes, in order to make meaningful comparisons. We have developed a dedicated, publicly available, splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window size on single local networks for training, to cover both donor and acceptor site information. We have applied NetAspGene to other Aspergilli, including Aspergillus nidulans, Aspergillus oryzae, and Aspergillus niger. Evaluation with independent data sets reveal that NetAspGene performs substantially better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene. PMID:18948220

Wang, Kai; Ussery, David Wayne; Brunak, Søren

2009-03-01

31

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. PMID:24155930

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

2013-01-01

32

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

PubMed

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 reading frame intact; however, TYMP protein was not detected by immunoblot analysis, suggesting that neither mutant allele is expressed as protein. The patient's fibroblasts showed gradual loss of the mitochondrial DNA-encoded subunit I of cytochrome-c oxidase, suggesting a progressive mitochondrial DNA defect in culture. PMID:19056268

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

2009-02-01

33

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

PubMed Central

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

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

2014-01-01

34

Characterization of human RNA splice signals by iterative functional selection of splice sites.  

PubMed Central

An iterative in vitro splicing strategy was employed to select for optimal 3' splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3' end of the randomized insert. Mutating the 3' terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly. PMID:10786844

Lund, M; Tange, T O; Dyhr-Mikkelsen, H; Hansen, J; Kjems, J

2000-01-01

35

Heteroduplex formation and S1 digestion for mapping alternative splicing sites.  

PubMed

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies. PMID:18949713

Ferreira, E N; Rangel, M C R; Pineda, P B; Vidal, D O; Camargo, A A; Souza, S J; Carraro, D M

2008-01-01

36

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

37

Dual-specificity splice sites function alternatively as 5 and 3 splice sites  

E-print Network

-scale sequencing projects and recent splicing- microarray studies, estimates of mammalian genes expressing multiple genome- wide, high-quality alignment of mRNA/EST and genome sequences and experimentally verified by RT of genome sequences and a large amount of mRNA/EST data, especially in human and mouse, genome

38

Differing patterns of selection in alternative and constitutive splice sites  

PubMed Central

In addition to allowing identification of putative functional elements as regions having reduced substitution rates, comparison of genome sequences can also provide insights into these elements at the nucleotide level, by indicating the pattern of tolerated substitutions. We created data sets of orthologous alternative and constitutive splice sites in mouse, rat, and human and analyzed the substitutions occurring within them. Our results illuminate differences between alternative and constitutive sites and, in particular, strongly support the idea that alternative sites are under selection to be weak. PMID:17556528

Garg, Kavita; Green, Phil

2007-01-01

39

Intrinsic differences between authentic and cryptic 5? splice sites  

PubMed Central

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 close to the authentic 5?ss. Statistical analysis of the strengths of the 5?ss using the Shapiro and Senapathy matrix revealed that authentic 5?ss have significantly higher score values than cryptic 5?ss, which in turn have higher values than the mutant ones. ?-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the ?-globin authentic and cryptic 5?ss, but not their surrounding context, determine the correct 5?ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5?ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5?ss-disrupting mutations at the mRNA level. PMID:14576320

Roca, Xavier; Sachidanandam, Ravi; Krainer, Adrian R.

2003-01-01

40

Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene.  

PubMed

Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype. PMID:15968391

Ding, Qiulan; Wu, Wenman; Fu, Qihua; Wang, Xuefeng; Hu, Yiqun; Wang, Hongli; Wang, Zhenyi

2005-06-01

41

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

PubMed Central

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

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

2014-01-01

42

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 PMID:8730289

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

43

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. PMID:22488673

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

44

U2AF1 mutations alter splice site recognition in hematological malignancies  

PubMed Central

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

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

2015-01-01

45

The spliced leader sequence of Trypanosoma brucei has a potential role as a cap donor structure.  

PubMed Central

Trypanosoma brucei brucei and other trypanosomatid species are unique among eucaryotes because transcription of their protein-coding genes is discontinuous. The 5' ends of their mRNAs consist of an identical 35-nucleotide spliced leader which is encoded at a separate locus from that for the body of the protein-coding transcript. We show here that the spliced leader transcript contains a 5' cap structure and suggest that at least one function of the spliced leader sequence is to provide a cap structure to trypanosome mRNAs. Images PMID:3837191

Lenardo, M J; Dorfman, D M; Donelson, J E

1985-01-01

46

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

47

Flap Donor Site Size Reduction with Substratum Horizontal Mattress Suture  

PubMed Central

BACKGROUND Closure of donor site of the flap has special problems. Reduction of this site will decrease the morbidity of operation. In this study, we present our experience in donor site size reduction. METHODS Between 2006 and 2008, 15 patients with skin and soft tissue defects underwent operation. In all patients, coverage of defect was performed with various flaps. Substratum horizontal mattress suture was used to reduce donor site dimensions. In all 15 patients, size of the flaps, the defect after the flap elevation and the scar size were measured. RESULTS The mean size of the flap, the defect after flap elevation, and the scar after 3 months were 43.9 cm2, 69.4 cm2, and 32.2 cm2, respectively. There was 46.5% reduction in the donor site after using this suture. CONCLUSION The substratum horizontal mattress suture was shown to de- crease the donor site dimensions and also its scar size in flap surgery. This suture is highly recommend in order to reduce donor site dimensions. PMID:25489499

Jalilimanesh, Mohammad

2013-01-01

48

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

PubMed

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

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

2014-03-01

49

The architecture of pre-mRNAs affects mechanisms of splice-site pairing  

E-print Network

nuclear ribonucleoprotein particle with the 5 splice site, branch-point-binding protein SF1 (received for review March 30, 2005) The exon intron architecture of genes determines whether com- ponents intron length Pre-mRNA splicing is an essential process that accounts for many aspects of regulated gene

Hertel, Klemens J.

50

Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons  

PubMed Central

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence. PMID:25030907

Szafranski, Karol; Fritsch, Claudia; Schumann, Frank; Siebel, Lisa; Sinha, Rileen; Hampe, Jochen; Hiller, Michael; Englert, Christoph; Huse, Klaus; Platzer, Matthias

2014-01-01

51

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles  

Microsoft Academic Search

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish

E C Landels; P M Green; I H Ellis; A H Fensom; M Bobrow

1992-01-01

52

A novel otoferlin splice-site mutation in siblings with auditory neuropathy spectrum disorder.  

PubMed

We characterize a novel otoferlin mutation discovered in a sibling pair diagnosed with auditory neuropathy spectrum disorder and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the otoferlin splice donor site of exon 28 (IVS28 + 1G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (0.78 ms) compared to subjects with sensorineural hearing loss (average: adults 0.71 ms, children 0.85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered. PMID:24135434

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

2013-01-01

53

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

PubMed Central

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

2011-01-01

54

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

PubMed

Mutations affecting splice sites comprise approximately 7.5?% of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9?%, 14.2?%, 5.2?%, 19.2?%, 1.8?% and 2.5?% of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations. PMID:25503412

Liang, Q; Xiang, M; Lu, Y; Ruan, Y; Ding, Q; Wang, X; Xi, X; Wang, H

2015-03-01

55

Detection of somatic TP53 splice site mutations in diffuse astrocytomas.  

PubMed

Alteration in TP53 is the most common genetic event reported for many tumors, including astrocytomas. The majority of studies, on analyzing TP53 mutations, have not included all splice junctions. Consequently, splice site mutations are thought to be relatively infrequent. TP53 were examined for mutations by polymerase chain reaction, single strand conformation polymorphism and direct sequencing in cases of diffuse astrocytomas. We found TP53 mutations in 17.8% (8 out of 45) of the tumors tested: 3 splicing, 3 missense and 2 silent mutations. We have shown that splice site mutations of TP53 are more frequent than previously reported. These findings emphasize the importance of thorough screening of TP53 mutations in gliomas. PMID:15914282

Uno, Miyuki; Oba-Shinjo, Sueli Mieko; de Aguiar, Paulo Henrique; Leite, Claudia C; Rosemberg, Sérgio; Miura, Flávio K; Junior, Raul Marino; Scaff, Milberto; Nagahashi Marie, Suely Kazue

2005-06-28

56

SplicingTypesAnno: Annotating and quantifying alternative splicing events for RNA-Seq data.  

PubMed

Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. PMID:25720307

Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

2015-04-01

57

Splice-site pairing is an intrinsically high fidelity process  

E-print Network

splicing accuracy, including core components of the spliceosome, as well as Isy1, Prp8p, Slu7p, and Sky1p (5­8). More recent studies have shown that Prp16 and Prp22p act as ATP-dependent proofreading factors

Hertel, Klemens J.

58

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles.  

PubMed

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish subjects (15 TSD carriers, four TSD patients, and one TSD fetus), five had mutations common in the Ashkenazi Jewish community, and 10 had the intron 9 splice site mutation. This is an unexpected result considering the diverse origin of the population of the British Isles. This mutation was not found in 28 control UK subjects or 11 Jewish carriers of known TSD mutations. Before attempting detection of unknown mutations, non-Jewish TSD carriers from the British Isles should be screened for the intron 9 donor splice site mutation as well as those mutations which predominate in the Jewish community. PMID:1387685

Landels, E C; Green, P M; Ellis, I H; Fensom, A H; Bobrow, M

1992-08-01

59

Novel Splice-Site Mutation in SMN1 Associated with a very Severe SMA-I Phenotype.  

PubMed

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of motor neurons and muscle weakness and atrophy. The majority of patients harbor homozygous SMN1 deletions, resulting in an SMN1-null genotype. A variable number of copies of SMN2, the centromeric copy of SMN1, fails to compensate for the absence of SMN1 but can act as a modifier. Less than 5 % of patients with SMA display intragenic mutations on the second allele, detectable by direct sequencing. The effects of these mutations are not easily predictable, hindering a clear correlation with the clinical phenotype. We describe a novel SMN1 mutation that affected the donor splice site of exon 7 and resulted in an unusually severe SMA phenotype with rapid fatal outcome in an Italian infant. PMID:25572663

Ronchi, Dario; Previtali, Stefano Carlo; Sora, Maria Grazia Natali; Barera, Graziano; Del Menico, Benedetta; Corti, Stefania; Bresolin, Nereo; Comi, Giacomo Pietro

2015-05-01

60

Requirement of a polypyrimidine tract for trans-splicing in trypanosomes: discriminating the PARP promoter from the immediately adjacent 3' splice acceptor site.  

PubMed Central

We studied sequence requirements for trans-splicing at the 3' splice acceptor site of a procyclic acidic repetitive protein (PARP) coding gene in trypanosomes. In transient CAT transfection assays with linker scanning (LS) mutants in a PARP promoter--3' splice acceptor site--CAT construct, minor differences in the sequence composition of the polypyrimidine tract (nt -36 to -5 with respect to the 3' splice acceptor site) severely affected the CAT activity. Analysis of steady-state CAT RNA in stably transformed trypanosomes revealed that the LS mutations had indeed affected the pre-mRNA splicing efficiency. The data indicate that mini-exon addition is not required simply for maturation of polycistronic pre-mRNA but is also essential for the generation of functional mRNA from monocistronic genes, since unspliced monocistronic pre-mRNA did not accumulate or allow synthesis of CAT. We postulate that mini-exon addition at polycistronically transcribed genes, which can have drastically different polypyrimidine tracts at each of their 3' splice acceptor sites, can occur with different efficiencies for each gene of the array thus affecting mRNA abundance. Images PMID:1935907

Huang, J; Van der Ploeg, L H

1991-01-01

61

Donor’s site evaluation after restoration with autografts or synthetic plugs in rabbits  

PubMed Central

AIM: To investigate donor site’s area histological and immunohistochemical knee cartilage appearances after resurfacing iatrogenic defects with biosynthetic plugs orautografts. METHODS: Thirty New Zealand White rabbits were used in this study. A full-thickness cylindrical defect of 4.5 mm (diameter) × 7 mm (depth) was created with a hand drill in the femoral groove of every animal. In Group A (n = 10) the defect of the donor site was repaired with a biosynthetic osteochondral plug, in Group B (n = 10) with an osteochondral autograft, while in Group C (control group of 10) rabbits were left untreated. RESULTS: Twenty-four weeks postoperatively, smooth articular cartilage was found macroscopically in some trocleas’ surfaces; in all others, an articular surface with discontinuities was observed. Twenty-eight out of 30 animals were found with predominantly viable chondrocytes leaving the remaining two -which were found only in the control group- with partially viable chondrocytes. However, histology revealed many statistical differences between the groups as far as the International Cartilage Repair Society (ICRS) categories are concerned. Immunofluoresence also revealed the presence of collagen II in all specimens of Group B, whereas in Group A collagen II was found in less specimens. In Group C collagen IIwas not found. CONCLUSION: The matrix, cell distribution, subchondral bone and cartilage mineralization ICRS categories showed statistically differences between the three groups. Group A was second, while group B received the best scores; the control group got the worst ICRS scores in these categories. So, the donor site area, when repairing osteochondral lesions with autografting systems, is better amended with osteochondral autograft rather than bone graft substitute implant. PMID:25232531

Intzoglou, Konstantinos S; Mastrokalos, Dimitrios S; Korres, Dimitrios S; Papaparaskeva, Kleo; Koulalis, Dimitrios; Babis, George C

2014-01-01

62

Nonconsensus Branch-Site Sequences in the in vitro Splicing of Transcripts of Mutant Rabbit beta -globin Genes  

Microsoft Academic Search

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

Richard A. Padgett; Maria M. Konarska; Markus Aebi; Horst Hornig; Charles Weissmann; Phillip A. Sharp

1985-01-01

63

Cross-kingdom patterns of alternative splicing and splice recognition  

PubMed Central

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

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

2008-01-01

64

Prediction of Splice Sites in Plant Pre-mRNA from Sequence Properties  

E-print Network

Prediction of Splice Sites in Plant Pre-mRNA from Sequence Properties Volker Brendel1 *, Ju et al., 1994; Brendel et al., 1998). Plant intron recognition by sequence inspection serves two primary goals. First, genome sequen- cing efforts produce copious raw sequence data that must initially

Brendel, Volker

65

Features of 5?-splice-site efficiency derived from disease-causing mutations and comparative genomics  

PubMed Central

Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5?-splice-site (5?ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies between 5?ss nucleotides as a conserved feature of the entire set of 5?ss. These dependencies are also conserved in human–mouse pairs of orthologous 5?ss. Many disease-associated 5?ss mutations disrupt these dependencies, as can some human SNPs that appear to alter splicing. The consistency of the evidence signifies the relevance of this approach and suggests that 5?ss SNPs play a role in complex diseases. PMID:18032726

Roca, Xavier; Olson, Andrew J.; Rao, Atmakuri R.; Enerly, Espen; Kristensen, Vessela N.; Børresen-Dale, Anne-Lise; Andresen, Brage S.; Krainer, Adrian R.; Sachidanandam, Ravi

2008-01-01

66

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier  

PubMed Central

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies. PMID:20949073

Hinzpeter, Alexandre; Aissat, Abdel; Sondo, Elvira; Costa, Catherine; Arous, Nicole; Gameiro, Christine; Martin, Natacha; Tarze, Agathe; Weiss, Laurence; de Becdelièvre, Alix; Costes, Bruno; Goossens, Michel; Galietta, Luis J.; Girodon, Emmanuelle; Fanen, Pascale

2010-01-01

67

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

68

Cross-kingdom patterns of alternative splicing and splice recognition  

E-print Network

Background: Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently ...

McGuire, Abigail Manson

69

A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signaling pathway and causes Lenz microphthalmia syndrome  

PubMed Central

Introduction Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. Methods and results Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T?A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. Conclusions We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway. PMID:24431331

Esmailpour, Taraneh; Riazifar, Hamidreza; Liu, Linan; Donkervoort, Sandra; Huang, Vincent H; Madaan, Shreshtha; Shoucri, Bassem M; Busch, Anke; Wu, Jie; Towbin, Alexander; Chadwick, Robert B; Sequeira, Adolfo; Vawter, Marquis P; Sun, Guoli; Johnston, Jennifer J; Biesecker, Leslie G; Kawaguchi, Riki; Sun, Hui; Kimonis, Virginia; Huang, Taosheng

2014-01-01

70

The Consensus 5' Splice Site Motif Inhibits mRNA Nuclear Export  

PubMed Central

In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3’terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5’splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3’cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3’terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5’splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise. PMID:25826302

Lee, Eliza S.; Akef, Abdalla; Mahadevan, Kohila; Palazzo, Alexander F.

2015-01-01

71

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

72

Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation  

Microsoft Academic Search

The formation of base-pairing between the branch-site (BS) sequence and the U2 snRNP is an important step in mRNA splicing. We developed a new algorithm to identify both the BS sequence and the polypyrimidine tract (PPT) and validated its predictions experimentally. To assess BS conservation between human and mouse, we assembled and analyzed 46 812 and 242 constitutively and alternatively

Guy Kol; Galit Lev-Maor; Gil Ast

2005-01-01

73

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

PubMed Central

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

2014-01-01

74

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

PubMed

Large-scale RNA sequencing has revealed a large number of long mRNA-like transcripts (lncRNAs) that do not code for proteins. The evolutionary history of these lncRNAs has been notoriously hard to study systematically due to their low level of sequence conservation that precludes comprehensive homology-based surveys and makes them nearly impossible to align. An increasing number of special cases, however, has been shown to be at least as old as the vertebrate lineage. Here we use the conservation of splice sites to trace the evolution of lncRNAs. We show that >85% of the human GENCODE lncRNAs were already present at the divergence of placental mammals and many hundreds of these RNAs date back even further. Nevertheless, we observe a fast turnover of intron/exon structures. We conclude that lncRNA genes are evolutionary ancient components of vertebrate genomes that show an unexpected and unprecedented evolutionary plasticity. We offer a public web service (http://splicemap.bioinf.uni-leipzig.de) that allows to retrieve sets of orthologous splice sites and to produce overview maps of evolutionarily conserved splice sites for visualization and further analysis. An electronic supplement containing the ncRNA data sets used in this study is available at http://www.bioinf.uni-leipzig.de/publications/supplements/12-001. PMID:25802408

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

2015-05-01

75

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

PubMed Central

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

Umen, J. G.; Guthrie, C.

