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1

[Coding region of far-red fluorescent protein katushka contains a strong donor splice site].  

PubMed

Computer analysis predicted a strong donor splice site within the 3'-part of the far-red fluorescent protein Katushka coding region. To test the functional activity of this site a model vector has been constructed. This vector encoded Katushka and green fluorescent protein TagGFP2 with a gene fragment of tafazzin in between. Normal splicing of this pre-mRNA should result in a frameshift between Katushka and TagGFP2. Alternatively, after splicing at internal katushka donor splice site appearance of Katushka-TagGFP2 fusion protein was expected. Expression of this construct in a mammalian cell culture led to bright red and green fluorescence. Therefore, katushka-specific donor splice site is functional. Disruption of this splice site by several silent substitutions resulted in red-only fluorescent cells that corresponded to normal splicing. The mutant katushka can be used for visualization of pre-mRNA splicing at single cell level by fluorescence microscopy and flow cytometry. PMID:21899059

Gurskaia, N G; Staroverov, D B; Fradkov, A F; Luk'ianov, K A

2011-01-01

2

The coding region of far-red fluorescent protein Katushka contains a strong donor splice site  

Microsoft Academic Search

Computer analysis of the sequence encoding the red fluorescent protein Katushka revealed a strong donor splice site in the\\u000a 3?-terminal region. A model vector encoding protein Katushka and green fluorescent protein TagGFP2 separated by a fragment\\u000a of the gene tafazzin, has been constructed for experimental verification of the functional activity of this site. Splicing\\u000a of this pre-mRNA should lead to

N. G. Gurskaya; D. B. Staroverov; A. F. Fradkov; K. A. Lukyanov

2011-01-01

3

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

PubMed

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

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

2015-01-01

4

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

PubMed Central

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

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

2013-01-01

5

Characterization of conserved tandem donor sites and intronic motifs required for alternative splicing in corticosteroid receptor genes.  

PubMed

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

Rivers, Caroline; Flynn, Andrea; Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

2009-11-01

6

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

SciTech Connect

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

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

1993-08-01

7

The HIV-1 5' LTR poly(A) site is inactivated by U1 snRNP interaction with the downstream major splice donor site.  

PubMed Central

The inactivity of the 5' long terminal repeat (LTR) poly(A) site immediately downstream of the cap site maximizes the production of HIV-1 transcripts. In this paper, we demonstrate that this inactivity is mediated by the interaction of the U1 snRNP with the major splice donor site (MSD). The inhibition of the HIV-1 poly(A) site by U1 snRNP relies on a series of delicately balanced RNA processing signals. These include the poly(A) site, the major splice donor site and the splice acceptor sites. The inherent efficiency of the HIV-1 poly(A) site allows maximal activity where there is no donor site (in the 3' LTR) but full inhibition by the downstream MSD (in the 5' LTR). The MSD must interact efficiently with U1 snRNP to completely inhibit the 5' LTR poly(A) site, whereas the splice acceptor sites are inefficient, allowing full-length genomic RNA production. PMID:9312033

Ashe, M P; Pearson, L H; Proudfoot, N J

1997-01-01

8

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

9

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

PubMed

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

2014-09-01

10

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

SciTech Connect

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

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

1994-09-01

11

Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA.  

PubMed

The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1-5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3' splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5' splice site of exon 7 in nonmuscle cells. The 5' splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5' splice site of exon 7 does not result from the sequences at the 3' end of intron 6 that block the use of the 3' splice site of exon 7. However, mutating two conserved nucleotides GU at the 5' splice site of exon 6 results in the efficient use of the 5' splice site of exon 7. In addition, a mutation that changes the 5' splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5' splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells. PMID:10024180

Chen, C D; Helfman, D M

1999-02-01

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

Alternative 5' splice site selection induced by heat shock.  

PubMed Central

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. Images PMID:8264624

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

1994-01-01

14

Cryptic splice sites and split genes.  

PubMed

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

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

2011-08-01

15

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

16

Selection against tandem splice sites affecting structured protein regions  

PubMed Central

Background Alternative selection of splice sites in tandem donors and acceptors is a major mode of alternative splicing. Here, we analyzed whether in-frame tandem sites leading to subtle mRNA insertions/deletions of 3, 6, or 9 nucleotides are under natural selection. Results We found multiple lines of evidence that the human protein coding sequences are under selection against such in-frame tandem splice events, indicating that these events are often deleterious. The strength of selection is not homogeneous within the coding sequence as protein regions that fold into a fixed 3D structure (intrinsically ordered) are under stronger selection, especially against sites with a strong minor splice site. Investigating structures of functional protein domains, we found that tandem acceptors are preferentially located at the domain surface and outside structural elements such as helices and sheets. Using three-species comparisons, we estimate that more than half of all mutations that create NAGNAG acceptors in the coding region have been eliminated by selection. Conclusion We estimate that ~2,400 introns are under selection against possessing a tandem site. PMID:18366714

2008-01-01

17

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

18

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

SciTech Connect

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCK[sub B] receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 aminio acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction. 28 refs., 5 figs., 1 tab.

Song, I.; Brown, D.R.; Wiltshire, R.N.; Gantz, I.; Trent, J.M.; Yamada, T. (Univ. of Michigan Medical Center, Ann Arbor, MI (United States))

1993-10-01

19

A novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion of 30 nucleotides into the mature mRNA in a case of vascular type of Ehlers–Danlos syndrome  

Microsoft Academic Search

Vascular type of Ehlers–Danlos syndrome (EDS) is the most severe type of EDS. It is an autosomal dominantly inherited disorder\\u000a that results from mutations within the ?1 type III collagen gene (COL3A1). We report a novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion\\u000a of 30 nucleotides into the mature mRNA

Hiroshi Okita; Yasunori Ikeda; Yoshihiko Mitsuhashi; Hiromi Namikawa; Yohei Kitamura; Yoichiro Hamasaki; Soji Yamazaki; Atsushi Hatamochi

2010-01-01

20

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

SciTech Connect

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

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

1992-10-01

21

Role of the 3? Splice Site in U12-Dependent Intron Splicing  

PubMed Central

U12-dependent introns containing alterations of the 3? splice site AC dinucleotide or alterations in the spacing between the branch site and the 3? splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5? splice site AU dinucleotide, any nucleotide could serve as the 3?-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3? splice site function. A branch site-to-3? splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3? splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3? splice site but that formation of the prespliceosomal complex A requires an active 3? splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3? splice site. PMID:11238930

Dietrich, Rosemary C.; Peris, Marian J.; Seyboldt, Andrew S.; Padgett, Richard A.