1996-01-01

76

Cwc21p promotes the second step conformation of the spliceosome and modulates 3? splice site selection  

PubMed Central

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

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

2015-01-01

77

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

78

Rational selection of flaps from the abdomen in breast reconstruction to reduce donor site morbidity  

Microsoft Academic Search

Reconstruction of the female breast following mastectomy has become commonplace. The number of donor sites have increased as the quest both for improving reconstruction and reducing morbidity continues. There are a number of donor sites which resemble breast tissue in terms of skin texture, suppleness and colour. The ‘gold standard’ for transfer in breast reconstruction, however, is the lower abdominal

Z. M. Arnez; U. Khan; D. Pogorelec; F. Planinsek

1999-01-01

79

BAP1 Missense Mutation c.2054 A>T (p.E685V) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site in a Human Mesothelioma Cell Line  

PubMed Central

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

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

2015-01-01

80

Conserved stem-loop structures in the HIV1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing  

Microsoft Academic Search

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs .W hether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previousl yb een examined. Here we have determined the secondary structure of the HIV-1\\/BRU RNA segment, containing the alternative A3, A4a, A4b, A4 ca nd A5 3' splice sites .S ite A3, required for tat mRNA

Sandrine Jacquenet; S. Bilodeau; L aurence Damier; Annie Mougin; C. M artin Stoltzfus; Christiane Branlant

2001-01-01

81

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

PubMed Central

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC3 (NM_003786.3:c.1783-1G>A), KLHDC1 (NM_172193.1:c.568-2A>G), HOOK1 (NM_015888.4:c.1662-1G>A), SMAD9 (NM_001127217.2:c.1004-1C>T), and DNAH9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC3, HOOK1. In ABCC3 and HOOK1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4–6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis. PMID:24498620

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

2013-01-01

82

Using multiple donor sites for enhanced flood estimation in ungauged catchments  

NASA Astrophysics Data System (ADS)

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

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

2014-08-01

83

Constant Splice-Isoform Ratios in Human Lymphoblastoid Cells Support the Concept of a Splico-Stat  

PubMed Central

Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. PMID:21220357

Kramer, Marcel; Huse, Klaus; Menzel, Uwe; Backhaus, Oliver; Rosenstiel, Philip; Schreiber, Stefan; Hampe, Jochen; Platzer, Matthias

2011-01-01

84

Correct mRNA Processing at a Mutant TT Splice Donor in FANCC Ameliorates the Clinical Phenotype in Patients and Is Enhanced by Delivery of Suppressor U1 snRNAs  

PubMed Central

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5? splice sites (5?ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5?ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5?ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5?ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations. PMID:20869034

Hartmann, Linda; Neveling, Kornelia; Borkens, Stephanie; Schneider, Hildegard; Freund, Marcel; Grassman, Elke; Theiss, Stephan; Wawer, Angela; Burdach, Stefan; Auerbach, Arleen D.; Schindler, Detlev; Hanenberg, Helmut; Schaal, Heiner

2010-01-01

85

Identification of rare alternative splicing events in MS/MS data reveals a significant fraction of alternative translation initiation sites  

PubMed Central

Integration of transcriptome data is a crucial step for the identification of rare protein variants in mass-spectrometry (MS) data with important consequences for all branches of biotechnology research. Here, we used Splooce, a database of splicing variants recently developed by us, to search MS data derived from a variety of human tumor cell lines. More than 800 new protein variants were identified whose corresponding MS spectra were specific to protein entries from Splooce. Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events. Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites. Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences. To make the dataset generated here available to the community in a more effective form, the Splooce portal (http://www.bioinformatics-brazil.org/splooce) was modified to report the alternative splicing events supported by MS data. PMID:25405079

Kroll, José E.; de Souza, Sandro J.

2014-01-01

86

Sequences homologous to 5' splice sites are required for the inhibitory activity of papillomavirus late 3' untranslated regions.  

PubMed

Expression of bovine papillomavirus type 1 (BPV-1) late genes is limited to terminally differentiated keratinocytes in an infected epithelium. We have previously shown that although the BPV-1 late polyadenylation site is functional in nonpermissive cells, a 53-nucleotide (nt) fragment of the late 3' untranslated region acts posttranscriptionally to reduce polyadenylated cytoplasmic RNA levels. This 53-nt fragment does not appear to function by destabilizing polyadenylated cytoplasmic RNA (P. A. Furth and C. C. Baker, J. Virol. 65:5806-5812, 1991). In this study, we used site-directed mutagenesis and deletion analysis to demonstrate that the sequence AAG/GUAAGU, which is identical to the consensus 5' splice site sequence, was both necessary and sufficient for the inhibitory activity of the 53-nt fragment. Furthermore, base pairing between the 5' end of the U1 small nuclear RNA and this 5' splice site-like sequence was shown to be required for the inhibitory activity in vivo. We have also further mapped the human papillomavirus type 16 late 3' inhibitory element (I. M. Kennedy, J. K. Haddow, and J. B. Clements, J. Virol. 65:2093-2097, 1991) to a 51-nt region containing four overlapping sequence motifs with partial homology to 5' splice sites. Mutation of each of these motifs demonstrated that only one of these motifs is required for the inhibitory activity. However, the presence of the other motifs may contribute to the full inhibitory activity of the element. No BPV-1 or human papillomavirus type 16 mRNAs which are spliced by using the potential 5' splice sites present in the viral late 3' untranslated regions have been identified. This suggests that the primary function of these 5' splice site-like sequences is the inhibition of late gene expression. The most likely mechanism of action of these elements is reduction of polyadenylation efficiency, perhaps through interference with 3'-terminal exon definition. PMID:8035806

Furth, P A; Choe, W T; Rex, J H; Byrne, J C; Baker, C C

1994-08-01

87

A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures  

Microsoft Academic Search

A clean data set of verified splice sites from Homo sapiens are reported as well as the standards used for the clean-up procedure. The sites were validated by: (i) standard cleaning procedures such as requiring consistency in the annotation of the gene structural elements, completeness of the coding regions and elimination of redundant sequences; (ii) clustering by decision trees coupled

Thangavel Alphonse Thanaraj

1999-01-01

88

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

PubMed

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 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), 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. PMID:23175611

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

2013-01-01

89

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

PubMed Central

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

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

2011-01-01

90

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

PubMed

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

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

2011-04-01

91

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

2012-09-15

92

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures  

SciTech Connect

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. 16 refs., 3 tabs.

Hunter, T.C.; Albertini, R.J.; O`Neill, J.P. [Univ. of Vermont Genetics Lab., Burlington, VT (United States)] [and others

1996-03-01

93

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

94

Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.  

PubMed Central

The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step. PMID:11780639

Collins, C A; Guthrie, C

2001-01-01

95

Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity  

PubMed Central

Background: Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity. Methods: The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG). Results: After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the “fish-net” patterning caused by STSG. Conclusion: Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing. PMID:25289241

Wang, Ying; Farinelli, William A.; Jiménez-Lozano, Joel; Franco, Walfre; Sakamoto, Fernanda H.; Cheung, Evelyn J.; Purschke, Martin; Doukas, Apostolos G.; Anderson, R. Rox

2013-01-01

96

A novel splice site mutation in EYA4 causes DFNA10 hearing loss.  

PubMed

Nonsyndromic autosomal dominant sensorineural hearing loss (SNHL) at the DFNA10 locus was described in two families in 2001. Causative mutations that affect the EyaHR domain of the 'Eyes absent 4' (EYA4) protein were identified. We report on the clinical and genetic analyses of an Australian family with nonsyndromic SNHL. Screening of the EYA4 gene showed the novel polypyrimidine tract variation ca. 1,282-12T > A that introduces a new 3' splice acceptor site. This is the first report of a point mutation in EYA4 that is hypothesized to lead to aberrant pre-mRNA splicing and human disease. The DFNA10 family described is only the fourth to be identified. One individual presented with apparently the same phenotype as other affected members of the family. However, genotyping illustrated that he did not share the DFNA10 disease haplotype. Detailed clinical investigation showed differences in the onset and severity of his hearing loss and thus he is presumed to represent a phenocopy, perhaps resulting from long-term exposure to loud noise. PMID:17568404

Hildebrand, Michael S; Coman, David; Yang, Tao; Gardner, R J McKinlay; Rose, Elizabeth; Smith, Richard J H; Bahlo, Melanie; Dahl, Hans-Henrik M

2007-07-15

97

Natural variation in the splice site strength of a clock gene and species-specific thermal adaptation  

PubMed Central

Summary We show that multiple suboptimal splice sites underlie the thermal sensitive splicing of the period (per) 3’-terminal intron (dmpi8) from D. melanogaster, enabling this species to prolong its midday ‘siesta’, a mechanism that likely diminishes the deleterious effects of heat during the longer summer days in temperate climates. In D. yakuba and D. santomea, which have a more ancestral distribution indigenous to Afro-equatorial regions wherein day length and temperature exhibit little fluctuation throughout the year, the splicing efficiencies of their per 3’-terminal introns do not exhibit thermal calibration, consistent with the little effect of temperature on the daily distribution of activity in these species. We propose that the weak splice sites on dmpi8 underlies a mechanism that facilitated the acclimation of the widely colonized D. melanogaster (and possibly D. simulans) to temperate climates and that natural selection operating at the level of splicing signals plays an important role in the thermal adaptation of life forms. PMID:19109911

Low, Kwang Huei; Lim, Cecilia; Ko, Hyuk Wan; Edery, Isaac

2008-01-01

98

The location of constitutional neurofibromatosis 2 (NF2) splice site mutations is associated with the severity of NF2  

PubMed Central

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1–5 had more severe disease than those with splice site mutations in exons 11–15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours. PMID:15994874

Baser, M; Kuramoto, L; Woods, R; Joe, H; Friedman, J; Wallace, A; Ramsden, R; Olschwang, S; Bijlsma, E; Kalamarides, M; Papi, L; Kato, R; Carroll, J; Lazaro, C; Joncourt, F; Parry, D; Rouleau, G; Evans, D

2005-01-01

99

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

100

U1-like snRNAs lacking complementarity to canonical 5? splice sites  

PubMed Central

We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5? splice site (5?SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA–protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes. PMID:16829670

Kyriakopoulou, Christina; Larsson, Pontus; Liu, Lei; Schuster, Jens; Söderbom, Fredrik; A. Kirsebom, Leif; Virtanen, Anders

2006-01-01

101

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. PMID:8953723

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

1996-01-01

102

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

PubMed

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

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

2015-01-01

103

Widespread recognition of 5? splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides  

PubMed Central

An established paradigm in pre-mRNA splicing is the recognition of the 5? splice site (5?ss) by canonical base-pairing to the 5? end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 5?ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 5?ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 5?ss or U1 RNA strand, which we term “bulge registers.” By combining experimental evidence with transcriptome-wide free-energy calculations of 5?ss/U1 base-pairing, we estimate that 10,248 5?ss (?5% of human 5?ss) in 6577 genes use bulge registers. Several of these 5?ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing. PMID:22588721

Roca, Xavier; Akerman, Martin; Gaus, Hans; Berdeja, Andrés; Bennett, C. Frank; Krainer, Adrian R.

2012-01-01

104

A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family.  

PubMed

With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI. PMID:25296581

Siddiqi, Saima; Ismail, Muhammad; Oostrik, Jaap; Munawar, Saba; Mansoor, Atika; Kremer, Hannie; Qamar, Raheel; Schraders, Margit

2014-12-01

105

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end.  

PubMed Central

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP. PMID:11911363

Lund, Mette; Kjems, Jørgen

2002-01-01

106

SFmap: a web server for motif analysis and prediction of splicing factor binding sites  

E-print Network

­ Israel Institute of Technology, Haifa 32000, Israel and 2 Cold Spring Harbor Laboratory, Cold Spring Alternative splicing (AS) is a post-transcriptional process considered to be responsible for the huge Alternative splicing (AS) is a post-transcriptional process that generates multiple mRNA isoforms from

Mandel-Gutfreund, Yael

107

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.  

PubMed

BackgroundMutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.MethodsIn this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234¿+¿1G¿>¿A, c.633¿+¿1G¿>¿A and c.1542¿+¿4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A¿>¿G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234¿+¿1G¿>¿A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.ConclusionsWe have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications. PMID:25491247

Matos, Liliana; Canals, Isaac; Dridi, Larbi; Choi, Yoo; Prata, Maria; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Pshezhetsky, Alexey V; Grinberg, Daniel; Alves, Sandra; Vilageliu, Lluïsa

2014-12-10

108

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 Central

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

2013-01-01

109

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

PubMed Central

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

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

2014-01-01

110

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

PubMed

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

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

2013-01-01

111

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

2009-01-01

112

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

113

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. PMID:23172291

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

2013-01-01

114

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

PubMed Central

Summary Split skin grafting is a widely used technique for reconstructing skin defects. Although a vast number of different coverage options for donor sites have become available in daily clinical practice, no optimum dressing material has been found to date. For this reason, we conducted a globally-distributed online survey to poll for the properties of such an “ideal” donor site dressing, possibly leading to an improved clinically-driven direction of future wound dressing developments. A total of 69 respondents from 34 countries took part in the questionnaire, resulting in a response rate of 13.8% (69/500) over a 1-month period. The majority of respondents rated the characteristics of an “ideal” donor site dressing to be either “essential” or “desirable” as follows: lack of adhesion to the wound bed (“essential”: 31/69, 44.9%; “desirable”: 30/69, 43.5%); pain-free dressing changes (“essential”: 38/69, 55.1%; “desirable”: 30/69, 43.5%); absorbency (“essential”: 27/69, 39.1%; “desirable”: 33/69, 47.8%); ease of removal (“essential”: 37/69, 53.6%; “desirable”: 27/69, 39.13%). With regard to the desired frequency of dressing changes, respondents preferred “no dressing change until the donor site has healed” (51/69, 73.9%) in the majority of cases, followed by “twice weekly” (10/69, 14.5%), “alternate days” (5/69, 7.2%) and “daily” (3/69, 4.3%). With regard to the design of the dressing material, the majority of participants preferred a one-piece (composite) dressing product (44/69, 63.8%). The majority of respondents also denied the current availability of an “ideal” donor site dressing (49/69, 71%). The strength of this study was the remarkable geographic distribution of responses; all parts of the world were included and participated. We believe that this globally conducted online survey has polled for the properties of the “ideal” donor site dressing and possibly will lead to an improved clinically-driven direction of future wound dressing development. PMID:24563639

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

2013-01-01

115

Aberrant 3? splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization  

PubMed Central

The frequency distribution of mutation-induced aberrant 3? splice sites (3?ss) in exons and introns is more complex than for 5? splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3?ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3?ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3?ss was achieved by the maximum entropy model. Almost one half of aberrant 3?ss was activated by AG-creating mutations and ?95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3?ss was characterized by higher purine content than for authentic sites, particularly in position ?3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position ?11. A newly developed online database of aberrant 3?ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools. PMID:16963498

Vo?echovský, Igor

2006-01-01

116

Transcriptome Sequencing Reveals Potential Mechanism of Cryptic 3' Splice Site Selection in SF3B1-mutated Cancers.  

PubMed

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation. PMID:25768983

DeBoever, Christopher; Ghia, Emanuela M; Shepard, Peter J; Rassenti, Laura; Barrett, Christian L; Jepsen, Kristen; Jamieson, Catriona H M; Carson, Dennis; Kipps, Thomas J; Frazer, Kelly A

2015-03-01

117

Transcriptome Sequencing Reveals Potential Mechanism of Cryptic 3’ Splice Site Selection in SF3B1-mutated Cancers  

PubMed Central

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3’ splice sites (3’SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3’ SSs and propose that cryptic 3’SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3’SSs are present at low frequency (<10%) relative to nearby canonical 3’SSs, we identified ten genes that preferred out-of-frame cryptic 3’SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3’SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation. PMID:25768983

DeBoever, Christopher; Ghia, Emanuela M.; Shepard, Peter J.; Rassenti, Laura; Barrett, Christian L.; Jepsen, Kristen; Jamieson, Catriona H. M.; Carson, Dennis; Kipps, Thomas J.; Frazer, Kelly A.

2015-01-01

118

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. PMID:23056301

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

2012-01-01

119

Evaluation of Apligraf persistence and basement membrane restoration in donor site wounds: a pilot study.  

PubMed

Apligraf is a bilayered tissue-engineered product consisting of a bovine collagen matrix with neonatal fibroblasts, overlaid by a stratified epithelium containing living keratinocytes. The United States Food and Drug Administration has approved its use for venous leg ulcers and neuropathic diabetic foot ulcers. Apligraf provides a dermal matrix and produces cytokines similar to the human skin. However, its mechanism of action and ultimate fate in host wounds are unclear. The aim of this study was to evaluate the persistence of Apligraf fibroblasts and keratinocytes in human acute partial-thickness wounds (split-thickness donor sites) treated with Apligraf. In an open-label, within-patient, three-centered, controlled pilot study, 10 patients were treated with Apligraf, Apligraf dermis only (without epidermis), and a polyurethane film for donor site wounds of the same size, depth, and anatomical location. Apligraf DNA persistence was the primary outcome measure. Basement membrane components, cosmetic outcome, time to wound healing, and safety parameters were secondary outcome measures. One week after the initial treatment, reverse transcription polymerase chain reaction analysis found that two Apligraf and two Apligraf dermis-only-treated sites had Apligraf DNA present. Four weeks posttreatment, only one Apligraf and one Apligraf dermis-only sites showed the presence of Apligraf DNA. There was no difference between the three treatment modalities in establishing basement membrane in donor site wounds. No differences in other secondary outcomes were found. Apligraf DNA persisted in a minority of patients at 4 weeks in acute partial-thickness wounds. Apligraf's success in speeding healing of acute wounds appears to be related to factors other than the persistence of donor DNA or effect on basement membrane restoration. PMID:16939570

Hu, Shasa; Kirsner, Robert S; Falanga, Vincent; Phillips, Tania; Eaglstein, William H

2006-01-01

120

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

PubMed

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

Schwöbel, Johannes; Ebert, Ralf-Uwe; Kühne, Ralph; Schüürmann, Gerrit

2009-07-15

121

Characterization of cyclin L1 and L2 interactions with CDK11 and splicing factors: influence of cyclin L isoforms on splice site selection.  