2001-01-01

22

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

PubMed

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

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

2013-02-01

23

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

PubMed

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

Misiek, Mathias; Hoffmeister, Dirk

2008-02-01

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

Assessing the fraction of short-distance tandem splice sites under purifying selection  

PubMed Central

Many alternative splice events result in subtle mRNA changes, and most of them occur at short-distance tandem donor and acceptor sites. The splicing mechanism of such tandem sites likely involves the stochastic selection of either splice site. While tandem splice events are frequent, it is unknown how many are functionally important. Here, we use phylogenetic conservation to address this question, focusing on tandems with a distance of 3–9 nucleotides. We show that previous contradicting results on whether alternative or constitutive tandem motifs are more conserved between species can be explained by a statistical paradox (Simpson's paradox). Applying methods that take biases into account, we found higher conservation of alternative tandems in mouse, dog, and even chicken, zebrafish, and Fugu genomes. We estimated a lower bound for the number of alternative sites that are under purifying (negative) selection. While the absolute number of conserved tandem motifs decreases with the evolutionary distance, the fraction under selection increases. Interestingly, a number of frameshifting tandems are under selection, suggesting a role in regulating mRNA and protein levels via nonsense-mediated decay (NMD). An analysis of the intronic flanks shows that purifying selection also acts on the intronic sequence. We propose that stochastic splice site selection can be an advantageous mechanism that allows constant splice variant ratios in situations where a deviation in this ratio is deleterious. PMID:18268022

Hiller, Michael; Szafranski, Karol; Sinha, Rileen; Huse, Klaus; Nikolajewa, Swetlana; Rosenstiel, Philip; Schreiber, Stefan; Backofen, Rolf; Platzer, Matthias

2008-01-01

26

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; Aguilo, Sira; Fernandez-Rebollo, Eduardo

2014-01-01

27

Three novel alternatively spliced isoforms of the human beta-site amyloid precursor protein cleaving enzyme (BACE) and their effect on amyloid beta-peptide production  

Microsoft Academic Search

Three novel alternatively spliced transcripts of the beta-site amyloid precursor protein cleaving enzyme (BACE) were cloned from human brain. Alternative splicing of the RNA occurs at an internal donor in exon 3 and\\/or an internal acceptor in exon 4. The splicing events lead to a deletion of 25 (BACE-I-476), 44 (BACE-I-457) and 69 (BACE-I-432) amino acids and the latter two

Hiroshi Tanahashi; Takeshi Tabira

2001-01-01

28

A novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion of 30 nucleotides into the mature mRNA in a case of vascular type of Ehlers-Danlos syndrome.  

PubMed

Vascular type of Ehlers-Danlos syndrome (EDS) is the most severe type of EDS. It is an autosomal dominantly inherited disorder that results from mutations within the alpha1 type III collagen gene (COL3A1). We report a novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion of 30 nucleotides into the mature mRNA in a case of vascular type of EDS. Since the age of approximately 8 months, the patient had had repeated episodes of purpura and gradually developed thin, translucent skin. She had a past history of pneumothorax. At the initial examination, she was found to have the characteristic facies, i.e., bird-like face, of the vascular type of EDS, thinning of skin over the limbs and trunk, and scattered purpura. The blood vessels under the skin could be clearly visualized. She showed hypermobility of the small joints of all the four limbs and acrogeric changes of the hands and feet. Analysis of the amount of collagen synthesized from cultured dermal fibroblasts by SDS-polyacrylamide gel electrophoresis and fluorography was conducted based on the clinical suspicion of the vascular type of EDS, and a marked reduction in the synthesis of type III collagen was observed. Genetic analysis of the COL3A1 revealed a novel point mutation at the donor splice-site of intron 42, which resulted in the inclusion of 30 nucleotides into the mature mRNA of one allele. PMID:19543901

Okita, Hiroshi; Ikeda, Yasunori; Mitsuhashi, Yoshihiko; Namikawa, Hiromi; Kitamura, Yohei; Hamasaki, Yoichiro; Yamazaki, Soji; Hatamochi, Atsushi

2010-07-01

29

Splice-site pairing is an intrinsically high fidelity process  

PubMed Central

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

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

2009-01-01

30

Method of predicting Splice Sites based on signal interactions  

PubMed Central

Background Predicting and proper ranking of canonical splice sites (SSs) is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE) and Intronic (ISE) Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand. PMID:16584568

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

2006-01-01

31

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  

Microsoft Academic Search

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

Yoko Hagiwara; Hisahide Nishio; Yoshihiko Kitoh; Yasuhiro Takeshima; Naoko Narita; Hiroko Wada; Mitsuhiro Yokoyama; Hajime Nakamura; Masafumi Matsuo

1994-01-01

32

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism.  

PubMed Central

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation. PMID:12212851

Nasim, Faiz-Ul Hassan; Hutchison, Stephen; Cordeau, Melanie; Chabot, Benoit

2002-01-01

33

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

34

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

PubMed Central

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

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

2013-01-01

35

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

36

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

PubMed

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

2014-12-01

37

Efficient internal exon recognition depends on near equal contributions from the 3? and 5? splice sites  

PubMed Central

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3? and 5? splice sites. However, it is unknown if the 3? or the 5? splice site dominates the exon recognition process. Here, we test the 3? and 5? splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3? and 5? splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3? or the 5? splice site strength alone. PMID:21795381

Shepard, Peter J.; Choi, Eun-A.; Busch, Anke; Hertel, Klemens J.

2011-01-01

38

Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus  

PubMed Central

Background Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. Results By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. Conclusion We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus. PMID:17617899

S?rensen, Annette Balle; Lund, Anders H; Kunder, Sandra; Quintanilla-Martinez, Leticia; Schmidt, Jorg; Wang, Bruce; Wabl, Matthias; Pedersen, Finn Skou

2007-01-01

39

A Novel Otoferlin Splice-Site Mutation in Siblings with Auditory Neuropathy Spectrum Disorder  

PubMed Central

We characterize a novel otoferlin (OTOF) mutation discovered in a sibling pair diagnosed with ANSD and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the OTOF splice donor site of exon 28 (IVS28+1 G>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 (.78 ms) compared to subjects with sensorineural hearing loss (SNHL) (average: adults .71 ms; children .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

40

The role of overlapping U1 and U11 5' splice site sequences in a negative regulator of splicing.  

PubMed Central

Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U15' splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U115' splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication. PMID:10094303

Hibbert, C S; Gontarek, R R; Beemon, K L

1999-01-01

41

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

E-print Network

Bungtown Road, Cold Spring Harbor, NY 11724; and Department of Biomedical Engineering, State University of New York, Stony Brook, NY 11794 Edited by James E. Dahlberg, University of Wisconsin Medical School vertebrate species, although most sites are not conserved, suggesting that their origin is recent. We discuss

42

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1.  

PubMed

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3' splice site 2 (A2) and 5'splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electrophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electrophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1. PMID:21329653

Tsuruno, Chikayuki; Ohe, Kenji; Kuramitsu, Madoka; Kohma, Takuya; Takahama, Youichi; Hamaguchi, Yukio; Hamaguchi, Isao; Okuma, Kazu

2011-03-25

43

Identification and characterization of a novel splice-site mutation in the Wilson disease gene.  

PubMed

This study aimed to identify aberrant transcripts of the new splice-site mutation c.3244-2A>C in the Wilson disease (WD) gene (ATPase, Cu++ transporting, beta polypeptide, ATP7B) and discuss its genotype and clinical phenotype. DNA and RNA were extracted from peripheral blood lymphocytes, amplified by polymerase chain reaction (PCR) and nested reverse transcription PCR (RT-nested PCR) to characterize the aberrant transcripts. RT-nested PCR product sequencing comparison showed that c.3244-2A>C splice-site mutation caused aberrant transcripts and formatted a new splice acceptor. Patient carrying the splice-site mutation c.3244-2A>C presented early onset age, severe clinical manifestations, and poor prognosis. WD patients with the splice-site mutation show severe clinical manifestations, indicating that aberrant transcripts have important implications for WD phenotype. PMID:25086856

Diao, Sheng-Peng; Hong, Ming-Fan; Huang, Ye-Qing; Wei, Zhi-Sheng; Su, Quan-Xi; Peng, Zhong-Xing; Yu, Qing-Yun; Liu, Ai-Qun; Chen, Jin; Hu, Li

2014-10-15

44

Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA.  

PubMed

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation. PMID:10094307

Selvakumar, M; Helfman, D M

1999-03-01

45

Atypical 5? Splice Sites Cause CFTR Exon 9 To Be Vulnerable to Skipping  

PubMed Central

The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3? splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3? splice sites, yet they are not skipped. Inspection of the 5? splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5? splice sites and determined the effect on exon skipping. Conversion of the upstream 5? splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5? splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5? splice site was consensus or not. These results suggested that the native downstream 5? splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y14), it skips exon 9 in vivo and has a nonconsensus downstream 5? splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y5) but is not skipped in vivo. Its downstream 5? splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5? splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5? splice sites may identify constitutive exons that are vulnerable to skipping. PMID:12068373

Hefferon, Timothy W.; Broackes-Carter, Fiona C.; Harris, Ann; Cutting, Garry R.

2002-01-01

46

The strength of the HIV-1 3' splice sites affects Rev function  

PubMed Central

Background The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. Results In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression. Conclusion Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs. PMID:17144911

Kammler, Susanne; Otte, Marianne; Hauber, Ilona; Kjems, J?rgen; Hauber, Joachim; Schaal, Heiner

2006-01-01

47

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

48

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

PubMed Central

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

Luukkonen, B G; Seraphin, B

1998-01-01

49

Open wound healing of the osseocutaneous fibula flap donor site.  

PubMed

Split skin grafts are the predominant method of closure for fibular flap donor sites. We present a novel approach to manage the donor site using the inter-related components of secondary intention healing: creation of a lattice to aid partial closure and compression dressings. The technique, which is widely used in dermatological surgery to manage cutaneous defects after operations for skin cancer, avoids the morbidity associated with the use of split skin grafts and enables early postoperative mobilisation. PMID:24894710

Fry, A M; Patterson, A; Orr, R L; Colver, G B

2014-11-01

50

Computer-assisted selection of donor sites for autologous grafts  

NASA Astrophysics Data System (ADS)

A new method is proposed for a precise planning of autologous bone grafts in cranio- and maxillofacial surgery. In patients with defects of the facial skeleton, autologous bone transplants can be harvested from various donor sites in the body. The preselection of a donor site depends i.a. on the morphological fit of the available bone mass and the shape of the part that is to be transplanted. A thorough planning and simulation of the surgical intervention based on 3D CT studies leads to a geometrical description and the volumetric characterization of the bone part to be resected and transplanted. Both, an optimal fit and a minimal lesion of the donor site are guidelines in this process. We use surface similarity and voxel similarity measures in order to select the optimal donor region for an individually designed transplant.

Krol, Zdzislaw; Zeilhofer, Hans-Florian U.; Sader, Robert; Hoffmann, Karl-Heinz; Gerhardt, Paul; Horch, Hans-Henning

1997-05-01

51

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

52

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

PubMed Central

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5? splice site recognition when this process is impaired as a result of the presence of noncanonical 5? splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5? splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5? splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA. PMID:10072385

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

1999-01-01

53

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

54

CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation.  

PubMed

Histone acetylation modulates alternative splicing of several hundred genes. Here, we tested the role of the histone acetyltransferase p300 in alternative splicing and showed that knockdown of p300 promotes inclusion of the fibronectin (FN1) alternative EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as p300 knockdown. Next we showed that p300 controls histone H4 acetylation along the FN1 gene. Consistently, p300 depletion and CRE deletion/mutation both reduced histone H4 acetylation on mini-gene reporters. Finally, we provide evidence that the effect of CRE inactivation on H4 acetylation and alternative splicing is counteracted by the inhibition of histone deacetylases. Together, these data suggest that histone acetylation could be one of the mechanisms how promoter and promoter binding proteins influence alternative splicing. PMID:25019513

Dušková, Eva; Hnilicová, Jarmila; Stan?k, David

2014-07-01

55

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

PubMed

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

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

2013-08-01

56

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Gouti?res syndrome  

PubMed Central

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

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

2013-01-01

57

Alterations of pre-mRNA splicing in cancer.  

PubMed

Recent genomewide analyses of alternative splicing (AS) indicate that up to 70% of human genes may have alternative splice forms, suggesting that AS together with various posttranslational modifications plays a major role in the production of proteome complexity. Splice-site selection under normal physiological conditions is regulated in the developmental stage in a tissue type-specific manner by changing the concentrations and the activity of splicing regulatory proteins. Whereas spliceosomal errors resulting in the production of aberrant transcripts rarely occur in normal cells, they seem to be an intrinsic property of cancer cells. Changes in splice-site selection have been observed in various types of cancer and may affect genes implicated in tumor progression (for example, CD44, MDM2, and FHIT) and in susceptibility to cancer (for example, BRCA1 and APC). Splicing defects can arise from inherited or somatic mutations in cis-acting regulatory elements (splice donor, acceptor and branch sites, and exonic and intronic splicing enhancers and silencers) or variations in the composition, concentration, localization, and activity of regulatory proteins. This may lead to altered efficiency of splice-site recognition, resulting in overexpression or down-regulation of certain splice variants, a switch in splice-site usage, or failure to recognize splice sites correctly, resulting in cancer-specific splice forms. At least in some cases, changes in splicing have been shown to play a functionally significant role in tumorigenesis, either by inactivating tumor suppressors or by gain of function of proteins promoting tumor development. Moreover, cancer-specific splicing events may generate novel epitopes that can be recognized by the host's immune system as cancer specific and may serve as targets for immunotherapy. Thus, the identification of cancer-specific splice forms provides a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention. PMID:15648050