PubMed

Although it has been reported that cyclin L1alpha and L2alpha proteins interact with CDK11(p110), the nature of the cyclin L transcripts, the formation of complexes between the five cyclin L and the three CDK11 protein isoforms, and the influence of these complexes on splicing have not been thoroughly investigated. Here we report that cyclin L1 and L2 genes generate 14 mRNA variants encoding six cyclin L proteins, one of which has not been described previously. Using cyclin L gene-specific antibodies, we demonstrate expression of multiple endogenous cyclin L proteins in human cell lines and mouse tissues. Moreover, we characterize interactions between CDK11(p110), mitosis-specific CDK11(p58), and apoptosis-specific CDK11(p46) with both cyclin Lalpha and -beta proteins and the co-elution of these proteins following size exclusion chromatography. We further establish that CDK11(p110) and associated cyclin Lalpha/beta proteins localize to splicing factor compartments and nucleoplasm and interact with serine/arginine-rich proteins. Importantly, we also determine the effect of CDK11-cyclin L complexes on pre-mRNA splicing. Preincubation of nuclear extracts with purified cyclin Lalpha and -beta isoforms depletes the extract of in vitro splicing activity. Ectopic expression of cyclin L1alpha, L1beta, L2alpha, or L2beta or active CDK11(p110) individually enhances intracellular intron splicing activity, whereas expression of CDK11(p58/p46) or kinase-dead CDK11(p110)represses splicing activity. Finally, we demonstrate that expression of cyclins Lalpha and -beta and CDK11(p110) strongly and differentially affects alternative splicing in vivo. Together, these data establish that CDK11(p110) interacts physically and functionally with cyclin Lalpha and -beta isoforms and SR proteins to regulate splicing. PMID:18216018

Loyer, Pascal; Trembley, Janeen H; Grenet, Jose A; Busson, Adeline; Corlu, Anne; Zhao, Wei; Kocak, Mehmet; Kidd, Vincent J; Lahti, Jill M

2008-03-21

122

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

2013-08-01

123

Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection.  

PubMed Central

By examining the first step of group II intron splicing in the absence of the second step, we have found that there is an interplay of three distinct reactions at the 5'-splice site: branching, reverse branching, and hydrolytic cleavage. This approach has yielded the first kinetic parameters describing eukaryotic branching and establishes that group II intron catalysis can proceed on a rapid timescale. The efficient reversibility of the first step is due to increased conformational organization in the branched intermediate and it has several important mechanistic implications. Reversibility in the first step requires that the second step of splicing serve as a kinetic trap, thus driving splicing to completion and coordinating the first and second step of splicing. Facile reverse branching also provides the intron with a proofreading mechanism to control the fidelity of 5'-splice site selection and it provides a kinetic basis for the apparent mobility of group II introns. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 FIGURE 6 PMID:7493317

Chin, K; Pyle, A M

1995-01-01

124

Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.  

PubMed

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism. PMID:25375251

Hamid, Fursham M; Makeyev, Eugene V

2014-11-01

125

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

126

Applying the Brakes to Multi-Site SR Protein Phosphorylation: Substrate-Induced Effects on the Splicing Kinase SRPK1†  

PubMed Central

To investigate how a protein kinase interacts with its protein substrate during extended, multi-site phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates approximately 10 serines in the arginine-serine-rich domain (RS domain) of the SR protein SRSF1 in a C-to-N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t1/2 = 0.1 sec) followed by slower, multi-site phosphorylation at the remaining serines (t1/2 = 15 sec). Mutagenesis experiments suggest that efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic network between the RS domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multi-site phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension and termination events associated with prolonged, multi-site phosphorylation. PMID:21728354

Aubol, Brandon E.; Adams, Joseph A.

2011-01-01

127

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

128

Heterogeneous Nuclear Ribonucleoprotein G Regulates Splice Site Selection by Binding to CC(A/C)-rich Regions in Pre-mRNA*S?  

PubMed Central

Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentration-dependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sex-specific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals. PMID:19282290

Heinrich, Bettina; Zhang, Zhaiyi; Raitskin, Oleg; Hiller, Michael; Benderska, Natalya; Hartmann, Annette M.; Bracco, Laurent; Elliott, David; Ben-Ari, Shani; Soreq, Hermona; Sperling, Joseph; Sperling, Ruth; Stamm, Stefan

2009-01-01

129

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

PubMed

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

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

2011-08-01

130

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

PubMed

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

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

2015-03-15

131

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

PubMed

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

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

2015-04-01

132

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. 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.

2013-01-01

133

The evolution of mRNA splicing in mammals  

E-print Network

In this thesis, I describe investigations into the evolution of splicing in mammals. I first investigate a small class of alternative splicing events, tandem splice sites, and show how they are used to introduce and remove ...

Merkin, Jason Jay

2014-01-01

134

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

PubMed

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

Acar, Mustafa; Yaz?c?, Demet; San, Turhan; Muluk, Nuray Bayar; Cingi, Cemal

2015-04-01

135

Improvement of the radial forearm donor site by prefabrication of fascial-split-thickness skin grafts.  

PubMed

A basic disadvantage of radial forearm flaps is the removal of skin from a functionally important and cosmetically exposed region. To minimize the donor-site morbidity of the radial forearm flap, we have thus far used a two-phase procedure for intraoral defect coverage in five patients: In a first step, a split-thickness skin graft is transplanted to the forearm fascia, which "takes" there over a period of 2 weeks. In step two, the prefabricated fascial-split-thickness skin flap can be raised with complete preservation of the forearm skin and microsurgically transplanted like a conventional radial flap. Performing this procedure, we have obtained the following results: (1) All skin grafts "took" completely on the forearm fascia. (2) Prefabricated fascial-split-thickness skin flaps could be raised without any problems, like conventional radial forearm flaps. (3) All flaps were excellently suited for defect coverage in the oral cavity as very thin and moldable grafts and "took" without any complications. (4) Tension-free primary closure of all forearm donor sites was achieved with only slight cosmetic and functional impairment. PMID:8764728

Wolff, K D; Ervens, J; Hoffmeister, B

1996-08-01

136

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. PMID:21622561

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

2011-01-01

137

Catalytic Site Components Common to Both Splicing Steps of a Group II Intron Author(s): Guillaume Chanfreau and Alain Jacquier  

E-print Network

Catalytic Site Components Common to Both Splicing Steps of a Group II Intron Author(s): GuillaumeManualand Handbook forEscherichia coli and Related Bacteria, J. H. Miller,Eds. (Cold Spring HarborLaboratoryPress, Cold Spring Har- bor, NY, 1992), pp. 2.3-2.43. 13. N. Saitou and M. Nei, Mol. Biol. Evol.4, 406 (1987

Chanfreau, Guillaume

138

1996 Oxford University Press 47094718Nucleic Acids Research, 1996, Vol. 24, No. 23 Logitlinear models for the prediction of splice sites in  

E-print Network

models for the prediction of splice sites in plant pre-mRNA sequences Jürgen Kleffe*, Klaus Hermann. INTRODUCTION An important aspect of eukaryotic genome research is scanning functionally unknown sequences of genomic sequence data, the majority of new sequences must be studied by statistical, computational methods

Brendel, Volker

139

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

PubMed

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

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

2015-04-15

140

Novel acceptor splice site mutation in the invariant AG of intron 6 of alpha-galactosidase A gene, causing Fabry disease. Mutations in brief no. 146. Online.  

PubMed

We found a novel acceptor splice site mutation in the invariant AG of intron 6 of alpha-galactosidase A (alpha-Gal A) gene (IVS6-1G->A) in a patient with Fabry disease by sequencing of genomic DNA. Sequencing of RT-PCR revealed the deletion of first base pair (c909del) of exon 7 in mRNA and a frameshift resulting in premature termination. This mutation gives rise to a rare aberrant splicing (Simultaneous 3' destruction and 3' creation). PMID:10200059

Matsumura, T; Osaka, H; Sugiyama, N; Kawanishi, C; Maruyama, Y; Suzuki, K; Onishi, H; Yamada, Y; Morita, M; Aoki, M; Kosaka, K

1998-01-01

141

mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain  

PubMed Central

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system. PMID:20452967

Hammer, Paul; Banck, Michaela S.; Amberg, Ronny; Wang, Cheng; Petznick, Gabriele; Luo, Shujun; Khrebtukova, Irina; Schroth, Gary P.; Beyerlein, Peter; Beutler, Andreas S.

2010-01-01

142

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

PubMed Central

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5? splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine “ladder.” A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition. PMID:19304800

Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

2009-01-01

143

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

PubMed

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

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

2015-01-15

144

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.  

PubMed Central

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4. PMID:11333022

Kammler, S; Leurs, C; Freund, M; Krummheuer, J; Seidel, K; Tange, T O; Lund, M K; Kjems, J; Scheid, A; Schaal, H

2001-01-01

145

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5? splice site  

PubMed Central

Recognition of the 5? splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem–loop of domain 1 and a complementary sequence at the 3? end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5? intron, we probed the solution structure of the ID3 stem–loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5? and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5? splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1–IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5? and O.i. introns and may help to fine-tune elements of recognition in group II introns. PMID:24243113

Popovi?, Milena; Greenbaum, Nancy L.

2014-01-01

146

Investigation of the low-affinity oxidation site for exogenous electron donors in the Mn-depleted photosystem II complexes  

Microsoft Academic Search

In the manganese-depleted photosystem II (PSII[?Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(?Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L. Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to YZ•

V. N. Kurashov; E. R. Lovyagina; D. Yu. Shkolnikov; M. K. Solntsev; M. D. Mamedov; B. K. Semin

2009-01-01

147

Complex pattern of alternative splicing in the normal uroporphyrinogen decarboxylase gene: implications for diagnosis of familial porphyria cutanea tarda.  

PubMed

We describe multiple alternative transcripts of uroporphyrinogen decarboxylase mRNA in normal individuals and patients with familial porphyria cutanea tarda. mRNA was reverse-transcribed, subjected to the polymerase chain reaction, and analyzed for nucleotide sequence. Seven different transcripts were characterized, and a cryptic splice acceptor site was identified in intron 1. In all mRNAs the exons abutted at previously defined exon boundaries. Characterization of the splice junctions in the genomic DNA showed that splice donor and acceptor sequences complied with the consensus sequences for these sites except for the splice acceptor sequences of exons 3 and 10. THese deviations were present in two normal individuals and one patient with familial porphyria cutanea tarda and were thus unable to explain the multiple aberrant uroporphyrinogen decarboxylase transcripts. We conclude that apparent deletions observed in transcripts derived from the uroporphyrinogen decarboxylase gene in patients with familial porphyria cutanea tarda should be interpreted with caution. PMID:7923766

McManus, J F; Begley, C G; Ratnaike, S

1994-10-01

148

Evolution of splicing regulatory networks in Drosophila  

PubMed Central

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

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

2014-01-01

149

A randomized, prospective, parallel group study of laparoscopic versus laparoendoscopic single site donor nephrectomy for kidney donation.  

PubMed

Few prospective, randomized studies have assessed the benefits of laparoendoscopic single site donor nephrectomy (LESS-DN) over laparoscopic donor nephrectomy (LDN). Our center initiated such a trial in January 2011, following subjects randomized to LESS-DN versus LDN from surgery through 5 years postdonation. Subjects complete recovery/satisfaction questionnaires at 2, 6 and 12 months postdonation; transplant recipient outcomes are also recorded. One hundred subjects (49 LESS-DN, 51 LDN) underwent surgery; donor demographics were similar between groups, and included a predominance of female, living-unrelated donors, mean age of 47 years who underwent left donor nephrectomy. Operative parameters (overall time, time to extraction, warm ischemia time, blood loss) were similar between groups. Conversion to hand-assist laparoscopy was required in 3 LESS-DN (6.1%) versus 2 LDN (3.9%; p?=?0.67). Questionnaires revealed that 97.2% of LESS-DN versus 79.5% of LDN (p?=?0.03) were 100% recovered by 2 months after donation. No significant difference was seen in satisfaction scores between the groups. Recipient outcomes were similar between groups. Our randomized trial comparing LESS donor nephrectomy to LDN confirms that LESS-DN offers a safe alternative to conventional LDN in terms of intra- and post-operative complications. LDN and LESS-DN offer similar recovery and satisfaction after donation. PMID:24934732

Aull, M J; Afaneh, C; Charlton, M; Serur, D; Douglas, M; Christos, P J; Kapur, S; Del Pizzo, J J

2014-07-01

150

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

PubMed Central

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

2014-01-01

151

Laser Doppler blood flow measurements of common cutaneous donor sites for reconstructive surgery.  

PubMed

The purpose of this study was to evaluate cutaneous blood flow in regions commonly used as donor sites in reconstructive surgery in order to better establish normal flow ranges. Flow was measured with the TSI Laserflo BPM 403 in 27 healthy volunteers and compared to the flow in uncomplicated postoperative autologous tissue transplants. The forehead produced the highest flow, with an average value of 6.50 +/- 0.31 (mean +/- SE), and the dorsalis pedis had the lowest flow, with an average value of 0.60 +/- 0.04. Gender differences were noted in the latissimus dorsi, pectoralis major, and rectus abdominis areas. There were no significant differences between smokers and nonsmokers, hand dominance, musculocutaneous and fasciocutaneous tissues, or supine and sitting body positions. Flow levels in volunteers were similar to those in postoperative surviving autologous tissue transplants. The site-specific flow and flow changes over long time periods (hours) have helped clinical monitoring of 77 patients in the last 24 months. In every case identified by the flowmeter as decreased perfusion, a definite etiology for low reduction was documented. Complications occurred in 12 patients, and the rate of salvaging compromised tissue has increased from 50 percent using temperature monitoring and clinical observation to 83 percent with the computerized laser Doppler flowmeter. PMID:2179975

Goldberg, J; Sepka, R S; Perona, B P; Pederson, W C; Klitzman, B

1990-04-01

152

Adhesive retention dressings are more comfortable than alginate dressings on split skin graft donor sites--a randomised controlled trial.  

PubMed Central

A prospective randomised trial examining the effectiveness, comparative comfort and ease of care of two different split skin graft donor site dressings was performed. One of the dressings was an alginate (Kaltostat), and the other an adhesive retention tape (Mefix). Alginates are the standard plastic surgical dressing, whereas the use of adhesive retention tapes as a donor site dressing presents a novel use of a readily available product. A total of 30 consecutive patients requiring split skin grafts were randomised to receive either alginate or retention donor site dressings. Dressings were assessed by interview and questionnaire at 24 h and 48 h and at 2 weeks, and by wound review at 2 weeks. Retention dressings were found to be more comfortable. They also required less nursing care and attention. The retention dressings allowed the patients easier mobility and a greater range of daily activities, especially washing. There was no significant difference in wound healing nor in complications. Adhesive retention tape applied directly to the split skin graft donor site wound is an effective, cheap and comfortable dressing requiring little postoperative care. PMID:11777142

Giele, H.; Tong, A.; Huddleston, S.

2001-01-01

153

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

PubMed

This study describes a new mechanism controlling the production of alternatively spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5' splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5' splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the ?3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue. PMID:24735995

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

2014-06-01

154

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

PubMed

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

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

2015-02-01

155

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Al= of a 3' Splice Site In Vivo  

E-print Network

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 ...

Ma, Long

156

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

PubMed

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome. PMID:25805857

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

2015-05-01

157

A new technique for harvesting costal cartilage with minimum sacrifice at the donor site.  

PubMed

With conventional procedures for harvesting costal cartilage, several large, full-thickness cartilage blocks are harvested from the chest wall and are cut, shaped, and joined to create the desired form. Many pieces of unused cartilage are discarded excluding those preserved for future use. Conventional procedures for costal cartilage harvesting are also associated with severe problems such as pain, deformity of the chest wall, and a long scar. We developed a new technique that permits only the necessary size and shape of cartilage to be directly harvested with the use of a chisel. With this technique, both sides and the bottom of the cartilage remain intact at the donor site. The anterior perichondrium can be harvested simultaneously. This technique was performed in 28 patients. The required quantities could be harvested in all patients without severe complications such as perforation of the pleura and excessive bleeding. The procedure required 30 min or less in all patients. The length of the skin incision was less than 3 cm in 25 patients and greater than 3 cm in two obese patients and a young man who had hard subcutaneous connective tissue. Pain intensity was markedly lower than that after conventional techniques. Twenty-six patients could walk 1 day after the operation. There were virtually no deformities of the thorax, even in children younger than 10 years. The structure of the reconstructed site was maintained during at least 2 years follow-up in all patients. Our technique for harvesting costal cartilage is associated with smaller scars, less pain, and less deformity of the chest wall than conventional procedures. In addition, it is minimally invasive and can be performed in a short time. PMID:16756249

Yotsuyanagi, Takatoshi; Mikami, Makoto; Yamauchi, Makoto; Higuma, Yuko; Urushidate, Satoshi; Ezoe, Kyouri

2006-01-01

158

Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA.  

PubMed Central

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use. Images PMID:8355693

Gilbert, E; Del Gatto, F; Champion-Arnaud, P; Gesnel, M C; Breathnach, R

1993-01-01

159

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

PubMed Central

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.

2013-01-01

160

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

PubMed

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

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

2015-01-01

161

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

2012-08-01

162

Long-term functional donor site morbidity of the free radial forearm flap in head and neck cancer survivors  

PubMed Central

Background To assess the functional donor site morbidity of the forearm free flap in patients surviving at least 2 years after ablative head and neck cancer surgery in a tertiary care centre. Methods This study involved nine long-term survivors (2 year post-operative) who had forearm free flaps to reconstruct head and neck defects. All flaps were raised from the non-dominant arm. The non-donor side acted as a control for all patients. Objective measurements were as follows: grip, tip pinch and key pinch strength measured with dynamometers; flexion, extension, radial and ulnar deviation and pronation and supination range of motion at the wrist measured with goniometry; A timed manual dexterity task was performed with a grooved pegboard test, and sensation of the radial nerve was tested with Semmes Weinstein monofilaments. Subjective measurements included a validated patient questionnaire of hand function and opinions of scar appearance as well as a validated scar assessment from two different observers. Results Pronation at the wrist, manual dexterity and sensation were found to be significantly reduced in the donor side compared to the non-donor side. Inter-rater agreement between the two observers was found to be poor, except for an acceptable correlation between overall scar opinions. No correlations were found between any subjective or objective items or between the patient’s and the observers’ subjective evaluations. Conclusions Donor site morbidity can be demonstrated with objective testing however this is accepted and well tolerated by head and neck cancer patients. PMID:24418459

2014-01-01

163

Dealing with the non-healing; donor site defect following latissimus dorsi myocutaneous flap in breast reconstruction  

Microsoft Academic Search

We present two cases of donor site morbidity following immediate breast reconstruction using the extended latissimus dorsi\\u000a myocutaneous flap. Early post operative dehiscence occurred in both cases, followed by a prolonged period of expectant and\\u000a conservative management. Plastic surgical intervention involved exploration and removal of significant epithelial lined cavities\\u000a and reconstruction using local flaps to solve this complication.