Kalnina, Zane; Zayakin, Pawel; Silina, Kar?na; Lin?, Aija

2005-04-01

58

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

SciTech Connect

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

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

1993-03-01

59

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

PubMed Central

Purpose To investigate the genetic basis for autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. Methods Patients underwent a detailed ophthalmic examination, including funduscopy, electroretinography (ERG), visual field testing, and optical coherence tomography. Genome-wide homozygosity mapping using a single nucleotide polymorphism array was performed to identify homozygous regions shared between the two affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico analysis was used to predict the effect of the mutation on splicing. Results The family included two affected individuals. Clinical findings included progressive deterioration of visual acuity, photophobia, defective color vision, loss of central visual fields, pigmentary deposits localized mainly in the peripheral retina, a thinned and atrophic macular region, retinal vessel attenuation, absent ERG cone responses, and reduced ERG rod responses. Homozygosity mapping revealed several homozygous intervals shared among the affected individuals. One, a 12Mb interval on chromosome 10, included the CDHR1 gene. Direct sequencing revealed a single base transversion, c.1485+2T>G, located in the conserved donor splice site of Intron 13. This mutation cosegregated with the disease in the family, and was not detected in 208 Israeli Christian Arab control chromosomes. In silico analysis predicted that this mutation eliminates the Intron 13 donor splice site. Conclusions Only three distinct pathogenic mutations of CDHR1 have been reported to date in patients with autosomal recessive retinal degeneration. Here we report a novel splice site mutation of CDHR1, c.1485+2T>G, underlying autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. This report expands the spectrum of pathogenic mutations of the CDHR1 gene. PMID:23233793

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

2012-01-01

60

Gene 230 (1999) 101109 Intron splice sites of Papilio glaucus PglRh3 corroborate  

E-print Network

Gene 230 (1999) 101­109 Intron splice sites of Papilio glaucus PglRh3 corroborate insect opsin-length cDNA clones encoding the PglRh3 opsin from the tiger swallowtail butterfly Papilio glaucus were; Papilio glaucus; PglRh3; Visual pigments 1. Introduction green- and green- or long

61

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

62

Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion  

PubMed Central

Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1. PMID:23519612

Liu, Yuying; Conaway, LaShardai; Rutherford Bethard, Jennifer; Al-Ayoubi, Adnan M.; Thompson Bradley, Amber; Zheng, Hui; Weed, Scott A.; Eblen, Scott T.

2013-01-01

63

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

64

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

PubMed Central

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. We 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 splice-donor-site mutation in IVS 1 (+1GT-->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% of ADA mRNA had normal sequence, and 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 < .002, 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. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8023852

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

1994-01-01

65

Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.  

PubMed

We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection. PMID:8036160

Tsukahara, T; Casciato, C; Helfman, D M

1994-06-25

66

Luc7p, a novel yeast U1 snRNP protein with a role in 5? splice site recognition  

PubMed Central

The characterization of a novel yeast-splicing factor, Luc7p, is presented. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine–serine and arginine–glutamic acid repeat sequences characteristic of a family of splicing factors. We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth. The composition of yeast U1 snRNP is altered in luc7 mutant strains. Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5? splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5? splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains. These data suggest that the loss of Luc7p disrupts U1 snRNP–CBC interaction, and that this interaction contributes to normal 5? splice site recognition. PMID:10500099

Fortes, Puri; Bilbao-Cortes, Daniel; Fornerod, Maarten; Rigaut, Guillaume; Raymond, Wendy; Seraphin, Bertrand; Mattaj, Iain W.

1999-01-01

67

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

68

A novel splice site mutation in the noncoding region of BRCA2: implications for Fanconi anemia and familial breast cancer diagnostics.  

PubMed

Fanconi anemia (FA) is a rare recessive disorder with chromosomal instability, congenital abnormalities, and a high cancer risk. The breast cancer susceptibility gene BRCA2 (FANCD1) is one of the 16 genes involved in this recessive disease. We have identified a novel mutation of the splice donor site of intron 1 in the noncoding region of BRCA2 in a Japanese FA family. This mutation may account for the FA phenotype in a patient originally reported to have biallelic mutations in BRCA2. Subsequent functional studies revealed that one of the mutations, K2729N, was a neutral change. As reported here, a more careful analysis resulted in the identification of a novel splice site mutation. Functional analysis using a mouse embryonic stem cell-based assay revealed that it causes aberrant splicing, reduced transcript levels and hypersensitivity to DNA damaging agents, suggesting that it is likely to be pathogenic. Although similar pathogenic variants in the noncoding region of BRCA1 and 2 were not identified in a cohort of 752 familial breast cancer cases, we still think this finding is relevant for mutation analysis in Hereditary Breast and Ovarian Cancer Syndrome families in a diagnostic setting. PMID:24395671

Bakker, Janine L; Thirthagiri, Eswary; van Mil, Saskia E; Adank, Muriel A; Ikeda, Hideyuki; Verheul, Henk M W; Meijers-Heijboer, Hanne; de Winter, Johan P; Sharan, Shyam K; Waisfisz, Quinten

2014-04-01

69

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

70

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides.  

PubMed Central

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation. PMID:9740126

Sha, M; Levy, T; Kois, P; Konarska, M M

1998-01-01

71

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

PubMed

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

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

2013-03-01

72

Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.  

PubMed

The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection. PMID:8029003

Chanfreau, G; Legrain, P; Dujon, B; Jacquier, A

1994-06-11

73

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

74

cis-elements involved in alternative splicing in the rat beta-tropomyosin gene: the 3'-splice site of the skeletal muscle exon 7 is the major site of blockage in nonmuscle cells.  

PubMed

We have been using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. The beta-TM gene spans 10 kb with 11 exons and encodes two distinct isoforms, namely skeletal muscle beta-TM and fibroblast TM-1. Exons 1-5, 8, and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts, as well as in smooth muscle cells, whereas exons 7 and 10 are used exclusively in skeletal muscle cells. Our previous studies localized the critical elements for regulated alternative splicing to sequences within exon 7 and the adjacent upstream intron. We also demonstrated that these sequences function, in part, to regulate splice-site selection in vivo by interacting with cellular factors that block the use of the skeletal muscle exon in nonmuscle cells (1). Here we have further characterized the critical cis-acting elements involved in alternative splice site selection. Our data demonstrate that exon 7 and its flanking intron sequences are sufficient to regulate the suppression of exon 7 in nonmuscle cells when flanked by heterologous exons derived from adenovirus. We have also shown by both in vivo and in vitro assays that the blockage of exon 7 in nonmuscle cells is primarily at its 3'-splice site. A model is presented for regulated alternative splicing in both skeletal muscle and nonmuscle cells. PMID:8233825

Guo, W; Helfman, D M

1993-10-11

75

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

76

Identification and characterization of a novel splice site mutation in the SERPING1 gene in a family with hereditary angioedema.  

PubMed

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant disease caused by mutations in SERPING1 gene. The main clinical feature of C1INH deficiency is the spontaneous edema of the subcutaneous and submucosal layers. More than 280 different mutations scattering the entire SERPING1 gene have been reported. We identified and characterized a new mutation in SERPING1 gene in a Spanish family with hereditary angioedema. The mutation (c.685 + 2 T > A) disrupts the donor splice site of intron 4 leading to the loss of exon 4 in mutant mRNA. We demonstrated that mutant mRNA is mostly degraded, probably by the surveillance pathway no-go mRNA decay. Bioinformatic analysis showed that the mutant protein, if produced, would be non-functional since the protein lacks a stretch of 45 amino acids affecting the functional RCL loop. Finally, we found a reduction of the wild-type mRNA expression in c.685 + 2 T > A carriers. PMID:24412907

Colobran, Roger; Lois, Sergio; de la Cruz, Xavier; Pujol-Borrell, Ricardo; Hernández-González, Manuel; Guilarte, Mar

2014-02-01

77

Complete human gene structure of obscurin: implications for isoform generation by differential splicing  

Microsoft Academic Search

The complete gene giant muscle protein obscurin, a modular protein composed largely of tandem Ig-domains, GDP\\/GTP exchange factor domains (GEF) for small G-proteins, and differentially spliced kinase domains, was analysed. The splice donor and acceptor sites of the 117 exons give important clues for potential splice pathways. The fusion of the conventional obscurin A, containing only the GEF domain, and

Atsushi Fukuzawa; Seraphina Idowu; Mathias Gautel

2005-01-01

78

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.

Kroll, Jose E.; de Souza, Sandro J.

2014-01-01

79

Splice-Site A Choice Targets Plasma-Membrane Ca2+-ATPase Isoform 2 to Hair Bundles  

PubMed Central

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport. PMID:16763025

Hill, Jennifer K.; Williams, Diane E.; LeMasurier, Meredith; Dumont, Rachel A.; Strehler, Emanuel E.; Gillespie, Peter G.

2007-01-01

80

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

81

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

PubMed Central

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

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

2012-01-01

82

Squamous cell carcinoma of the pectoralis major myocutaneous flap donor site.  

PubMed

The pectoralis major myocutaneous flap is considered a workhorse flap in the reconstruction of head and neck defects after cancer ablative surgeries and remains one of the most widely used reconstructive options. Complications at the donor site after the use of this flap, although rare, do occur and are usually restricted to minor infections, hematoma, and seroma formation. Metastasis to the flap donor site is a rare complication with limited documentation. Metastasis at the donor site usually follows local recurrence at the primary site, supporting the probable hypothesis of re-establishment of lymphatic drainage to the primary site by the flap pedicle. Tumor implantation, although a probable cause for metastasis at the donor site, cannot be confidently distinguished from other mechanisms, such as hematogenous spread or lymphogenous metastasis. This report describes a case that supports a seeding or tumor implantation mechanism of metastasis exclusively. PMID:24560174

Mohan, A Mathan; Balaguhan, B; Krishna, Vinod; Nagarjuna, Muralidhara

2014-07-01

83

Achieving Direct Closure of the Anterolateral Thigh Flap Donor Site—An Algorithmic Approach  

PubMed Central

Background: Minimizing donor-site morbidity after free flap harvest is of paramount importance. In this article, we share our experience with achieving primary closure of 58 anterolateral thigh (ALT) free flap donor sites using a simple algorithm in cases where primary closure would otherwise have not been possible. Methods: Between 2004 and 2010, 58 patients who underwent free ALT flap reconstruction were included in the study. The inclusion criteria were those who had flap width requirements that were wider than 16% of the thigh circumference and had achieved direct primary closure of the donor site by the use of our technique. Results: Primary closure of the donor sites was facilitated in all cases by the use of 3 distinct techniques. This included the use of the V-Y advancement technique in 13 patients, split skin paddle technique in 7 patients, and the tubed skin paddle design in 38 patients. No episodes of postoperative wound dehiscence at the donor site were encountered; however, 2 cases were complicated by superficial wound infections that settled with a course of antibiotics. Conclusions: Direct primary closure of the ALT donor site can be facilitated by the use of our simple algorithm. Certain strategies need to be adopted at the design stage; however, the techniques used are simple and reliable, produce superior cosmetic results at the donor site, save time, and spare the patient the morbidity associated with the harvest of a skin graft.

Pachón Suárez, Jaime Eduardo; Sadigh, Parviz Lionel; Shih, Hsiang-Shun; Hsieh, Ching-Hua

2014-01-01

84

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

85

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

PubMed Central

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

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

1997-01-01

86

Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.  

PubMed

Ophthalmological and molecular genetic studies were performed in a consanguineous family with individuals showing either retinitis pigmentosa (RP) or cone-rod dystrophy (CRD). Assuming pseudodominant (recessive) inheritance of allelic defects, linkage analysis positioned the causal gene at 1p21-p13 (lod score 4.22), a genomic segment known to harbor the ABCR gene involved in Stargardt's disease (STGD) and age-related macular degeneration (AMD). We completed the exon-intron structure of the ABCR gene and detected a severe homozygous 5[prime] splice site mutation, IVS30+1G->T, in the four RP patients. The five CRD patients in this family are compound heterozygotes for the IVS30+1G->T mutation and a 5[prime] splice site mutation in intron 40 (IVS40+5G->A). Both splice site mutations were found heterozygously in two unrelated STGD patients, but not in 100 control individuals. In these patients the second mutation was either a missense mutation or unknown. Since thus far no STGD patients have been reported to carry two ABCR null alleles and taking into account that the RP phenotype is more severe than the STGD phenotype, we hypothesize that the intron 30 splice site mutation represents a true null allele. Since the intron 30 mutation is found heterozygously in the CRD patients, the IVS40+5G->A mutation probably renders the exon 40 5[prime] splice site partially functional. These results show that mutations in the ABCR gene not only result in STGD and AMD, but can also cause autosomal recessive RP and CRD. Since the heterozygote frequency for ABCR mutations is estimated at 0.02, mutations in ABCR might be an important cause of autosomal recessive and sporadic forms of RP and CRD. PMID:9466990

Cremers, F P; van de Pol, D J; van Driel, M; den Hollander, A I; van Haren, F J; Knoers, N V; Tijmes, N; Bergen, A A; Rohrschneider, K; Blankenagel, A; Pinckers, A J; Deutman, A F; Hoyng, C B

1998-03-01

87

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.

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

2013-01-01

88

"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.  

PubMed Central

A beta+-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the beta+-thalassemia gene (GGA) encodes glycine. The function of this beta+-thalassemia gene was compared to the function of the normal human beta-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype. Images PMID:6572978

Goldsmith, M E; Humphries, R K; Ley, T; Cline, A; Kantor, J A; Nienhuis, A W

1983-01-01

89

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

PubMed

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

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

2011-02-01

90

Rapid healing of MRSA infection at the suprafascial radial donor site  

Microsoft Academic Search

Abstract.Methicillin-resistant Staphylococcus aureus (MRSA) infection at the radial suprafascial donor site resulted in significant loss of the skin graft, but no tendon exposure, in two patients. The complication was successfully managed with wound debridement, appropriate antibiotics, a negative-pressure wound dressing and early partial-thickness skin grafting. The suprafascial dissection creates a donor site that resists both skin graft loss and tendon

C. M. E. Avery; C. Harrop

2002-01-01

91

Probabilistic simple splicing systems  

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

92

The use of silver coated dressings on donor site wounds: a prospective, controlled matched pair study.  