Mashuk Khan; Garrick A. Georgeu; Niri Niranjan

2010-01-01

164

Functional analysis of splicing mutations in MYO7A and USH2A genes.  

PubMed

Usher syndrome is defined by the association of sensorineural hearing loss, retinitis pigmentosa and variable vestibular dysfunction. Many disease-causative mutations have been identified in MYO7A and USH2A genes, which play a major role in Usher syndrome type I and type II, respectively. The pathogenic nature of mutations that lead to premature stop codons is not questioned; nevertheless, additional studies are needed to verify the pathogenicity of some changes such as those putatively involved in the splice process. Five putative splice-site variants were detected in our cohort of patients: c.2283-1G>T and c.5856G>A in MYO7A and c.1841-2A>G, c.2167+5G>A and c.5298+1G>C in the USH2A gene. In this study, we analyze these changes with bioinformatic tools and investigate the expression of MYO7A and USH2A transcripts through hybrid minigene assays. Our study showed that all five mutations abolished the consensus splice site producing the skipping of involved exons. In addition, for variant c.2167+5G>A, a new donor splice site was observed. Our data reveal the pathogenic nature of the analyzed variants. The fact that splicing mutations led to in-frame or out-of-frame alterations cannot explain phenotypic differences, thus, genotype-phenotype correlations cannot be inferred. PMID:20497194

Jaijo, T; Aller, E; Aparisi, M J; García-García, G; Hernan, I; Gamundi, M J; Nájera, C; Carballo, M; Millán, J M

2011-03-01

165

Donor site morbidity following iliac crest bone harvesting for cervical fusion: a comparison between minimally invasive and open techniques  

PubMed Central

We have studied the occurrence of donor site morbidity, cosmesis and overall satisfaction with graft procedure in 76 patients who had undergone iliac crest bone harvesting for anterior cervical discectomy and fusion (ACDF). Totally 24 patients underwent an open procedure and 52 a minimally invasive trephine harvesting method. Although our study demonstrated substantial donor site pain and its effect on ambulation in both groups, this was of limited duration. Two patients, one in each group, suffered long-term pain that was eventually resolved. Totally 8.3% of patients in the open group suffered minor complications and 11.5% in the trephine group. There were two cases of meralgia parasthetica. There were no major complications in either group. There was no statistically significant difference in morbidity between the open and trephine groups. There was a trend towards significance (P = 0.076) for pain at the donor site, with less pain reported by patients who underwent the trephine procedure for harvesting. PMID:18389294

Pollock, Raymond; Bhatia, Chandra; Chuter, Graham; Lingutla, Kiran; Budithi, Chakravarty; Krishna, Manoj

2008-01-01

166

Evaluation of Human Amniotic Membrane as a Wound Dressing for Split-Thickness Skin-Graft Donor Sites  

PubMed Central

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

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

2014-01-01

167

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

PubMed

Nipple reconstruction is of importance in achieving the best possible aesthetic outcome after breast reconstruction. Nipple sharing is a common technique; this study focused on the potential morbidity at the donor nipple. Between 2008 and 2012, 26 patients underwent nipple sharing at our institution. The donor nipple was examined before and after the procedure (mean follow-up of 21 months). Sensitivity, projection, diameter, and patient satisfaction were evaluated. The sensitivity in the donor nipple decreased, albeit insignificantly, from 1.2 g/mm2 (0.8-1.6) to 1.8 g/mm2 (0.8-4.8) (p=0.054, n=26). The projection due to graft removal decreased from 8.0 mm (6.8-10.0) to 4.5 mm (4.0-5.0) (p=0.001). Of the patients, 88% were "very satisfied" or "somewhat satisfied" with the sensitivity and 89% with the symmetry between the donor and reconstructed nipple. At least 60% of the patients were "very satisfied" with all aesthetic outcome parameters (projection, appearance, naturalness, color, and shape). All patients would agree to undergo this procedure again, if necessary. Nipple sharing was associated with minimal morbidity at the donor nipple. The postoperative projection was adequate. Regardless of whether simultaneous mastopexy was performed, the loss of sensitivity was minimal and presumably imperceptible to the patient. By using no sutures after graft removal and letting the donor nipple heal spontaneously, scarring was minimized and the natural appearance and good sensitivity of the donor nipple were preserved. PMID:25465146

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

2015-02-01

168

Differential 3? splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP  

PubMed Central

Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF65 with the 3? splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF65 crosslinking. Furthermore, blocking recognition of a Tra2-?1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF65 crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation. PMID:19244360

Martins de Araújo, Mafalda; Bonnal, Sophie; Hastings, Michelle L.; Krainer, Adrian R.; Valcárcel, Juan

2009-01-01

169

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. PMID:18388132

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

2008-01-01

170

Lipoprotein lipase deficiency due to a 3' splice site mutation in intron 6 of the lipoprotein lipase gene  

Microsoft Academic Search

In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C+A mutation in position -3 at the acceptor splice script showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control

B. Holzl; R. Huber; B. Paulweber; J. R. Patsch

171

Functional study of SR splicing factors in a cellular genetic system  

E-print Network

Because SR proteins affect alternative splicing in a dosage-proteins are known to affect alternative splicing in a dosage-proteins, however, U2AF does not seem to affect 3’ splice site choice in a dosage

Lin, Shengrong

2007-01-01

172

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

PubMed

Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406], showed that the N-terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N-termini of transthyretin evolved by successive shifts of the 3' splice site of intron 1 in the 3' direction, resulting in successive shortening of the 5' end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N-terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection. PMID:9208931

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

1997-06-01

173

DNA donors for sequencing at Celera, Craig VenterSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Craig Venter DNAi Location:Genome>the project>players>private project The private project's DNA donors Craig Venter, the leader of the private genome effort at Celera Genomics, talks about the sources of the DNA used in their sequence.

2008-10-06

174

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

2013-01-01

175

Expression studies of a novel splice site mutation in the LIPH gene identified in a Japanese patient with autosomal recessive woolly hair.  

PubMed

Autosomal recessive woolly hair (ARWH) is characterized by short and tightly curled scalp hair without any obvious complications. The disease is known to be caused by either lipase H (LIPH) or LPAR6 genes. Proteins encoded by these two genes are closely related to each other in a lipid-signaling pathway that is believed to play crucial roles in hair follicle development and hair growth. In the Japanese population, most affected individuals with ARWH have been shown to carry two prevalent founder mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.742C>A (p.His248Asn), while other LIPH mutations have been occasionally identified. In this study, we analyzed a Japanese patient with ARWH, and identified compound heterozygous mutations in the LIPH gene, c.736T>A (p.Cys246Ser) and c.982+5G>T. The latter one was a novel splice site mutation in intron 7. Expression studies using blood-derived RNA from the patient detected the LIPH transcript from the c.736T>A mutant allele, but not from the c.982+5G>T mutant allele. Furthermore, in vitro transcription assay in cultured cells showed that the mutation c.982+5G>T caused an aberrant splicing event, leading to a frame-shift and a premature termination codon (p.Met328Serfs*41). To the best of our knowledge, this is the second splice site mutation in the LIPH gene, and our findings further expand the spectrum of the LIPH mutations underlying ARWH. PMID:25271093

Hayashi, Ryota; Inui, Shigeki; Farooq, Muhammad; Ito, Masaaki; Shimomura, Yutaka

2014-10-01

176

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome.  

PubMed

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40% of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1. PMID:24590738

Kim, Hui Ram; Song, Mee Hyun; Kim, Min-A; Kim, Ye-Ri; Lee, Kyu-Yup; Sonn, Jong Kyung; Lee, Jaetae; Choi, Jae Young; Kim, Un-Kyung

2014-07-01

177

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

PubMed

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

Brenner, Maria; Hilliard, Carol; Peel, Glynis; Crispino, Gloria; Geraghty, Ruth; O?Callaghan, Gill

2015-01-01

178

Skin Thickness of the Anterior, Anteromedial, and Anterolateral Thigh: A Cadaveric Study for Split-Skin Graft Donor Sites  

PubMed Central

Background The depth of graft harvest and the residual dermis available for reepithelization primarily influence the healing of split-skin graft donor sites. When the thigh region is chosen, the authors hypothesize based on thickness measurements that the anterolateral region is the optimal donor site. Methods Full-thickness skin specimens were sampled from the anteromedial, anterior, and anterolateral regions of human cadavers. Skin specimens were cut perpendicularly with a custom-made precision apparatus to avoid the overestimation of thickness measurements. The combined epidermal and dermal thicknesses (overall skin thickness) were measured using a digital calliper. The specimens were histologically stained to visualize their basement membrane, and microscopy images were captured. Since the epidermal thickness varies across the specimen, a stereological method was used to eliminate observer bias. Results Epidermal thickness represented 2.5% to 9.9% of the overall skin thickness. There was a significant difference in epidermal thickness from one region to another (P<0.05). The anterolateral thigh region had the most consistent and highest mean epidermal thickness (60±3.2 µm). We observed that overall skin thickness increased laterally from the anteromedial region to the anterior and anterolateral regions of the thigh. The overall skin thickness measured 1,032±435 µm in the anteromedial region compared to 1,220±257 µm in the anterolateral region. Conclusions Based on skin thickness measurements, the anterolateral thigh had the thickest epidermal and dermal layers. We suggest that the anterolateral thigh region is the optimal donor site for split-skin graft harvests from the thigh. PMID:25396179

Ward, John; Quondamatteo, Fabio; Dockery, Peter; Kelly, John L

2014-01-01

179

An essential splice site mutation (c.317+1G>A) in the TSHR gene leads to severe thyroid dysgenesis.  

PubMed

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and 2% of cases have familial origin. Our aim in this study was to determine the genetic alterations in two siblings with CH coming from a consanguineous family. Because CH is often inherited in autosomal recessive manner in consanguineous/multicase-families, we first performed genetic linkage studies to all known causative CH loci followed by conventional sequencing of the linked gene. The family showed potential linkage to the TSHR locus, and we detected an essential splice site mutation (c.317+1G>A) in both siblings. RT-PCR analysis confirmed the functionality of the mutation. The mutation was homozygous in the cases whereas heterozygous in carrier parents and an unaffected sibling. Here we conclude that thyroid agenesis in both siblings in this study originates from c.317+1G>A splice site mutation in the TSHR gene, and this study underlines the importance of detailed molecular genetic studies in the definitive diagnosis and classification of CH. PMID:24859513

Cangul, Hakan; Saglam, Halil; Saglam, Yaman; Eren, Erdal; Dogan, Durmus; Kendall, Michaela; Tarim, Omer; Maher, Eamonn R; Barrett, Timothy G

2014-09-01

180

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

181

Splicing Programs and Cancer  

PubMed Central

Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets. PMID:22132318

Germann, Sophie; Gratadou, Lise; Dutertre, Martin; Auboeuf, Didier

2012-01-01

182

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. PMID:24083206

Di Bari, Roberto; Coronelli, Roberto

2013-01-01

183

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. PMID:21203346

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

184

An RNA map predicting Nova-dependent splicing regulation  

Microsoft Academic Search

Nova proteins are a neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova's effect to inhibit or

Jernej Ule; Giovanni Stefani; Aldo Mele; Matteo Ruggiu; Xuning Wang; Bahar Taneri; Terry Gaasterland; Benjamin J. Blencowe; Robert B. Darnell

2006-01-01

185

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

2012-01-01

186

A 46,XY Female DSD Patient with Bilateral Gonadoblastoma, a Novel SRY Missense Mutation Combined with a WT1 KTS Splice-Site Mutation  

PubMed Central

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

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.

2012-01-01

187

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. PMID:23836433

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

2013-09-01

188

The public Human Genome Project's DNA donors, Eric LanderSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Eric Lander DNAi Location: Genome>The Project>players>Public consortium The public's DNA donors Eric Lander, director of the Whitehead Institute Center for Genome Research, explains where the DNA donors for the first reference sequence came from.

2008-10-06

189

Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.  

PubMed

The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2? splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2?-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes. PMID:25665175

Dal Mas, Andrea; Fortugno, Paola; Donadon, Irving; Levati, Lauretta; Castiglia, Daniele; Pagani, Franco

2015-05-01

190

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

PubMed Central

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

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

1993-01-01

191

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 PMID:8950674

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

192

Molecular genetics of the glycophorin gene family, the antigens for MNSs blood groups: multiple gene rearrangements and modulation of splice site usage result in extensive diversification.  

PubMed

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteins exhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSs blood groups; thus individuals bearing variant glycophorins can be readily identified by serological typing. Examination of the wide array of variants of these antigens showed that they include many forms, possibly made evident by lack of constraints due to the apparent dispensability of the parent molecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversions coupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature is the shuffling of sequences within two specific exons (one of which is silent), homologous in the two parent genes. This has resulted in expression of a mosaic of sequences within this region, leading to polymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique among human gene families. It could have occurred by chance rearrangements among closely linked genes and been driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibility complex (MHC). In the glycophorin family the small size of the region within which gene interactions have occurred and the participation of essentially only two alleles makes this relatively simpler system more focused and easier to dissect and describe molecularly. PMID:8535438

Blumenfeld, O O; Huang, C H

1995-01-01

193

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

PubMed

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

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

2015-01-01

194

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

PubMed Central

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

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

2015-01-01

195

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

NASA Astrophysics Data System (ADS)

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

Plotch, Stephen J.; Krug, Robert M.

1986-08-01

196

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. PMID:18945809

Hebeisen, Michaël; Drysdale, John; Roy, Richard

2008-01-01

197

SOX2 modulates alternative splicing in transitional cell carcinoma.  

PubMed

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor. PMID:20138825

Tung, Chun-Liang; Hou, Pei-Hsuan; Kao, Yung-Ling; Huang, Yu-Wen; Shen, Chiung-Chun; Cheng, Yi-Hsin; Wu, Shu-Fen; Lee, Moon-Sing; Li, Chin

2010-03-12

198

Direct Repression of Splicing by transformer-2  

PubMed Central

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

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

2003-01-01

199

Design of Novel Chalcogen-Containing Organic Metals: Extensively Conjugated Electron Donors and Acceptors With Reduced On-Site Coulomb Repulsion  

Microsoft Academic Search

This review describes the design of extensively conjugated electron donors and acceptors based on structural modifications of tetrathiafulvalene (TTF) and tetracyanoquinodimethane (TCNQ), respectively. These molecules have the advantage of reduced on-site Coulomb repulsion which is one of the prerequisites for components of organic metals. The construction of such large compounds is currently accomplished by insertion of an extensively ?-conjugated system

Fumio Ogura; Tetsuo Otsubo; Yoshio Aso

1992-01-01

200

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 39 Splice Site In Vivo  

E-print Network

) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature- sensitive lethality and caused in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1 of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 39 splice

Horvitz, H. Robert

201

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

PubMed Central

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

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

2013-01-01

202

BRCA1 exon 11 a model of long exon splicing regulation  

PubMed Central

BRCA1 exon 11 is one of the biggest human exons, spanning 3426 bases. This gene is potentially involved in DNA repair as well as cell growth and cell cycle control. Exon 11 is regulated at the splicing level producing three main different combinations of BRCA1 mature transcripts; one including the whole of exon 11 (full isoform), one skipping the entire exon (D11 isoform), and one including only 117 base pairs of exon 11 (D11q isoform). Using minigene and deletion analyses, we have previously described important splicing regulatory sequences located at the beginning of this exon (5? end). We have now found additional important sequences located at its 3? end. In particular, we describe the presence of a strong splicing enhancer adjacent to the downstream 5? splice site, which minimizes competition from an upstream 5? splice site and so ensures long exon inclusion. Analyses of the proteins binding these RNA sequences have revealed that Tra2beta and hnRNP L are involved in the regulation of BRCA1 exon 11 by influencing the recognition of donor sites. Interestingly, BRCA1 exon 11 carrying deletion of the regulatory sequences bound by these factors also showed unexpected responses to up- or downregulation of these regulatory proteins, suggesting that they can also bind elsewhere in this large exon and elicit different effects on its recognition.   The identification of sequences and proteins relevant for the regulation of BRCA1 exon 11 now provides better knowledge on how this exon is recognized and may represent an important step toward understanding how large exons are regulated. PMID:24658338

Raponi, Michela; Smith, Lindsay D; Silipo, Marco; Stuani, Cristiana; Buratti, Emanuele; Baralle, Diana

2014-01-01

203

Defective pre-mRNA splicing in PKD1 due to presumed missense and synonymous mutations causing autosomal dominant polycystic disease.  

PubMed

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G>A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A>T (p.G109G) and c.11257C>A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level. PMID:24907393

Gonzalez-Paredes, Francisco J; Ramos-Trujillo, Elena; Claverie-Martin, Felix

2014-08-10

204

Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals.  

PubMed

We propose a framework for modeling sequence motifs based on the maximum entropy principle (MEP). We recommend approximating short sequence motif distributions with the maximum entropy distribution (MED) consistent with low-order marginal constraints estimated from available data, which may include dependencies between nonadjacent as well as adjacent positions. Many maximum entropy models (MEMs) are specified by simply changing the set of constraints. Such models can be utilized to discriminate between signals and decoys. Classification performance using different MEMs gives insight into the relative importance of dependencies between different positions. We apply our framework to large datasets of RNA splicing signals. Our best models out-perform previous probabilistic models in the discrimination of human 5' (donor) and 3' (acceptor) splice sites from decoys. Finally, we discuss mechanistically motivated ways of comparing models. PMID:15285897

Yeo, Gene; Burge, Christopher B

2004-01-01

205

HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation  

SciTech Connect

The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore)] [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France)] [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom)] [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom)] [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France)] [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

2012-07-20

206

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements  

PubMed Central

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

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

2014-01-01

207

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. PMID:19330717

2009-01-01

208

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

2012-04-01

209

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. PMID:24058716

Davidson, Andrew; Jina, N. Hamesh; Marsh, Clive; Than, Martin; Simcock, Jeremy W.