PubMed

Acticoat, a new silver-coated dressing, produces a moist healing environment along with the sustained release of ionic silver for improved microbial control. These properties suggest that Acticoat might be a useful donor site dressing. However, there are no human studies which assess Acticoat for this use. The purpose of this study was to compare the healing of human skin graft donor sites dressed with Acticoat, to the healing of those dressed with Allevyn, an occlusive moist-healing environment material, which is our standard donor site dressing. In burn patients who had undergone burn excision and grafting, identical side-by-side split thickness donor site wound pairs were dressed with Allevyn and Acticoat. Re-epithelialization was directly assessed daily by a single observer from post-operative day 6 onward, and by four independent observers who rated the extent of re-epithelialization by viewing standardized digital images of the wounds that had been obtained on post-operative days 6, 8, 10,and 12. Donor sites were swabbed for bacterial culture on days 3, 6, and 9. Subsequently, each study donor site scar was rated by a blinded observer using the Vancouver Scar Scale at 1, 2, and 3 months. Sixteen paired sites in 15 patients (3 female, 12 male) were studied. Donor sites dressed with Allevyn were >90% re-epithelialized at a mean of 9.1+/-1.6 days while donor sites dressed with Acticoat required a mean of 14.5+/-6.7 days to achieve >90% re-epithelialization (P=0.004). The Allevyn sites had significantly greater estimated re-epithelialization at days 6, 8, 10 and 12 than the Acticoat sites based on the observations of the digital images. There were no significant differences in the incidence of positive bacterial cultures with either dressing at days 3, 6, and 9. Donor sites dressed with Acticoat had significantly worse scars at 1 and 2 months but this difference resolved by 3 months. Our findings do not support the use of Acticoat as a skin graft donor site dressing. PMID:11525858

Innes, M E; Umraw, N; Fish, J S; Gomez, M; Cartotto, R C

2001-09-01

93

A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis  

PubMed Central

Background Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). Case Presentation We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G>A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G>A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A>G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. Conclusions We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor. The possible phenotypical implications of this mutation are discussed. PMID:21708027

2011-01-01

94

Genome-wide survey of allele-specific splicing in humans  

PubMed Central

Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST) and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array data, including several examples for which there is experimental evidence of polymorphisms affecting splicing in the literature. We also present a set of novel allele-specific splicing candidates and discuss the strengths and weaknesses of alternative technologies for inferring the effect of sequence variants on mRNA splicing. PMID:18518984

Nembaware, Victoria; Lupindo, Bukiwe; Schouest, Katherine; Spillane, Charles; Scheffler, Konrad; Seoighe, Cathal

2008-01-01

95

Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.  

PubMed Central

The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection. Images PMID:8029003

Chanfreau, G; Legrain, P; Dujon, B; Jacquier, A

1994-01-01

96

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

PubMed Central

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

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

2013-01-01

97

Propeller flaps in the closure of free fibula flap donor site skin defects.  

PubMed

The free fibula is a versatile and commonly used free flap in microvascular reconstruction. It allows for reconstruction of both bone and soft tissue defects. In head and neck reconstruction, the skin paddle harvested along with the flap allows for the reconstruction of skin or oral mucosal defects. After skin paddle harvest, the donor site can be closed primarily or with skin grafts. Grafting the donor area is the common method used. However, this could lead to delayed healing because of the poor graft over the area of peroneal tendons. Propeller flaps have been extensively reported for closure of leg skin defects. We report a series of 10 patients in whom we used a local propeller flap for the closure of the fibula flap skin donor site. The donor defects could be satisfactorily closed without the need of a skin graft in 9 patients. This method is simple, reliable, and suitable for closing small to medium defects. PMID:23364675

Sharma, Mohit; Balasubramanian, Deepak; Thankappan, Krishnakumar; Sampathirao, Chandrasekhararao Leelamohan; Mathew, Jimmy; Chavre, Sachin; Iyer, Subramania

2013-07-01

98

The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex  

NASA Astrophysics Data System (ADS)

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

1995-07-01

99

The missing puzzle piece: splicing mutations  

PubMed Central

Proper gene splicing is highly dependent on the correct recognition of exons. Among the elements allowing this process are the “cis” (conserved sequences) and “trans” (snRNP, splicing factors) elements. Splicing mutations are related with a number of genetic disorders and usually induce exon skipping, form new exon/intron boundaries or activate new cryptic exons as a result of alterations at donor/acceptor sites. They constitute more than 9% of the currently published mutations, but this value is highly underestimated as many of the potential mutations are located in the “cis” elements and should be confirmed experimentally. The most commonly detected splicing mutations are located at donor (5’) and acceptor (3’) sites. Mutations at the branch point are rare (only over a dozen are known to date), and are mostly searched and detected when no alteration has been detected in the sequenced exons and UTRs. Polypyrimidine tract mutations are equally rare. High throughput technologies, as well as traditional Sanger sequencing, allow detection of many changes in intronic sequences and intron/exon boundaries. However, the assessment whether a mutation affects exon recognition and results in a genetic disorder has to be conducted using molecular biology methods: in vitro transcription of the sequence of interest cloned into a plasmid, with and without alterations, or mutation analysis via a hybrid minigene system. Even though microarrays and new generation sequencing methods pose difficulties in detecting novel branch point mutations, these tools seem appropriate to expand the mutation detection panel especially for diagnostic purposes. PMID:24294354

Lewandowska, Marzena A

2013-01-01

100

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

101

Coupling transcription and alternative splicing.  

PubMed

Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation. PMID:18380347

Kornblihtt, Alberto R

2007-01-01

102

Association of LILRA2 (ILT1, LIR7) splice site polymorphism with systemic lupus erythematosus and microscopic polyangiitis.  

PubMed

Leukocyte immunoglobulin-like receptors (LILRs) are inhibitory, stimulatory or soluble receptors encoded within the leukocyte receptor complex. Some LILRs are extensively polymorphic, and exhibit evidence for balancing selection and association with disease susceptibility. LILRA2 (LIR7/ILT1) is an activating receptor highly expressed in inflammatory tissues, and is involved in granulocyte and macrophage activation. In this study, we examined the association of LILRA2 and adjacently located LILRA1 with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and microscopic polyangiitis (MPA). Polymorphism screening detected a LILRA2 SNP (rs2241524 G>A) that disrupts splice acceptor site of intron 6. Case-control association studies on 273 Japanese SLE, 296 RA, 50 MPA and 284 healthy individuals revealed increase of genotype A/A in SLE (12.1%, odds ratio (OR) 1.82, 95% confidence interval (CI) 1.02-3.24, P=0.041) and in MPA (16.0%, OR 2.52, 95% CI 1.07-5.96, P=0.049) compared with healthy individuals (7.0%). The risk allele caused an activation of a cryptic splice acceptor site that would lead to a novel LILRA2 isoform lacking three amino acids in the linker region (Delta 419-421). Flow cytometry indicated that this isoform was expressed on the surface of monocytes. These findings suggested that LILRA2 Delta 419-421 isoform encoded by the splice site SNP may play a role in SLE and MPA. PMID:18273033

Mamegano, K; Kuroki, K; Miyashita, R; Kusaoi, M; Kobayashi, S; Matsuta, K; Maenaka, K; Colonna, M; Ozaki, S; Hashimoto, H; Takasaki, Y; Tokunaga, K; Tsuchiya, N

2008-04-01

103

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

PubMed Central

More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5? splice site (5?ss), even in the presence of a stronger 5?ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5?ss. This enhancing effect depended on the strength of the downstream 5?ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5?ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within ?20 nt downstream of the 5?ss. For most metazoans, the strength of the 5?ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular. PMID:19911040