2013-01-01

210

Design, synthesis, and testing of potential antisickling agents. 5. Disubstituted benzoic acids designed for the donor site and proline salicylates designed for the acceptor site.  

PubMed

This paper reports the discovery of a new class of potent antigelling agents. The new compounds, disubstituted benzoic acid derivatives, were designed by using molecular modeling experiments. These molecules contain functional groups positioned to interact with several polar amino acid residues near the Val-6 beta mutation site (donor site) in HbS. The compounds also contain a hydrophobic group designed to occupy a nonpolar ara on the surface of the protein created by several hydrophobic amino acids. The synthesis and testing of these new molecules using a standard solubility assay is reported. A structural comparison is made between one of the most active antigelling agents, compound 13, which has little effect on the oxygen affinity of Hb in solution, and bezafibrate, a known antilipidemic drug that is progelling and has a very potent effect on decreasing Hb oxygen affinity (Perutz, M. F.; Poyart, C. Lancet 1983, 2, 881-882). We also report the synthesis and testing of a series of proline-salicylate molecules designed to react covalently at the mutation acceptor area. This class of molecules did not show significant activity. PMID:6094807

Abraham, D J; Gazze, D M; Kennedy, P E; Mokotoff, M

1984-12-01

211

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. PMID:17616531

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

2011-01-01

212

A mouse splice-site mutant and individuals with atypical chromosome 22q11.2 deletions demonstrate the crucial role for crkl in craniofacial and pharyngeal development.  

PubMed

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

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

2014-12-01

213

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

PubMed Central

The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P2) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P2 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome. Electronic supplementary material The online version of this article (doi:10.1007/s00335-010-9281-7) contains supplementary material, which is available to authorized users. PMID:20872266

Bothwell, Susan P.; Farber, Leslie W.; Hoagland, Adam

2010-01-01

214

The doublesex Splicing Enhancer Components Tra2 and Rbp1 Also Repress Splicing through an Intronic Silencer?  

PubMed Central

The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression. PMID:17101798

Qi, Junlin; Su, Shihuang; Mattox, William

2007-01-01

215

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3' Splice Site In Vivo  

E-print Network

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 ...

Ma, Long

216

Conserved Alternative Splicing Patterns and Splicing Signals in the Drosophila Sodium Channel Gene Para  

PubMed Central

We cloned genomic DNA corresponding to the Drosophila virilis homologue of para, a gene encoding a sodium channel ?-subunit, and obtained many partial cDNA clones from embryos and adults. Para protein has been well conserved, and the optional elements at six different sites of alternative splicing in D. melanogaster are present in D. virilis, in addition to one new optional exon. Among 31 different splice-types observed in D. virilis, the stage-specific pattern of alternative splicing seen in D. melanogaster is also conserved. Comparison of genomic DNA sequence revealed three aspects that vary between alternatively and constitutively used exon sequences. Sixteen short blocks (10-75 bp), the only recognizably conserved intron sequence, were disproportionately associated with alternatively used splice sites. Silent site substitutions were found much less frequently in alternative than constitutive exon elements, and the degree of match to the Drosophila splice site consensus tended to be lower at less frequently selected alternative splice junctions. This study shows that the developmentally regulated variability of para products is highly conserved and therefore likely to be of functional significance and suggests that a variety of different sequence-dependent mechanisms may regulate this pattern of alternative splicing. PMID:8536968

Thackeray, J. R.; Ganetzky, B.

1995-01-01

217

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

PubMed Central

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

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

2013-01-01

218

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

PubMed

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

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

2015-01-01

219

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

PubMed Central

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

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

2014-01-01

220

The evolution, impact and properties of exonic splice enhancers  

PubMed Central

Background In humans, much of the information specifying splice sites is not at the splice site. Exonic splice enhancers are one of the principle non-splice site motifs. Four high-throughput studies have provided a compendium of motifs that function as exonic splice enhancers, but only one, RESCUE-ESE, has been generally employed to examine the properties of enhancers. Here we consider these four datasets to ask whether there is any consensus on the properties and impacts of exonic splice enhancers. Results While only about 1% of all the identified hexamer motifs are common to all analyses we can define reasonably sized sets that are found in most datasets. These consensus intersection datasets we presume reflect the true properties of exonic splice enhancers. Given prior evidence for the properties of enhancers and splice-associated mutations, we ask for all datasets whether the exonic splice enhancers considered are purine enriched; enriched near exon boundaries; able to predict trends in relative codon usage; slow evolving at synonymous sites; rare in SNPs; associated with weak splice sites; and enriched near longer introns. While the intersect datasets match expectations, only one original dataset, RESCUE-ESE, does. Unexpectedly, a fully experimental dataset identifies motifs that commonly behave opposite to the consensus, for example, being enriched in exon cores where splice-associated mutations are rare. Conclusions Prior analyses that used the RESCUE-ESE set of hexamers captured the properties of consensus exonic splice enhancers. We estimate that at least 4% of synonymous mutations are deleterious owing to an effect on enhancer functioning. PMID:24359918

2013-01-01

221

Suppression of HPV-16 late L1 5?-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs  

PubMed Central

Human papillomavirus type 16 (HPV-16) 5?-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5?-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

2013-01-01

222

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

223

Electron donor concentrations in sediments and sediment properties at the agricultural chemicals team research site near New Providence, Iowa, 2006-07  

USGS Publications Warehouse

The concentrations of electron donors in aquifer sediments are important to the understanding of the fate and transport of redox-sensitive constituents in groundwater, such as nitrate. For a study by the U.S. Geological Survey National Water-Quality Assessment Program, 50 sediment samples were collected from below the water table from 11 boreholes at the U.S. Geological Survey Agricultural Chemicals Team research site near New Providence, Iowa, during 2006-07. All samples were analyzed for gravel, sand (coarse, medium, and fine), silt, clay, Munsell soil color, inorganic carbon content, and for the following electron donors: organic carbon, ferrous iron, and inorganic sulfide. A subset of 14 sediment samples also was analyzed for organic sulfur, but all of these samples had concentrations less than the method detection limit; therefore, the presence of this potential electron donor was not considered further. X-ray diffraction analyses provided important semi-quantitative information of well-crystallized dominant minerals within the sediments that might be contributing electron donors.

Maharjan, Bijesh; Korom, Scott F.; Smith, Erik A.

2013-01-01

224

Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1.  

PubMed

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Sequencing of the CYP11B1 gene showed two new base substitutions, a conservative 954 G-->C transversion at the last base of exon 5 (T318T), and a IVS8 + 4A-->G transition in intron 8. In addition, two polymorphisms were found in exons 1 and 2. The genetically female patient was raised as a male because of severe pseudohermaphroditism. Glucocorticoid-suppressive treatment encountered difficulties in equilibration and compliance, resulting in uncontrolled hypertension with pronounced hypertrophic cardiomyopathy. At 42 yr of age the occurrence of central retinal vein occlusion with permanent loss of left eye vision led to the decision to perform bilateral laparoscopic adrenalectomy. Surgery was followed by normalization of blood pressure and good compliance with glucocorticoid and androgen substitutive therapies. In vitro, adrenal cells in culture and isolated mitochondria showed extremely low 11beta-hydroxylase activity. Analysis of adrenal CYP11B1 messenger ribonucleic acid (mRNA) by RT-PCR and sequencing showed the expression of a shorter mRNA that lacked exon 8 and did not contain either the exon 5 mutation or the exon 1 and 2 polymorphisms. This suggested that one CYP11B1 allele carried the intron 8 mutation, responsible for skipping exon 8. The other allele carried the exon 5 mutation, and its mRNA was not detectable. Western blot analysis showed weak expression of a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus, bilateral adrenalectomy in this patient allowed effective treatment of severe hypertension and helped in understanding the mechanisms and physiopathological consequences of two novel mutations of CYP11B1. PMID:11095433

Chabre, O; Portrat-Doyen, S; Chaffanjon, P; Vivier, J; Liakos, P; Labat-Moleur, F; Chambaz, E; Morel, Y; Defaye, G

2000-11-01

225

Heritability of alternative splicing in the human genome  

PubMed Central

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

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

2007-01-01

226

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

PubMed

Diffuse palmoplantar keratoderma (DPPK) is an autosomal-dominant genodermatosis characterized by restricted, uniform hyperkeratosis on the palm and sole epidermis. DPPK is normally associated with dominant-negative mutations in the keratin-encoding gene, KRT1. We report a heterozygous novel point mutation in the exon 6 splice donor site of KRT1 (c.1254G>C) by next-generation sequencing, resulting in the formation of two alternative transcripts, which segregates with DPPK in a four-generation Chinese family. This results in both the complete loss of exon 6 and the simultaneous utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent deletion of 42 amino acids and the insertion of 18 amino acids into the protein's 2B domain. This is the first report of a novel splice donor site mutation with aberrant splicing and the formation of two alternative transcripts causing DPPK. This study also demonstrates the value of next-generation sequencing in the identification of novel disease-causing mutations. PMID:25429721

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

2015-02-01

227

Genetic analysis of autosomal recessive osteopetrosis in Chuvashiya: the unique splice site mutation in TCIRG1 gene spread by the founder effect  

PubMed Central

The rare malignant disorder autosomal recessive osteopetrosis (OPTB) is one of the most prevalent autosomal recessive diseases in the Chuvash Republic of Russia. The purpose of this study was to determine the underlying molecular cause of osteopetrosis in Chuvashiya and to reveal the factors causing the unusual high frequency of the disease in this region. Having assumed a founder effect, we performed linkage disequilibrium (LD) mapping of the OPTB locus at the TCIRG1 region and found a unique splice site mutation c.807+5G>A in all Chuvashian OPTB patients studied. We then analyzed the mutational change in mRNA and detected an intron insertion within the mutant transcript, resulting in a frameshift and premature stop-codon formation (p.Leu271AspfsX231). A decreased expression of the mutant transcript was also detected, which may have been the result of nonsense-mediated decay. Real-time qPCR and MLPA® melting curve analysis-based systems were designed and used for c.807+5G>A mutation screening. In addition to analyzing the gene frequency in Chuvashiya, we also estimated three other populations in the Volga-Ural region (Mari, Udmurt and Bashkir). We found a 1.68% prevalence in Chuvashiya (calculated disease frequency, 1/3500 newborns) and a 0.84% in the Mari population (1/14?000 newborns). The haplotype analysis revealed that all OPTB cases in Chuvashians and Marians originated from a single mutational event and the age of the mutation in Chuvashians was estimated to be approximately 890 years. PMID:19172990

Bliznetz, Elena A; Tverskaya, Svetlana M; Zinchenko, Rena A; Abrukova, Anna V; Savaskina, Ekaterina N; Nikulin, Maxim V; Kirillov, Alexander G; Ginter, Evgeny K; Polyakov, Alexander V

2009-01-01

228

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. PMID:20158892

2010-01-01

229

Laparoendoscopic single-site (LESS) vs laparoscopic living-donor nephrectomy: a systematic review and meta-analysis.  

PubMed

The aim of this study was to provide a systematic review and meta-analysis of reports comparing laparoendoscopic single-site (LESS) living-donor nephrectomy (LDN) vs standard laparoscopic LDN (LLDN). A systematic review of the literature was performed in September 2013 using PubMed, Scopus, Ovid and The Cochrane library databases. Article selection proceeded according to the search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-analyses criteria. Weighted mean differences (WMDs) were used to measure continuous variables and odds ratios (ORs) to measure categorical ones. Nine publications meeting eligibility criteria were identified, including 461 LESS LDN and 1006 LLDN cases. There were more left-side cases in the LESS LDN group (96.5% vs 88.6%, P < 0.001). Meta-analysis of extractable data showed that LLDN had a shorter operative time (WMD 15.06?min, 95% confidence interval [CI] 4.9-25.1; P = 0.003), without a significant difference in warm ischaemia time (WMD 0.41?min, 95% CI -0.02 to 0.84; P = 0.06). Estimated blood loss was lower for LESS LDN (WMD -22.09?mL, 95% CI -29.5 to -14.6; P < 0.001); however, this difference was not clinically significant. There was a greater likelihood of conversion for LESS LDN (OR 13.21, 95% CI 4.65-37.53; P < 0.001). Hospital stay was similar (WMD -0.11 days, 95% CI -0.33 to 0.12; P = 0.35), as well as the visual analogue pain score at discharge (WMD -0.31, 95% CI -0.96 to 0.35; P = 0.36), but the analgesic requirement was lower for LESS LDN (WMD -2.58?mg, 95% CI -5.01 to -0.15; P = 0.04). Moreover, there was no difference in the postoperative complication rate (OR 1.00, 95% CI 0.65-1.54; P = 0.99). Renal function of the recipient, as based on creatinine levels at 1 month, showed similar outcomes between groups (WMD 0.10?mg/dL, -0.09 to 0.29; P = 0.29). In conclusion, LESS LDN represents an emerging option for living kidney donation. This procedure offers comparable surgical and early functional outcomes to the conventional LLDN, with a lower analgesic requirement. However, it is more technically challenging than LLDN, as shown by a greater likelihood of conversion. The role of LESS LDN remains to be defined. PMID:24588876

Autorino, Riccardo; Brandao, Luis Felipe; Sankari, Bashir; Zargar, Homayoun; Laydner, Humberto; Akça, Oktay; De Sio, Marco; Mirone, Vincenzo; Chueh, Shih-Chieh J; Kaouk, Jihad H

2015-02-01

230

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences  

Microsoft Academic Search

A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that

Michael Krawczak; Jochen Reiss; David N. Cooper

1992-01-01

231

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] [Thomas Jefferson Univ., Philadelphia, PA (United States); and others

1996-06-14

232

Visualizing RNA splicing in vivo Gayatri Gowrishankar and Jianghong Rao*  

E-print Network

the completely unexpected discovery that RNA from a unicellular protozoan called Tetrahymena thermophila could perform auto-splicing.1 This discovery jump- started widespread research into the structure, function atom at the 59-splice site and forms a 39,59-phosphodiester bond to the first nucleotide of the intron

Rao, Jianghong

233

Complex Alternative Splicing  

PubMed Central

Alternative splicing is a powerful means of controlling gene expression and increasing protein diversity. Most genes express a limited number of mRNA isoforms, but there are several examples of genes that use alternative splicing to generate hundreds, thousands, and even tens of thousands of isoforms. Collectively such genes are considered to undergo complex alternative splicing. The best example is the Drosophila Down syndrome cell adhesion molecule (Dscam) gene, which can generate 38,016 isoforms by the alternative splicing of 95 variable exons. In this review, we will describe several genes that use complex alternative splicing to generate large repertoires of mRNAs and what is known about the mechanisms by which they do so. PMID:18380340

Park, Jung Woo; Graveley, Brenton R.

2015-01-01

234

Functionally antagonistic sequences are required for normal autoregulation of Drosophila tra-2 pre-mRNA splicing  

PubMed Central

Expression of functional TRA-2 protein in the male germline of Drosophila is regulated through a negative feedback mechanism in which a specific TRA-2 isoform represses splicing of the M1 intron in the TRA-2 pre-mRNA. We have previously shown that the mechanism of M1 splicing repression is conserved between distantly related Drosophila species. Using transgenic fly strains, we have examined the effects on regulation of mutations in two conserved features of the M1 intron. Our results show that TRA-2-dependent repression of M1 splicing depends on the presence of a suboptimal non-consensus 3? splice site. Substitution of this 3? splice site with a strong splice site resulted in TRA-2 independent splicing, while substitution with an unrelated weak 3? splice site was compatible with repression, implying that reduced basal splicing efficiency is important for regulation. A second conserved element internal to the intron was found to be essential for efficient M1 splicing in the soma where the intron is not normally retained. We show that the role of this element is to enhance splicing and overcome the reduction in efficiency caused by the intron’s suboptimal 3? splice site. Our results indicate that antagonistic elements in the M1 intron act together to establish a context that is permissive for repression of splicing by TRA-2 while allowing efficient splicing in the absence of a repressor. PMID:11452026

Chandler, Dawn S.; McGuffin, M. Elaine; Mattox, William

2001-01-01

235

Evidence for Widespread Association of Mammalian Splicing and Conserved Long-Range RNA Structures  

E-print Network

splice sites. As an example, we validated the RNA structure in the human SF1 gene using mini-genes in HEK splicing; SF1; HNRNPK; ZFX; ZIP7; SLC39A7; ZNF384; SRSF7; PRPF39 Abstract Pre-mRNA structure impacts many cellular processes, including splicing in genes associated with disease. The contemporary paradigm of RNA

Pervouchine, Dmitri D.

236

Splicing regulators: targets and drugs  

PubMed Central

Silencing of splicing regulators by RNA interference, combined with splicing-specific microarrays, has revealed a complex network of distinct alternative splicing events in Drosophila, while a high-throughput screen of more than 6,000 compounds has identified drugs that interfere specifically and directly with one class of splicing regulators in human cells. PMID:16356274

Yeo, Gene Wei-Ming

2005-01-01

237

The splicing landscape is globally reprogrammed during male meiosis  

PubMed Central

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ?150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2?, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2? and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5? splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle. PMID:24038356

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

2013-01-01

238

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii  

PubMed Central

Background Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes. PMID:20163725

2010-01-01

239

Conditional Control of Alternative Splicing through Light-Triggered Splice-Switching Oligonucleotides.  

PubMed

The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF ? ON and ON ? OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals. PMID:25734836

Hemphill, James; Liu, Qingyang; Uprety, Rajendra; Samanta, Subhas; Tsang, Michael; Juliano, Rudolph L; Deiters, Alexander

2015-03-18

240

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

PubMed

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

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

2015-04-20

241

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

PubMed

The aim of this study was to assess the objective and subjective morbidity after reconstruction of alveolar bone defects with mandibular symphyseal bone grafts in patients with cleft lip and palate. One hundred and eleven patients born between 1995 and 1999, who had undergone chin bone harvesting for alveolar cleft reconstruction in the period from 2000 through 2011, were included. A survey of medical records was conducted. Subjective morbidity after reconstruction was assessed using a questionnaire. Medical records revealed few postoperative incidents; 5.6% reported persistent sensory disturbances in the donor area. Postoperative pain averaged 3.6 ± 2.1 (scale 0-10). The overall satisfaction with the surgical result was 8.7 ± 1.7 (scale 0-10). This study revealed that chin bone harvesting for reconstruction of alveolar defects in patients with cleft lip and palate is a safe and predictable procedure, highly appreciated by the patients, and characterized by only minor postoperative incidents. Patients must be informed of the risk of sensory disturbances in the donor area. PMID:24183738

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

2014-04-01

242

Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing  

PubMed Central

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

Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

2012-01-01

243

Human Genomic Sequences That Inhibit Splicing  

Microsoft Academic Search

Mammalian genes are characterized by relatively small exons surrounded by variable lengths of intronic sequence. Sequences similar to the splice signals that define the 5* and 3* boundaries of these exons are also present in abundance throughout the surrounding introns. What causes the real sites to be distinguished from the multitude of pseudosites in pre-mRNA is unclear. Much progress has

WILLIAM G. FAIRBROTHER; LAWRENCE A. CHASIN

2000-01-01

244

Multifunctional In Situ Photopolymerized Semi-Interpenetrating Network System is an Effective Donor Site Dressing: A Cross Comparison Study in a Swine Model  

PubMed Central

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

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

2012-01-01

245

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

PubMed Central

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

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

2013-01-01

246

Splicing regulation as a potential genetic modifier  

Microsoft Academic Search

Inherited diseases are associated with profound phenotypic variability, which is affected strongly by genetic modifiers. The splicing machinery could be one such modifying system, through a mechanism involving splicing motifs and their interaction with a complex repertoire of splicing factors. Mutations in splicing motifs and changes in levels of splicing factors can result in different splicing patterns. Changes in the

Malka Nissim-Rafinia; Batsheva Kerem

2002-01-01

247

WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo  

SciTech Connect

Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

Markus, M. Andrea [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Heinrich, Bettina [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Raitskin, Oleg [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Adams, David J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Mangs, Helena [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Goy, Christine [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Ladomery, Michael [Centre for Research in Biomedicine, Bristol Genomics Research Institute, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY (United Kingdom); Sperling, Ruth [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Stamm, Stefan [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Morris, Brian J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia)]. E-mail: brianm@medsci.usyd.edu.au

2006-10-15

248

Alternative splicing generates secretory isoforms of human CD1.  

PubMed Central

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

Woolfson, A; Milstein, C

1994-01-01

249

Splice form-dependence of ?-neurexin/neuroligin binding interactions  

PubMed Central

Alternatively spliced ?-neurexins (?-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a ?-NRX/NL synaptic recognition code. We have utilized surface plasmon resonance to measure binding affinities between all sixty combinations of alternatively spliced ectodomains of ?-NRXs 1–3 and NLs 1–3. Binding was observed for all ?-NRX/NL pairs. The presence of the NL1 B splice insertion lowers ?-NRX binding affinity by ~2-fold, while ?-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated ?-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new ?-strand that replaces the ?10-strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in ?-NRX/NL mediated synaptic recognition. PMID:20624592

Koehnke, Jesko; Katsamba, Phinikoula S.; Ahlsen, Goran; Bahna, Fabiana; Vendome, Jeremie; Honig, Barry; Shapiro, Lawrence; Jin, Xiangshu

2010-01-01

250

Modeling the active sites of non-heme diiron metalloproteins with sterically hindered carboxylates and syn N-Donor ligands  

E-print Network

Chapter 1. Different Synthetic Approaches to Modeling the Active Sites of Carboxylate-Bridged Non-Heme Diiron Enzymes Carboxylate-bridged non-heme diiron enzymes activate dioxygen to perform a variety of biological functions. ...

Friedle, Simone, 1976-

2009-01-01

251

Oxidation of substrates tethered to N-donor ligands for modeling non-heme diiron enzyme active sites  

E-print Network

Chapter 1. Modeling Carboxylate-Rich Diiron Sites of Dioxygen-Dependent Non-Heme Enzymes Carboxylate-bridged diiron centers are employed in a variety of biological systems to activate dioxygen for substrate oxidation, and ...

Carson, Emily Carrig, 1978-

2005-01-01

252

The splice of life: Alternative splicing and neurological disease  

Microsoft Academic Search

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we

B. Kate Dredge; Alexandros D. Polydorides; Robert B. Darnell

2001-01-01

253

Functional studies on the ATM intronic splicing processing element  

PubMed Central

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

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

2005-01-01

254

Unexpected CEP290 mRNA Splicing in a Humanized Knock-In Mouse Model for Leber Congenital Amaurosis  

PubMed Central

Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations. PMID:24223178

Garanto, Alejandro; van Beersum, Sylvia E. C.; Peters, Theo A.; Roepman, Ronald; Cremers, Frans P. M.; Collin, Rob W. J.

2013-01-01

255

Unexpected CEP290 mRNA splicing in a humanized knock-in mouse model for Leber congenital amaurosis.  

PubMed

Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations. PMID:24223178

Garanto, Alejandro; van Beersum, Sylvia E C; Peters, Theo A; Roepman, Ronald; Cremers, Frans P M; Collin, Rob W J

2013-01-01

256

Donor Tag Game  

MedlinePLUS

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

257

Splice assembly tool and method of splicing  

DOEpatents

A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

Silva, Frank A. (Basking Ridge, NJ)

1980-01-01

258

Biogeochemical Modeling of In Situ U(VI) Reduction and Immobilization with Emulsified Vegetable Oil as the Electron Donor at a Field Site in Oak Ridge, Tennessee  

NASA Astrophysics Data System (ADS)

A comprehensive biogeochemical model was developed to quantitatively describe the coupled hydrologic, geochemical and microbiological processes that occurred following injection of emulsified vegetable oil (EVO) as the electron donor to immobilize U(VI) at the Oak Ridge Integrated Field Research Challenge site (ORIFRC) in Tennessee. The model couples the degradation of EVO, production and oxidation of long-chain fatty acids (LCFA), glycerol, hydrogen and acetate, reduction of nitrate, manganese, ferrous iron, sulfate and uranium, and methanoganesis with growth of multiple microbial groups. The model describes the evolution of geochemistry and microbial populations not only in the aqueous phase as typically observed, but also in the mineral phase and therefore enables us to evaluate the applicability of rates from the literature for field scale assessment, estimate the retention and degradation rates of EVO and LCFA, and assess the influence of the coupled processes on fate and transport of U(VI). Our results suggested that syntrophic bacteria or metal reducers might catalyze LCFA oxidation in the downstream locations when sulfate was consumed, and competition between methanogens and others for electron donors and slow growth of methanogen might contribute to the sustained reducing condition. Among the large amount of hydrologic, geochemical and microbiological parameter values, the initial biomass, and the interactions (e.g., inhibition) of the microbial functional groups, and the rate and extent of Mn and Fe oxide reduction appear as the major sources of uncertainty. Our model provides a platform to conduct numerical experiments to study these interactions, and could be useful for further iterative experimental and modeling investigations into the bioreductive immobiliztion of radionuclide and metal contaminants in the subsurface.

Tang, G.; Parker, J.; Wu, W.; Schadt, C. W.; Watson, D. B.; Brooks, S. C.; Orifrc Team

2011-12-01

259

FOA Lecture 6: Fiber Optic Splices  

NSDL National Science Digital Library

This is the sixth lecture in the FOA series on fiber optics, covering splices. In this lecture the presenter discusses the uses of splices, types of splices such as fusion splices and mechanical splices, and the processes used for making each type of splice. The video also discusses cleaving, which is the key to getting good splices. Running time for the lecture is 9:03. Flash is required to view the video.

260

Tissue-specific splicing mutation in acute intermittent porphyria.  

PubMed Central

An inherited deficiency of porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G----A) in the canonical 5' splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using oligonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families. Images PMID:2563167

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

1989-01-01

261

Splicing-Related Features of Introns Serve to Propel Evolution  

PubMed Central

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution. PMID:23516505

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

2013-01-01

262

Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster  

PubMed Central

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster. PMID:21233220

Hartmann, Britta; Castelo, Robert; Miñana, Belén; Peden, Erin; Blanchette, Marco; Rio, Donald C.; Singh, Ravinder; Valcárcel, Juan

2011-01-01

263

A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.  

PubMed

X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development. PMID:25443657

Gray, P E A; Logan, G J; Alexander, I E; Poulton, S; Roscioli, T; Ziegler, J

2015-02-01

264

Management of young blood donors.  

PubMed

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

Newman, Bruce H

2014-07-01

265

Management of Young Blood Donors  

PubMed Central

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

Newman, Bruce H.

2014-01-01

266

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

PubMed Central

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

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

2011-01-01

267

RNA splicing mutation in an aberrantly rearranged immunoglobulin lambda I gene.  

PubMed Central

The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma. We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain. This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene. The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied. The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining. In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions. Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing. Images PMID:6171827

Hozumi, N; Wu, G E; Murialdo, H; Roberts, L; Vetter, D; Fife, W L; Whiteley, M; Sadowski, P

1981-01-01

268

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

PubMed

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

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

2014-04-28

269

Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis.  

PubMed

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes. PMID:25372122

Burkhardt, Alyssa; Buchanan, Alex; Cumbie, Jason S; Savory, Elizabeth A; Chang, Jeff H; Day, Brad

2015-03-01

270

Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy  

PubMed Central

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

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

2013-01-01

271

The landscape of alternative splicing in cervical squamous cell carcinoma  

PubMed Central

Background Alternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. In this study, we identified alternative splicing events in four pairs of cervical squamous cell carcinoma (CSCC) and adjacent nontumor tissues using RNA sequencing. Methods The transcripts of the four paired samples were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted to detect the alternative spliced genes-related signal pathways. The alternative spliced genes were validated by reverse transcription-polymerase chain reaction (RT-PCR). Results There were 35 common alternative spliced genes in the four CSCC samples; they were novel and CSCC specific. Sixteen pathways were significantly enriched (P<0.05). One novel 5?AS site in the KLHDC7B gene, encoding kelch domain-containing 7B, and an exon-skipping site in the SYCP2 gene, encoding synaptonemal complex 2, were validated by RT-PCR. The KLHDC7B gene with 5?AS was found in 67.5% (27/40) of CSCC samples and was significantly related with cellular differentiation and tumor size. The exon-skipping site of the SYCP2 gene was found in 35.0% (14/40) of CSCC samples and was significantly related with depth of cervical invasion. Conclusion The KLHDC7B and the SYCP2 genes with alternative spliced events might be involved in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC. PMID:25565867

Guo, Peng; Wang, Dan; Wu, Jun; Yang, Junjun; Ren, Tong; Zhu, Baoli; Xiang, Yang

2015-01-01

272

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo  

PubMed Central

In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2r?null mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the ?-globin gene using nanoparticles carrying ?-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- ?-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo. PMID:23076379

McNeer, Nicole A.; Schleifman, Erica B.; Cuthbert, Amy; Brehm, Michael; Jackson, Andrew; Cheng, Christopher; Anandalingam, Kavitha; Kumar, Priti; Shultz, Leonard D.; Greiner, Dale L.

2013-01-01

273

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

PubMed

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

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

1999-10-01

274

TRAP150 activates splicing in composite terminal exons  

PubMed Central

The spliceosomal factor TRAP150 is essential for pre-mRNA splicing in vivo and, when overexpressed, it enhances splicing efficiency. In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5? splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3? splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression. PMID:25326322

Lee, Kuo-Ming; Tarn, Woan-Yuh

2014-01-01

275

Committee of Donor Agencies for Small Enterprise Development  

NSDL National Science Digital Library

Established in 1979, the Committee of Donor Agencies promotes the development of small enterprise in developing countries. This site offers numerous working and research papers about the Committee of Donor Agencies and its members. Showcased on the site are the Committee's Donor Business Development Services Case Studies. The Case studies are browseable by several categories including Region, Country, Theme, and Member Agency. Also provided are the Donor Committee Guidelines and links to member agencies's sites.

276

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

PubMed

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV. PMID:25098561

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

2014-11-01

277

Living Donor Liver Transplantation  

MedlinePLUS

... the donor's blood type matches the recipient’s blood type. Next, the transplant team will measure liver and kidney function as well as red cell, white cell, and platelet counts. The donor is also tested for viruses ... the donor’s and recipient’s blood types are compatible, the donor will get a physical ...

278

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution  

PubMed Central

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3? splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ?15-fold increase in the frequency of three base pair gaps at 3? splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences. PMID:22235189

Bradley, Robert K.; Merkin, Jason; Lambert, Nicole J.; Burge, Christopher B.

2012-01-01

279

Global analysis of alternative splicing regulation by insulin and wingless signaling in Drosophila cells  

PubMed Central

Background Despite the prevalence and biological relevance of both signaling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signaling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis using splicing-sensitive microarrays of changes in alternative splicing induced by activation of two distinct signaling pathways, insulin and wingless, in Drosophila cells in culture. Results Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and post-transcriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related to carbohydrate metabolism and cellular signaling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5' splice sites. Conclusions Taken together, our data indicate that signaling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for crosstalk between different signaling pathways. PMID:19178699

Hartmann, Britta; Castelo, Robert; Blanchette, Marco; Boue, Stephanie; Rio, Donald C; Valcárcel, Juan

2009-01-01

280

Basal splicing factors regulate the stability of mature mRNAs in trypanosomes.  

PubMed

Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3' UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3' UTR bind the splicing factors and control their stability. PMID:23283975

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

2013-02-15

281

Extensive aggregation of ?-synuclein and tau in juvenile-onset neuroaxonal dystrophy: an autopsied individual with a novel mutation in the PLA2G6 gene-splicing site  

PubMed Central

Background Infantile neuroaxonal dystrophy (INAD) is a rare autosomal-recessive neurodegenerative disorder. Patients with INAD usually show neurological symptoms with infant onset and die in childhood. Recently, it was reported that mutations in the PLA2G6 gene cause INAD, but neuropathological analysis of genetically confirmed individuals with neuroaxonal dystrophy has been limited. Results Here, we report a Japanese individual with neuroaxonal dystrophy associated with compound heterozygous mutations in the PLA2G6 gene. A novel splice-site mutation resulting in skipping and missense mutations (p.R538C) in exon 9 was identified in the patient. This patient initially presented with cerebellar ataxia at the age of 3 years, which was followed by symptoms of mental retardation, extrapyramidal signs, and epileptic seizure. The patient survived until 20 years of age. Neuropathological findings were characterized by numerous axonal spheroids, brain iron deposition, cerebellar neuronal loss, phosphorylated alpha-synuclein-positive Lewy bodies (LBs), and phosphorylated-tau-positive neurofibrillary tangles. In particular, LB pathology exhibited a unique distribution with extremely severe cortical involvement. Conclusions Our results support a genetic clinical view that compound heterozygous mutations with potential residual protein function are associated with a relatively mild phenotype. Moreover, the severe LB pathology suggests that dysfunction of the PLA2G6 gene primarily contributes to LB formation. PMID:24252552

2013-01-01

282

Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop  

PubMed Central

In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNAThr species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNAThr species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site. PMID:21846775

Hirata, Akira; Kitajima, Tsubasa; Hori, Hiroyuki

2011-01-01

283

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

PubMed

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

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

2015-03-17

284

Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism  

USGS Publications Warehouse

The gene encoding IgH ? has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated ?-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory ? transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory ? transcript resulted in two ?-H chains, which incorporated C?1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory ? mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.

2012-01-01

285

The fibronectin gene as a model for splicing and transcription studies  

Microsoft Academic Search

The fibronectm (FN) gene has become paradigmatic to illustrate genome evolution by exon shuffling, generation of protein diversity by alterna- tive mRNA splicing, and topological coordination between transcription and splicing. Alternative splic- ing in three sites of the primary transcript gives rise to multiple FN polypeptides. This process is cell type-, development- and age-regulated. The differ- ent FN variants seem

A. R. KORNBUHrF; C. C. PESCE; C. R. ALONSO; P. CRAMER; A. SREBROW; S. WERBA; A. F. MURO; Ingenieria Gen

286

Alternative splicing of Alu exons--two arms are better than one  

Microsoft Academic Search

Alus, primate-specific retroelements, are the most abundant repetitive elements in the human genome. They are composed of two related but distinct monomers, left and right arms. Intronic Alu ele- ments may acquire mutations that generate func- tional splice sites, a process called exonization. Most exonizations occur in right arms of antisense Alu elements, and are alternatively spliced. Here we show

Nurit Gal-Mark; Schraga Schwartz; Gil Ast

2008-01-01

287

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

PubMed

Hepatitis B virus (HBV) is generally classified into eight genotypes (A to H) based on genomic sequence divergence. The sequence variation among the different HBV genotypes suggests that the spliced RNAs should be different from genotype to genotype. However, the cis-acting element involved in the modulation of the distinct expression profiles of spliced HBV RNAs remains unidentified. Moreover, the biological role of splicing in the life cycle of HBV is not yet understood. In this study, spliced RNAs generated from genotypes A and D were carefully characterized in transfected HepG2 cells. The species and frequency of the spliced RNAs were dramatically different in the two genotypes. Of note, a population of multiply spliced RNAs with intron 2067-2350 excision was identified in HBV genotype A-transfected HepG2 cells, but not in genotype D transfected HepG2 cells. Further, we found a single nucleotide difference (2335) located within the polypyrimidine tract of the splice acceptor site 2350 between the two genotypes, and a single base substitution at 2335 was able to convert the splicing pattern of genotype D (or genotype A) to that of genotype A (or genotype D). These findings suggest that different unique splice sites may be preferentially used in different HBV genotypes resulting in distinct populations of spliced RNAs. The possible significance of the distinct spliced RNAs generated from the different HBV genotypes in HBV infection is discussed. PMID:23501362

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

2013-06-01

288

Simultaneous detection of Hb constant spring (?142, TAA>CAA, ?2) and the ?2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran.  