Ram, Oren; Eyras, Eduardo; Ast, Gil

2009-01-01

104

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

PubMed Central

The Bactigras® paraffin tulle coated with chlorhexidine is normally used for the treatment of donor-site wounds in burn patients who received split-thickness skin grafts in several centers. It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD®, a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras® or the Telfa AMD® wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (?90%) was significantly shorter in patients treated with the Telfa AMD® compared to the Bactigras® group (14.00 ± 3.05 vs. 9.25 ± 1.88 days for Bactigras® and Telfa AMD® groups, respectively, p < 0.001). The average pain score was also significantly lower in the Telfa AMD® group (1.57 ± 0.55 vs. 4.70 ± 1.16, p < 0.001). There was no difference in the cost of treatment between the groups (4.64 ± 1.97 vs. 5.72 ± 2.54 USD, p = 0.19). This study indicated that the Telfa AMD® was an effective dressing for the treatment of donor-site wounds. PMID:21954342

Muangman, Pornprom; Nitimonton, Sooksan; Aramwit, Pornanong

2011-01-01

105

The chin as a donor site in early secondary osteoplasty: a retrospective clinical and radiological evaluation.  

PubMed

26 unilateral cleft palate patients received an autogenous bicortical chin bone graft for early reconstruction of the alveolar process. In the evaluation of the donor site, 4% of the anterior teeth showed a negative pulpal sensibility, less than 1% a peri-apical granuloma and 12% pulp canal obliteration. The tooth buds of the canines showed developmental disturbances in 6%. Exposure of unerupted canines should be avoided, and a 5 mm safety margin is advised. Based on its architecture, topographic accessibility, minimal post-operative morbidity and absence of visible scars, the chin can be considered to be a very useful donor site in bone grafting procedures. PMID:1613107

Hoppenreijs, T J; Nijdam, E S; Freihofer, H P

1992-04-01

106

Spliced leader RNA trans-splicing in dinoflagellates  

PubMed Central

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

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

2007-01-01

107

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

108

Enhanced cryoprecipitate for skin graft and donor site wound healing in pigs.  

PubMed

Due to similarities in skin characteristics, the authors hypothesise that a pig model would most accurately show the ability of autologous, enhanced cryoprecipitate (eCryo) to improve the wound healing of split-thickness skin grafts (STSGs) and corresponding donor sites. Fifty-two STSGs (5 × 5 cm) were fashioned and treated according to a randomised protocol with an autologous eCryo-treated and a control group. Macroscopic assessment, histological evaluation and cellular composition were completed at days 7, 14, 21 and 28. Thirty-two donor sites were also created and assessed in a similar manner. Histologic analysis showed enhancement of healing over all time points for eCryo-treated donor sites. All other results showed no statistically significant improvement with the use of eCryo. Autologous cryoprecipitate appears to be a safe, inexpensive and easy-to-use alternative to fibrin glue, which carries risks and is, in many cases, prohibitively expensive. Further studies are necessary to evaluate the full potential of eCryo. Interestingly, eCryo application may improve donor site aesthetic appearance. We believe that a pig model most reliably simulates eCryo's behaviour in humans to accurately reflect its future clinical applicability. PMID:22905755

Vetter, Thomas Sebastian; Mowlds, Donald S; Scholz, Thomas; Nam, Su Bong; Lin, Fritz; Owens, John W; Dey, Dilip; Wirth, Garrett A; Evans, Gregory R D

2014-04-01

109

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

110

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

Vorechovsky, Igor

2006-01-01

111

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

112

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

PubMed Central

The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

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

2010-01-01

113

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

Microsoft Academic Search

Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin\\u000a gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined\\u000a by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689),

Jana Kralovicova; Igor Vorechovsky

2010-01-01

114

Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5? Splice Site Choice  

PubMed Central

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.

2014-01-01

115

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

116

Redirecting splicing with bifunctional oligonucleotides  

PubMed Central

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5? splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants. PMID:24375754

Brosseau, Jean-Philippe; Lucier, Jean-François; Lamarche, Andrée-Anne; Shkreta, Lulzim; Gendron, Daniel; Lapointe, Elvy; Thibault, Philippe; Paquet, Éric; Perreault, Jean-Pierre; Abou Elela, Sherif; Chabot, Benoit

2014-01-01

117

Splicing- and cleavage-independent requirement of RNA polymerase II CTD for mRNA release from the transcription site  

PubMed Central

Eukaryotic cells have a surveillance mechanism that identifies aberrantly processed pre-mRNAs and prevents their flow to the cytoplasm by tethering them near the site of transcription. Here we provide evidence that mRNA release from the transcription site requires the heptad repeat structure of the C-terminal domain (CTD) of RNA polymerase II. The mammalian CTD, which is essential for normal co-transcriptional maturation of mRNA precursors, comprises 52 heptad repeats. We show that a truncated CTD containing 31 repeats (heptads 1–23, 36–38, and 48–52) is sufficient to support transcription, splicing, cleavage, and polyadenylation. Yet, the resulting mRNAs are mostly retained in the vicinity of the gene after transcriptional shutoff. The retained mRNAs maintain the ability to recruit components of the exon junction complex and the nuclear exosome subunit Rrp6p, suggesting that binding of these proteins is not sufficient for RNA release. We propose that the missing heptads in the truncated CTD mutant are required for binding of proteins implicated in a final co-transcriptional maturation of spliced and 3? end cleaved and polyadenylated mRNAs into export-competent ribonucleoprotein particles. PMID:17938247

Custódio, Noélia; Vivo, Maria; Antoniou, Michael; Carmo-Fonseca, Maria

2007-01-01

118

Comparison of scalp and abdomen as split-thickness skin graft donor sites for aural stenosis repair.  

PubMed

To evaluate and compare the scalp and the abdomen as split-thickness skin graft donor sites for aural stenosis repair. A total of 34 patients with aural stenosis were included in the study. All the patients underwent meatoplasty using split-thickness skin grafts. Among them, the skin graft donor site was the scalp in 11 patients and the abdomen in the other 23 patients. The surgical team followed the patients in the outpatient department for at least 6 months after surgery. Evaluations concerned healing of the donor site, hair regeneration of the donor site, survival of split-thickness skin grafts, reoccurrence of aural stenosis and hair growth in the ear canal. The incidences of reoccurrence of aural stenosis in the two groups were compared. Subjective scar evaluation of the donor sites was performed using the Patient Scar Assessment Scale (PASA). The scale items were pain, itching, color, stiffness, thickness and irregularity. All the scalp and abdominal donor sites healed well with no sign of infection. Hair regrowth and reepithelialization was observed at all the scalp donor sites. Pink discoloration was observed at the scalp donor sites in six patients 2-3 months after surgery and disappeared 6-9 months after surgery. Scars were observed at the scalp donor sites in two patients 6 months after surgery. No alopecia was observed at the scalp donor sites. The scars and pink discoloration were hidden in the hair. Scars and/or discoloration were observed at all the abdominal donor sites 12 months after surgery. All the scalp and abdominal skin grafts survived with no sign of infection. Hair growth was observed in the ear canals in two patients in the scalp group. The incidences of reoccurrence of aural stenosis were 0 % (0/23) in the abdominal group and 9.1 % (1/11) in the scalp group, respectively (Chi square test, p > 0.05). The PASA values about color, stiffness, thickness and irregularity were higher in the abdominal group than in the scalp group (Mann-Whitney U test, p < 0.001). The PASA values about pain and itching were the same (Mann-Whitney U test, p > 0.05). The scalp meets most requirements of an ideal donor site of skin grafts for aural stenosis. The advantages of scalp as a donor site include easy accessibility in the operative field, simple postoperative care, low risk of infection, rapid wound healing, minimal interference with rehabilitation, and minimal scar formation. PMID:24057102