PubMed

Hb Constant Spring (Hb CS, codon 142, TAA>CAA, ?2) (HBA2:c.427T>C) and ?2 IVS-I donor site (GAGGTGAGG>GAGG?- - - - -) (HBA2:c.95+2_95+6delTGAGG) are nondeletional ?-thalassemia (?-thal) mutations found all over the world. Identification of ?-thal genotypes in at-risk couples for severe anemia or in highly heterogeneous populations requires rapid, accurate and cost-effective genotyping methods. In this study, a pair of primers were used to specifically amplify an 883 bp fragment from the ?2-globin gene in order to simultaneously identify these two mutations by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We determined the genotypic frequencies of Hb CS and the ?2 IVS-I donor site mutations after amplification and enzymatic digestion with Tru9I in 238 northern Iranian samples referred for ?-thal testing. Hb CS and the ?2 IVS-I donor site mutations accounted for 21 (8.8%) and 29 (12.2%) of the nondeletional cases. This genotyping assay has proven to be a rapid, reliable and useful diagnostic tool for simultaneous detection of these two anomalies for genetic counseling or further prenatal diagnosis. PMID:22356652

Akhavan-Niaki, Haleh; Banihashemi, Ali; Mostafazadeh, Amrollah; Kholghi Oskooei, Vahid; Azizi, Mandana; Youssefi Kamangar, Reza; Elmi, Maryam Mitra

2012-01-01

289

Splicing defects caused by exonic mutations in PKD1 as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease.  

PubMed

The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD. PMID:25757501

Claverie-Martin, Felix; Gonzalez-Paredes, Francisco J; Ramos-Trujillo, Elena

2015-04-01

290

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

PubMed Central

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

Xu, Dong-Qing; Mattox, William

2006-01-01

291

Detection and measurement of alternative splicing using splicing-sensitive microarrays  

Microsoft Academic Search

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting

Karpagam Srinivasan; Lily Shiue; Justin D. Hayes; Ross Centers; Sean Fitzwater; Rebecca Loewen; Lillian R. Edmondson; Jessica Bryant; Michael Smith; Claire Rommelfanger; Valerie Welch; Tyson A. Clark; Charles W. Sugnet; Kenneth J. Howe; Yael Mandel-Gutfreund; Manuel Ares

2005-01-01

292

Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo.  

PubMed

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5' splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3' splice site. The 5' and 3' splice site complexes are thought to be joined together by protein-protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF(65). Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5' and 3' splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival. PMID:25605964

Becerra, Soraya; Montes, Marta; Hernández-Munain, Cristina; Suñé, Carlos

2015-03-01

293

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.  

PubMed

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton. PMID:20212022

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

294

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates  

PubMed Central

Pre-mRNA 5? spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ?250,000 high-quality reads corresponding to 8790 genes, ?58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of “infrequently trans-spliced” genes, accounting for ?28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing ?80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of ±3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca2+ homeostasis, and actin cytoskeleton. PMID:20212022

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B.; Macmil, Simone L.; Roe, Bruce A.; Zeller, Robert W.; Satou, Yutaka; Hastings, Kenneth E.M.

2010-01-01

295

Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis  

PubMed Central

Background Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing. Results Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes. Conclusions We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote. PMID:24393432

2014-01-01

296

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing  

PubMed Central

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3? splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation. PMID:19729513

Nojima, Takayuki; Oshiro-Ideue, Takako; Nakanoya, Hiroto; Kawamura, Hidenobu; Morimoto, Tomomi; Kawaguchi, Yasushi; Kataoka, Naoyuki; Hagiwara, Masatoshi

2009-01-01

297

Regulation of neurexin 1? tertiary structure and ligand binding through alternative splicing  

PubMed Central

Summary Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a “splice insert signaling code”. In particular, neurexin 1? carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1?+SS#4 reveals dramatic rearrangements to the “hyper-variable surface”, the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop ?10-?11 into a helix rearranging the binding site for neuroligins, and 3) rearranges the Ca2+-binding site required for ligand binding increasing its affinity. Our structures reveal the mechanism by which neurexin 1? isoforms acquire neuroligin splice isoform selectivity. PMID:18334217

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

2008-01-01

298

The primary donor cation P + rad in photosynthetic reaction centers of site-directed mutants of Rhodobacter sphaeroides: g-tensor shifts revealed by high-field EPR at 360 GHz/12.8 T  

NASA Astrophysics Data System (ADS)

The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P + rad in reaction centers of site-directed mutants of Rhodobacter ( Rb.) sphaeroides has been obtained at a microwave frequency ?=360 GHz and a magnetic field B0=12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g-tensors at T=160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g-tensor with the principal components gxx=2.00335(3), gyy=2.00236(2) and gzz=2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g-tensor anisotropy ? g= gxx- gzz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer.

Fuchs, Martin R.; Schnegg, Alexander; Plato, Martin; Schulz, Claudia; Müh, Frank; Lubitz, Wolfgang; Möbius, Klaus

2003-11-01

299

Splicing and alternative splicing in rice and humans.  

PubMed

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

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-09-01

300

Splicing and alternative splicing in rice and humans  

PubMed Central

Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. [BMB Reports 2013; 46(9): 439-447] PMID:24064058

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-01-01

301

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

PubMed Central

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

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

2007-01-01

302

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution  

E-print Network

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3? splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist ...

Bradley, Robert K.

303

SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.  

PubMed

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

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

2014-06-01

304

Targeting RNA Splicing for Disease Therapy  

PubMed Central

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

Havens, Mallory A.; Duelli, Dominik M.

2013-01-01

305

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

PubMed

Replacement of mRNA 5' UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3' of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare. PMID:20142326

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

2010-04-01

306

Promoter-proximal polyadenylation sites reduce transcription activity  

PubMed Central

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ?500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

2012-01-01

307

Phylogenetic and Molecular Characterization of the Splicing Factor RBM4  

PubMed Central

The mammalian multi-functional RNA-binding motif 4 (RBM4) protein regulates alterative splicing of precursor mRNAs and thereby affects pancreas and muscle cell differentiation. RBM4 homologs exist in all metazoan lineages. The C-terminal unstructured domain of RBM4 is evolutionarily divergent and contains stretches of low-complexity sequences, including single amino acid and/or dipeptide repeats. Here we examined the splicing activity, phosphorylation potential, and subcellular localization of RBM4 homologs from a wide range of species. The results show that these RBM4 homologs exert different effects on 5? splice site utilization and exon selection, and exhibit different subnuclear localization patterns. Therefore, the C-terminal domain of RBM4 may contribute to functional divergence between homologs. On the other hand, analysis of chimeric human RBM4 proteins containing heterologous sequences at the C-terminus revealed that the N-terminal RNA binding domain of RBM4 could have a dominant role in determining splicing outcome. Finally, all RBM4 homologs examined could be phosphorylated by an SR protein kinase, suggesting that they are regulated by a conserved mechanism in different species. This study offers a first clue to functional evolution of a splicing factor. PMID:23527094

Wu, Jhe-Rong; Chen, Hung-Hsi; Yu, Hsin-Yi; Tarn, Woan-Yuh

2013-01-01

308

Structure of the branched intermediate in protein splicing.  

PubMed

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

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

2014-06-10

309

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica  

PubMed Central

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3?-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment. PMID:22368181

Kabran, Philomène; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuvéglise, Cécile

2012-01-01

310

Our Future Donors  

ERIC Educational Resources Information Center

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

Miller, Richard E.

2004-01-01

311

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

NASA Astrophysics Data System (ADS)

The properties of serum albumin obtained from donors and from paranoid schizophrenia patients were studied with the fluorescent probe K-35 (N-carboxyphenylimide of dimethylaminonaphthalic acid) and time-resolved fluorescence spectroscopy on the SR beam station of the S-60 synchrotron of the Lebedev Physical Institute. The mean fluorescence quantum yield of K-35 in patients serum was decreased significantly by 25-60% comparing with donors. The analysis of pre-exponential factors of fluorescence decay using "amplitude standard" method has shown that in patient sera the fraction of K-35 molecules bound with albumin and inaccessible to fluorescence quenchers ("bright" K-35 molecules with ?1=8.0±0.4 ns) is 1.2-3 times less than in the donor sera. The fraction of K-35 molecules with partly quenched fluorescence ( ?2=1.44±0.22 ns) was significantly increased in schizophrenia patients. The results obtained suggest that the properties of binding region in serum albumin molecules of acute paranoid schizophrenia patients change significantly.

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

2000-06-01

312

Validation of predicted mRNA splicing mutations using high-throughput transcriptome data  

PubMed Central

Interpretation of variants present in complete genomes or exomes reveals numerous sequence changes, only a fraction of which are likely to be pathogenic. Mutations have been traditionally inferred from allele frequencies and inheritance patterns in such data. Variants predicted to alter mRNA splicing can be validated by manual inspection of transcriptome sequencing data, however this approach is intractable for large datasets. These abnormal mRNA splicing patterns are characterized by reads demonstrating either exon skipping, cryptic splice site use, and high levels of intron inclusion, or combinations of these properties. We present, Veridical, an in silico method for the automatic validation of DNA sequencing variants that alter mRNA splicing. Veridical performs statistically valid comparisons of the normalized read counts of abnormal RNA species in mutant versus non-mutant tissues. This leverages large numbers of control samples to corroborate the consequences of predicted splicing variants in complete genomes and exomes. PMID:24741438

Rogan, Peter K.

2014-01-01

313

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

SciTech Connect

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

Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A. [Universite de Paris (France)] [and others

1995-09-20

314

Exon junction complex enhances translation of spliced mRNAs at multiple steps.  

PubMed

Translation of spliced mRNAs is enhanced by exon junction complex (EJC), which is deposited on mRNAs as a result of splicing. Although this phenomenon itself is well known, the underlying molecular mechanism remains poorly understood. Here we show, using siRNAs against Y14 and eIF4AIII and spliced or intronless constructs that contain different types of internal ribosome entry sites (IRESes), that Y14 and eIF4AIII increase translation of spliced mRNAs before and after formation of the 80S ribosome complex, respectively. These results suggest that EJC modulates translation of spliced mRNA at multiple steps. PMID:19409878

Lee, Hyung Chul; Choe, Junho; Chi, Sung-Gil; Kim, Yoon Ki

2009-07-01

315

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

PubMed Central

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ?E10, ?E12, ?E33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame. PMID:24918291

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

2014-01-01

316

Becoming a Donor  

MedlinePLUS

... by Organ and Gender. > U.S. Waiting List Candidate Data HOW TO BECOME A DONOR The most important thing to do is to sign up as an organ and tissue donor in your state's donor registry. To cover all bases, it's also helpful to: Designate your decision on ...

317

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

PubMed

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

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

2014-01-01

318

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

PubMed Central

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

Grodecká, Lucie; Lockerová, Pavla; Rav?uková, Barbora; Buratti, Emanuele; Baralle, Francisco E.; Dušek, Ladislav; Freiberger, Tomáš

2014-01-01

319

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

PubMed

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

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

2014-01-01

320

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

PubMed Central

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

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

2014-01-01

321

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

PubMed Central

The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5’ splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the “spf/ash” mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations. PMID:25853564

Viecelli, Hiu Man; Rüfenacht, Veronique; Pérez, Belén; Ugarte, Magdalena; Häberle, Johannes; Thöny, Beat; Desviat, Lourdes Ruiz

2015-01-01

322

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

PubMed

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

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

2014-10-13

323

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays  

PubMed Central

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5? splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921

Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel

2006-01-01

324

Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome.  

PubMed

Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. PMID:25586593

Madan, Vikas; Kanojia, Deepika; Li, Jia; Okamoto, Ryoko; Sato-Otsubo, Aiko; Kohlmann, Alexander; Sanada, Masashi; Grossmann, Vera; Sundaresan, Janani; Shiraishi, Yuichi; Miyano, Satoru; Thol, Felicitas; Ganser, Arnold; Yang, Henry; Haferlach, Torsten; Ogawa, Seishi; Koeffler, H Phillip

2015-01-01

325

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

PubMed Central

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

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

2012-01-01

326

A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression.  

PubMed

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3' splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3' splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3' splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3' splice site at position 5639 in the late region. Optimization of the position 3358 3' splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3' splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression. PMID:16140776

Rush, Margaret; Zhao, Xiaomin; Schwartz, Stefan

2005-09-01

327

Splicing and Multifactorial Analysis of Intronic BRCA1 and BRCA2 Sequence Variants Identifies Clinically Significant Splicing Aberrations up to 12 Nucleotides from the Intron/Exon Boundary  

PubMed Central

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twentysix intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants. PMID:21394826

Whiley, Phillip J.; Guidugli, Lucia; Walker, Logan C.; Healey, Sue; Thompson, Bryony A.; Lakhani, Sunil R.; Da Silva, Leonard M.; Tavtigian, Sean V.; Goldgar, David E.; Brown, Melissa A.; Couch, Fergus J.; Spurdle1, Amanda B.

2015-01-01

328

Performance of Reinforced Concrete Column Lap Splices  

E-print Network

Cantilevered reinforced concrete columns with a lap splice of the longitudinal reinforcement near the base can induce high moment demands on the splice region when lateral loads are present on the structure. Code design specifications typically...

Alberson, Ryan M.

2010-01-14

329

Selecting unrelated donors  

PubMed Central

TEASER You are selecting an unrelated donor for a 40 year old man with AML in CR2. Which donor would you select? FULL CASE You are selecting an unrelated donor for a 40 year old man with AML in second complete remission with high-risk cytogenetics. He is CMV seronegative with blood type A?. He is an only child. The search coordinators have identified the following options: OptionAge/sexHLA matching (9/10 matched donors)CMV statusBlood typeYour ResponseDonor 150 FSingle allele mismatch at HLA-ACMV ?A+24%Donor 230 MSingle allele mismatch at HLA-CCMV +O+22%Donor 330 MSingle antigen mismatch at HLA-DQCMV +A+54% PMID:19203734

2015-01-01

330

Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe  

SciTech Connect

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

1994-09-01

331

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

PubMed

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

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

2014-01-01

332

Preparation of yeast whole cell splicing extract.  

PubMed

Pre-mRNA splicing, the removal of introns from pre-messenger RNA, is an essential step in eukaryotic gene expression. In humans, it has been estimated that 60 % of noninfectious diseases are caused by errors in splicing, making the study of pre-mRNA splicing a high priority from a health perspective. Pre-mRNA splicing is also complicated: the molecular machine that catalyzes the reaction, the spliceosome, is composed of five small nuclear RNAs, and over 100 proteins, making splicing one of the most complex processes in the cell.An important tool for studying pre-mRNA splicing is the in vitro splicing assay. With an in vitro assay, it is possible to test the function of each splicing component by removing the endogenous version and replacing it (or reconstituting it) with a modified one. This assay relies on the ability to produce an extract-either whole cell or nuclear-that contains all of the activities required to convert pre-mRNA to mRNA. To date, splicing extracts have only been produced from human and S. cerevisiae (yeast) cells. We describe a method to produce whole cell extracts from yeast that support splicing with efficiencies up to 90 %. These extracts have been used to reconstitute snRNAs, screen small molecule libraries for splicing inhibitors, and purify a variety of splicing complexes. PMID:24549660

Dunn, Elizabeth A; Rader, Stephen D

2014-01-01

333

Splicing in disease: disruption of the splicing code and the decoding machinery  

Microsoft Academic Search

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases,

Guey-Shin Wang; Thomas A. Cooper

2007-01-01

334

Transition splices and cost comparison  

NASA Technical Reports Server (NTRS)

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

Remedios, M. D.

1972-01-01

335

Influenza Viruses and mRNA Splicing: Doing More with Less  

PubMed Central

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

Dubois, Julia

2014-01-01

336

Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein  

PubMed Central

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (?9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that ?9-CETP protein is expressed in the liver but fails to circulate in the blood. PMID:24393849

Suhy, Adam; Hartmann, Katherine; Newman, Leslie; Papp, Audrey; Toneff, Thomas; Hook, Vivian; Sadee, Wolfgang

2014-01-01

337

Son maintains accurate splicing for a subset of human pre-mRNAs  

PubMed Central

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression. PMID:22193954

Sharma, Alok; Markey, Michael; Torres-Muñoz, Keshia; Varia, Sapna; Kadakia, Madhavi; Bubulya, Athanasios; Bubulya, Paula A.

2011-01-01

338

Determinants of Nam8-dependent splicing of meiotic pre-mRNAs  

PubMed Central

Nam8, a component of yeast U1 snRNP, is optional for mitotic growth but required during meiosis, because Nam8 collaborates with Mer1 to promote splicing of essential meiotic mRNAs AMA1, MER2 and MER3. Here, we identify SPO22 and PCH2 as novel targets of Nam8-dependent meiotic splicing. Whereas SPO22 splicing is co-dependent on Mer1, PCH2 is not. The SPO22 intron has a non-consensus 5? splice site (5?SS) that dictates its Nam8/Mer1-dependence. SPO22 splicing relies on Mer1 recognition, via its KH domain, of an intronic enhancer 5?-AYACCCUY. Mutagenesis of KH and the enhancer highlights Arg214 and Gln243 and the CCC triplet as essential for Mer1 activity. The Nam8-dependent PCH2 pre-mRNA has a consensus 5?SS and lacks a Mer1 enhancer. For PCH2, a long 5? exon and a non-consensus intron branchpoint dictate Nam8-dependence. Our results implicate Nam8 in two distinct meiotic splicing regulons. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. The leader, tail and RRM1 are dispensable for splicing meiotic targets and unnecessary for vegetative Nam8 function in multiple synthetic lethal genetic backgrounds. Nam8 activity is enfeebled by alanine mutations in the putative RNA binding sites of the RRM2 and RRM3 domains. PMID:21208980

Qiu, Zhicheng R.; Schwer, Beate; Shuman, Stewart

2011-01-01

339

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly  

PubMed Central

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7+ (spslu7+) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3? splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3? splice site distance, intron length, and the impact of its A/U content at the 5? end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly. PMID:23754748

Banerjee, Shataparna; Khandelia, Piyush; Melangath, Geetha; Bashir, Samirul; Nagampalli, Vijaykrishna

2013-01-01

340

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

PubMed Central

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

2014-01-01

341

Pre-mRNA Splicing  

Microsoft Academic Search

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most

Melissa S. Jurica; Melissa J. Moore

2003-01-01

342

Correspondence Alternative Splicing at NAGNAG  

E-print Network

splicing at NAGNAGs mainly results in the insertion/deletion of one amino acid. While such subtle events]: biases towards intron phase 1 and single amino acid insertions/deletions, correlation of amino acid. Meanwhile, Akerman and Mandel-Gutfreund found a high conservation of the intronic flanking regions [5

Will, Sebastian

343

Circular DNA and Splicing Systems  

Microsoft Academic Search

Circular strings representing DNA molecules and certain recombinant behaviour are formalized. Various actions of splicing schemes on linear and circular DNA molecules are examined. It is shown that there is a difference in the regularity result of Culik and Harju [1] between the linear and circular strings. A consequence of this result is that a conjecture of Head [4] that

Rani Siromoney; K. G. Subramanian; V. Rajkumar Dare

1992-01-01

344

Crowd Around: Expanding Your Donor Pool with Crowdfunding  

ERIC Educational Resources Information Center

At most institutions, annual fund-giving is down. Crowdfunding sites allow people with a great idea or worthy cause to bypass traditional funding methods and take their case directly to web-savvy investors and donors. This article describes how higher education institutions are expanding their donor pool through such crowdfunding sites as USEED,…

Jarrell, Andrea

2013-01-01

345

Digging up Classroom Dollars on DonorsChoose  

ERIC Educational Resources Information Center

Back in 2000, Charles Best was teaching at Wings Academy, an alternative high school in the Bronx, when he got the idea for a Web site where teachers could solicit donations for class projects. With help from his students, DonorsChoose.org soon was born. Last year, the site won Amazon.com's Nonprofit Innovation Award. So far, DonorsChoose has…

Curriculum Review, 2006

2006-01-01

346

The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing  

E-print Network

factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). WeRNP complex and the SF1/U2AF65/U2AF35 protein complex recognize the 59 and 39 splice sites of an intronThe Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing

Horvitz, H. Robert

347

Computational analysis reveals a correlation of exon-skipping events with splicing, transcription and epigenetic factors  

PubMed Central

Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, ‘skipping’, exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon–intron boundary establishment leading to skipping events. PMID:24369421

Ye, Zhenqing; Chen, Zhong; Lan, Xun; Hara, Stephen; Sunkel, Benjamin; Huang, Tim H.-M.; Elnitski, Laura; Wang, Qianben; Jin, Victor X.

2014-01-01

348

The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element  

PubMed Central

The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3? splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding. PMID:11598017

Tange, Thomas Ø.; Damgaard, Christian K.; Guth, Sabine; Valcárcel, Juan; Kjems, Jørgen

2001-01-01

349

Identification and functional characterization of a novel splicing mutation in RP gene PRPF31  

PubMed Central

A six-generation Chinese family with autosomal dominant retinitis pigmentosa (adRP) was identified and characterized. Genome-wide linkage analysis linked the family to markers D19S601 to D19S605, which span the PRPF31 gene on chromosome 19q13.33–13.43 (RP11) (LOD = 5.03). Direct DNA sequence analysis identified a novel splicing mutation (IVS1 +1 G > T) in affected family members, but not in unaffected family members and 200 normal controls. The splicing mutation occurs at the splicing donor of intron 1. Real time PCR with lymphoblastoid RNA samples from family members showed that in comparison to normal family members, the splicing mutation reduced the expression level of the PRPF31 mRNA by 57% in symptomatic patients and by 28% in clinically asymptomatic carriers. Our studies identify a novel splicing mutation in PRPF31 associated with adRP and suggest that the penetrance of RP11 mutations may be correlated with the expression level of the PRPF31 mRNA. PMID:18177735

Liu, Jingyu; Dai, Xiaohua; Wang, Xu; Cui, Xin; Sheng, Jiqun; Jiang, Xueqing; Tu, Xin; Tang, Zhaohui; Bai, Yan; Liu, Mugen; Wang, Qing K.

2009-01-01

350

A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts[W  

PubMed Central

Chloroplast genomes in land plants harbor ?20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein–protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity. PMID:17693527

Watkins, Kenneth P.; Kroeger, Tiffany S.; Cooke, Amy M.; Williams-Carrier, Rosalind E.; Friso, Giulia; Belcher, Susan E.; van Wijk, Klaas J.; Barkan, Alice

2007-01-01

351

Rich Donors, Poor Countries  

ERIC Educational Resources Information Center

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

Thomas, M. A.

2012-01-01

352

Living donor kidney exchange.  

PubMed

Living donor kidney exchange, also referred to as kidney paired donation (KPD), is a relatively new transplant modality that is growing by leaps and bounds in the U.S. From its first realization as an exchange of kidneys between two incompatible donor/recipient pairs, KPD has expanded to include compatible pairs, nondirected donors, three-way and larger exchanges, and living/deceased donor exchanges. Innovations both clinical (transporting organs instead of donors, and improved HLA screening) and mathematical (simulation to test policies, optimization to find better and more matches) have made this modality even more useful and accessible. There are several independent multi-center paired donation registries and many more single-center registries operating in the U.S., but incompatible pairs are most likely to match when they participate in the largest possible paired exchange pool; a single, unified KPD program in the United States would likely best serve patients in search of matches. PMID:22755420

Gentry, Sommer; Segev, Dorry L

2011-01-01

353

HMMSplicer: A Tool for Efficient and Sensitive Discovery of Known and Novel Splice Junctions in RNA-Seq Data  

PubMed Central

Background High-throughput sequencing of an organism's transcriptome, or RNA-Seq, is a valuable and versatile new strategy for capturing snapshots of gene expression. However, transcriptome sequencing creates a new class of alignment problem: mapping short reads that span exon-exon junctions back to the reference genome, especially in the case where a splice junction is previously unknown. Methodology/Principal Findings Here we introduce HMMSplicer, an accurate and efficient algorithm for discovering canonical and non-canonical splice junctions in short read datasets. HMMSplicer identifies more splice junctions than currently available algorithms when tested on publicly available A. thaliana, P. falciparum, and H. sapiens datasets without a reduction in specificity. Conclusions/Significance HMMSplicer was found to perform especially well in compact genomes and on genes with low expression levels, alternative splice isoforms, or non-canonical splice junctions. Because HHMSplicer does not rely on pre-built gene models, the products of inexact splicing are also detected. For H. sapiens, we find 3.6% of 3? splice sites and 1.4% of 5? splice sites are inexact, typically differing by 3 bases in either direction. In addition, HMMSplicer provides a score for every predicted junction allowing the user to set a threshold to tune false positive rates depending on the needs of the experiment. HMMSplicer is implemented in Python. Code and documentation are freely available at http://derisilab.ucsf.edu/software/hmmsplicer. PMID:21079731

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

2010-01-01

354

Single-Channel Properties of Recombinant AMPA Receptors Depend on RNA Editing, Splice Variation, and Subunit Composition  

Microsoft Academic Search

Non-NMDA glutamate receptor subunits of the AMPA- preferring subfamily combine to form ion channels with heter- ogeneous functional properties. We have investigated the ef- fects of RNA editing at the Q\\/R site, splice variation of the \\

Geoffrey T. Swanson; Sunjeev K. Kamboj; Stuart G. Cull-Candy

1997-01-01

355

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors  

Microsoft Academic Search

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction.

Johann Soret; Nadia Bakkour; Sophie Maire; Sébastien Durand; Latifa Zekri; Mathieu Gabut; Weronika Fic; Gilles Divita; Christian Rivalle; Daniel Dauzonne; Chi Hung Nguyen; Philippe Jeanteur; Jamal Tazi

2005-01-01

356

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis*  

PubMed Central

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade ?-propeller fold linked to a C-terminal ?-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (?1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the ?1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases. PMID:20466731

Chuankhayan, Phimonphan; Hsieh, Chih-Yu; Huang, Yen-Chieh; Hsieh, Yi-You; Guan, Hong-Hsiang; Hsieh, Yin-Cheng; Tien, Yueh-Chu; Chen, Chung-De; Chiang, Chien-Min; Chen, Chun-Jung

2010-01-01

357

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts  

PubMed Central

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts. PMID:19116412

Sanford, Jeremy R.; Wang, Xin; Mort, Matthew; VanDuyn, Natalia; Cooper, David N.; Mooney, Sean D.; Edenberg, Howard J.; Liu, Yunlong

2009-01-01

358

RNA Splicing in Plant Mitochondria  

Microsoft Academic Search

\\u000a Abstract\\u000a \\u000a Introns within the mitochondrial genes of land plants belong mostly to the group II category of ribozyme mobile retroelements,\\u000a although a minor number (particularly in nonvascular plants) are members of the group I intron family of “homing” ribozymes.\\u000a In vascular plants, the mitochondrial introns often lack conventional structural features, and their degenerate nature is\\u000a correlated with unusual splicing pathways.

Linda Bonen

359

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing  

PubMed Central

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3? and 5? splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. PMID:24960161

Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H.; Selbach, Matthias; Barton, Paul J.R.; Cook, Stuart A.; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

2014-01-01

360

Tissue-specific alternative splicing analysis reveals the diversity of chromosome 18 transcriptome.  

PubMed

The Chromosome-centric Human Proteome Project (C-HPP) is aimed to identify the variety of protein products and transcripts of the number of chromosomes. The Russian part of C-HPP is devoted to the study of the human chromosome 18. Using widely accepted Tophat and SpliceGrapher, a tool for accurate splice sites and alternative mRNA isoforms prediction, we performed the extensive mining of the splice variants of chromosome 18 transcripts and encoded protein products in liver, brain, lung, kidney, blood, testis, derma, and skeletal muscles. About 6.1 billion of the reads represented by 450 billion of the bases have been analyzed. The relative frequencies of splice events as well as gene expression profiles in normal tissues are evaluated. Using ExPASy PROSITE, the novel features and possible functional sites of previously unknown splice variants were highlighted. A set of unique proteotypic peptides enabling the identification of novel alternative protein species using mass-spectrometry is constructed. The revealed data will be integrated into the gene-centric knowledgebase of the Russian part of C-HPP available at http://kb18.ru and http://www.splicing.zz.mu/. PMID:24320163

Shargunov, Alexander V; Krasnov, George S; Ponomarenko, Elena A; Lisitsa, Andrey V; Shurdov, Mikhail A; Zverev, Vitaliy V; Archakov, Alexander I; Blinov, Vladimir M

2014-01-01

361

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1  

PubMed Central

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity. PMID:22210893

Sumanasekera, Chiranthani; Kelemen, Olga; Beullens, Monique; Aubol, Brandon E.; Adams, Joseph A.; Sunkara, Manjula; Morris, Andrew; Bollen, Mathieu; Andreadis, Athena; Stamm, Stefan

2012-01-01

362

Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program  

SciTech Connect

A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

2006-06-15

363

Marginal kidney donor  

PubMed Central

Renal transplantation is the treatment of choice for a medically eligible patient with end stage renal disease. The number of renal transplants has increased rapidly over the last two decades. However, the demand for organs has increased even more. This disparity between the availability of organs and waitlisted patients for transplants has forced many transplant centers across the world to use marginal kidneys and donors. We performed a Medline search to establish the current status of marginal kidney donors in the world. Transplant programs using marginal deceased renal grafts is well established. The focus is now on efforts to improve their results. Utilization of non-heart-beating donors is still in a plateau phase and comprises a minor percentage of deceased donations. The main concern is primary non-function of the renal graft apart from legal and ethical issues. Transplants with living donors outnumbered cadaveric transplants at many centers in the last decade. There has been an increased use of marginal living kidney donors with some acceptable medical risks. Our primary concern is the safety of the living donor. There is not enough scientific data available to quantify the risks involved for such donation. The definition of marginal living donor is still not clear and there are no uniform recommendations. The decision must be tailored to each donor who in turn should be actively involved at all levels of the decision-making process. In the current circumstances, our responsibility is very crucial in making decisions for either accepting or rejecting a marginal living donor. PMID:19718332

Gopalakrishnan, Ganesh; Gourabathini, Siva Prasad

2007-01-01

364

FOA Lecture 5: Splices and Connectors  

NSDL National Science Digital Library

This is the fifth lecture in the FOA series on fiber optics, covering splices and termination. This lecture covers the differences and similarities in splices and connectors and where they are used. Lectures 6 and 7 cover much more detail on fiber optic splices and connectors respectively. Those are available to view separately. Running time for the lecture is 12:21. Flash is required to view the video.

365

ASD: a bioinformatics resource on alternative splicing  

Microsoft Academic Search

Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on alternative splicing. We present the continuation and upgrade of the ASD (T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) NucleicAcidsRes.32,D64-D69)thatconsistsofcom- putationally and manually generated data. Its largest parts

Stefan Stamm; Jean-jack M. Riethoven; Vincent Le Texier; Chellappa Gopalakrishnan; Vasudev Kumanduri; Yesheng Tang; Nuno L. Barbosa-morais; Thangavel Alphonse Thanaraj

2006-01-01

366

Statistical and Computational Studies on Alternative Splicing  

Microsoft Academic Search

\\u000a The accumulating genome sequences and other high-throughput data have shed light on the extent and importance of alternative\\u000a splicing in functional regulation. Alternative splicing dramatically increases the transcriptome and proteome diversity of\\u000a higher organisms by producing multiple splice variants from different combinations of exons. It has an important role in many\\u000a biological processes including nervous system development and programmed cell

Liang Chen

367

Toward predicting self-splicing and protein-facilitated splicing of group I introns  

PubMed Central

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence. PMID:18768647

Vicens, Quentin; Paukstelis, Paul J.; Westhof, Eric; Lambowitz, Alan M.; Cech, Thomas R.

2008-01-01

368

Alternative Splicing in the Fly and the Worm: Splicing Databases for Drosophila melanogaster and Caenorhabditis elegans  

Microsoft Academic Search

Alternative splicing is a widespread cellular phenomenon, which regulates gene expression in eukaryotic genomes. Availability of accumulating transcript and genomic sequence data has lead to generation of a wide-range of alternative splicing databases. Generally the available databases focus on mammalian transcriptomes. Here, we present two new alternative splicing databases for the model organisms, Drosophila melanogaster and Caenorhabditis elengans. Databases presented

Bahar Taneri; Ben Snyder; Alexey Novoradovsky; Terry Gaasterland

2011-01-01

369

Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible  

PubMed Central

Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

Lareau, Liana F.; Brenner, Steven E.

2015-01-01

370

Studies of exon scrambling and mutually exclusive alternative splicing  

E-print Network

The goals of this thesis work were to study two special alternative splicing events: exon scrambling at the RNA splicing level and mutually exclusive alternative splicing (MEAS) by computational and experimental methods. ...

Kong, Rong, 1979-

2005-01-01

371

Global regulation of alternative splicing during myogenic differentiation  

E-print Network

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely ...

Bland, Christopher S.

372

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2014 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2014-07-01

373

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2010 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2010-07-01

374

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2011 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2011-07-01

375

30 CFR 57.12088 - Splicing trailing cables.  

Code of Federal Regulations, 2012 CFR

...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...

2012-07-01

376

Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation  

SciTech Connect

In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

Gesnel, Marie-Claude [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Theoleyre, Sandrine [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Del Gatto-Konczak, Fabienne [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France); Faculte des Sciences, Universite de Nantes, Nantes (France); Breathnach, Richard [INSERM, U601, Institut de Biologie-CHR, 9 quai Moncousu, 44093 Nantes Cedex 1 (France) and Faculte des Sciences, Universite de Nantes, Nantes (France)]. E-mail: breathna@nantes.inserm.fr

2007-07-13

377

3'UTR-located ALU elements: donors of potential miRNA target sites and mediators of network miRNA-based regulatory interactions.  

PubMed

Recent research data reveal complex, network-based interactions between mobile elements and regulatory systems of eukaryotic cells. In this article, we focus on regulatory interactions between Alu elements and micro RNAs (miRNAs). Our results show that the majority of the Alu sequences inserted in 3'UTRs of analyzed human genes carry strong potential target sites for at least 53 different miRNAs. Thus, 3'UTR-located Alu elements may play the role of mobile regulatory modules that supply binding sites for miRNA regulation. Their abundance and ability to distribute a set of certain miRNA target sites may have an important role in establishment, extension, network organization, and, as we suppose - in the regulation and environment-dependent activation/inactivation of some elements of the miRNA regulatory system, as well as for a larger scale RNA-based regulatory interactions. The Alu-miRNA connection may be crucial especially for the primate/human evolution. PMID:19455205

Daskalova, Evelina; Baev, Vesselin; Rusinov, Ventsislav; Minkov, Ivan

2006-01-01

378

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.  

PubMed

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. PMID:24751650

McNeil, Bonnie A; Zimmerly, Steven

2014-06-01

379

Living-donor liver transplantation: evaluation of donor and recipient  

Microsoft Academic Search

Living-donor liver transplantation (LDLT) in adults has been expanded after becoming the standard for children in many transplant centres. Advantages of LDLT include thorough donor screening, optimization of timing for transplantation and minimal cold ischaemia time. However, the risk of donor morbidity and mortality must be considered. The preoperative evaluation of the donor typically is performed in consecutive stages. Specific

Peter Sauer; Peter Schemmer; Waldemar Uhl; Jens Encke

2004-01-01

380

Reduced Mobility of the Alternate Splicing Factor (ASF) through the Nucleoplasm and Steady State Speckle Compartments  

Microsoft Academic Search

Compartmentalization of the nucleus is now recognized as an important level of regulation influenc- ing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concen- tration to sites

Michael J. Kruhlak; Melody A. Lever; Wolfgang Fischle; Eric Verdin; David P. Bazett-Jones; Michael J. Hendzel

2000-01-01

381

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression  

PubMed Central

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3?splice site, and the other involves an exonic splicing silencer (ESS) situated 3? to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator. PMID:23818605

Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L.; Feng, Ying

2013-01-01

382

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression.  

PubMed

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem-loop structure located downstream of the 3'splice site, and the other involves an exonic splicing silencer (ESS) situated 3' to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator. PMID:23818605

Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L; Feng, Ying

2013-07-16

383

Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds  

SciTech Connect

Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.

Doerk, T.; Wulbrand, U.; Tuemmler, B. (Medizinische Hochschule Hannover (Germany))

1993-03-01

384

The Effect of Donor Age on Corneal Transplantation Outcome: Results of the Cornea Donor Study  

PubMed Central

Objective To determine whether graft survival over a 5-year follow-up period using corneal tissue from donors older than 65 years of age is similar to graft survival using corneas from younger donors. Design Multi-center prospective, double-masked, controlled clinical trial Participants 1090 subjects undergoing corneal transplantation for a moderate risk condition (principally Fuchs’ dystrophy or pseudophakic corneal edema); 11 subjects with ineligible diagnoses were not included Methods 43 participating eye banks provided corneas from donors in the age range of 12 to 75 with endothelial cell densities of 2300 to 3300 cells/mm2, using a random approach without respect to recipient factors.