Du, Qiang; Zhang, Tianyu

2014-08-01

119

Ehlers-Danlos syndrome type IV: phenotypic consequences of a splicing mutation in one COL3A1 allele  

Microsoft Academic Search

The features of a child with Ehlers-Danlos syndrome type IV (EDS IV) resulting from a mutation in one COL3A1 allele were studied. The child was heterozygous for a G- to A-transition at the splice donor site of intron 41. It resulted in the splicing out of the exon 41 encoded sequence from alpha 1(III) mRNA and the deletion of 36

D O Sillence; A A Chiodo; P E Campbell; W G Cole

1991-01-01

120

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

121

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons.  

PubMed

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation. PMID:2675974

Danenberg, P V; Shea, L C; Danenberg, K

1989-08-01

122

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.

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

2014-01-01

123

A new case of frontotemporal dementia and parkinsonism resulting from an intron 10 +3-splice site mutation in the tau gene: clinical and pathological features.  

PubMed

Hereditary frontotemporal dementia and parkinsonism (FTDP) linked to chromosome 17 (FTDP-17) constitutes a new form of tauopathy, and mutations in the tau gene have recently been reported in some affected families. This report presents clinical and neuropathological data from a member of a British family (SOT 254) with a history of dementia and movement disorder. The medical history of the affected patient, a woman aged 44 years, was reviewed, and a detailed post-mortem examination of the brain was undertaken. A panel of well characterized phosphorylation-dependent and independent anti-tau antibodies was used to assess tau pathology, and inclusions were examined by electron microscopy. Neuronal loss and gliosis were widely distributed, but most severe in neocortical regions, and were associated with abundant neuronal and glial tau inclusions which consisted of a mixture of paired helical filaments (PHFs), similar to those in Alzheimer's disease, and distinct twisted ribbon-like filaments. Genomic DNA was obtained from post-mortem tissue from the index patient, and blood from two unaffected members of the same family. For the index case only, sequencing of intronic sequences flanking exon 10 of the tau gene identified a G to A transition at position +3 of the splice-donor site downstream of exon 10, identical to that reported in multiple system tauopathy with presenile dementia (MSTD). The clinical, neuropathological and genetic findings strongly suggest that SOT 254 represents a new example of FTDP-17 resulting from a mutation in the tau gene. These results are compared with those reported for other FTDP-17 families, i.e. for MSTD. PMID:10931371

Tolnay, M; Grazia Spillantini, M; Rizzini, C; Eccles, D; Lowe, J; Ellison, D

2000-08-01

124

Egg Membrane as a New Biological Dressing in Split-Thickness Skin Graft Donor Sites: A Preliminary Clinical Evaluation  

Microsoft Academic Search

Background: This preliminary investigation attempted to determine the effectiveness of egg membranes as a new biological dressing to promote infection-free heal- ing and provide pain relief over split-thickness skin graft (STSG) donor sites. Methods: Eighteen patients, with 28 STSG donor sites who were admitted to the LinKou Burn Center from August 1997 to July 1999, were selected for this trial.

Jui-Yung Yang; Shiow-Shuh Chuang; Wen-Guei Yang; Pei-Kwei Tsay

125

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

Microsoft Academic Search

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

Paravee Katavetin; Pisut Katavetin; Suttipong Wacharasindhu; Vorasuk Shotelersuk

2006-01-01

126

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

127

Computer-based planning of optimal donor sites for autologous osseous grafts  

NASA Astrophysics Data System (ADS)

Bone graft surgery is often necessary for reconstruction of craniofacial defects after trauma, tumor, infection or congenital malformation. In this operative technique the removed or missing bone segment is filled with a bone graft. The mainstay of the craniofacial reconstruction rests with the replacement of the defected bone by autogeneous bone grafts. To achieve sufficient incorporation of the autograft into the host bone, precise planning and simulation of the surgical intervention is required. The major problem is to determine as accurately as possible the donor site where the graft should be dissected from and to define the shape of the desired transplant. A computer-aided method for semi-automatic selection of optimal donor sites for autografts in craniofacial reconstructive surgery has been developed. The non-automatic step of graft design and constraint setting is followed by a fully automatic procedure to find the best fitting position. In extension to preceding work, a new optimization approach based on the Levenberg-Marquardt method has been implemented and embedded into our computer-based surgical planning system. This new technique enables, once the pre-processing step has been performed, selection of the optimal donor site in time less than one minute. The method has been applied during surgery planning step in more than 20 cases. The postoperative observations have shown that functional results, such as speech and chewing ability as well as restoration of bony continuity were clearly better compared to conventionally planned operations. Moreover, in most cases the duration of the surgical interventions has been distinctly reduced.

Krol, Zdzislaw; Chlebiej, Michal; Zerfass, Peter; Zeilhofer, Hans-Florian U.; Sader, Robert; Mikolajczak, Pawel; Keeve, Erwin

2002-05-01

128

Impaired hippocampal plasticity and errors in cognitive performance in mice with maladaptive AChE splice site  

E-print Network

, The Hebrew University of Jerusalem, Israel 91904 Keywords: acetylcholinesterase, cognition errors, LTP performance. Under stress, alternative splicing changes priority from synaptic acetylcholinesterase (ACh

Hochner, Binyamin

129

A spontaneous Fatp4/Scl27a4 splice site mutation in a new murine model for congenital ichthyosis.  

PubMed

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3'-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation. PMID:23226340

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

2012-01-01

130

Alternative splicing and retinal degeneration.  

PubMed

Alternative splicing is highly regulated in tissue-specific and development-specific patterns, and it has been estimated that 15% of disease-causing point mutations affect pre-mRNA splicing. In this review, we consider the cis-acting splice site and trans-acting splicing factor mutations that affect pre-mRNA splicing and contribute to retinal degeneration. Numerous splice site mutations have been identified in retinitis pigmentosa (RP) and various cone-rod dystrophies. Mutations in alternatively spliced retina-specific exons of the widely expressed RPGR and COL2A1 genes lead primarily to X-linked RP and ocular variants of Stickler syndrome, respectively. Furthermore, mutations in general pre-mRNA splicing factors, such as PRPF31, PRPF8, and PRPF3, predominantly cause autosomal dominant RP. These findings suggest an important role for pre-mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule-based therapies, has the potential to lead to new treatments for retinal degenerative diseases. PMID:23647439

Liu, M M; Zack, D J

2013-08-01

131

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

PubMed

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

Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

2005-06-01

132

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

PubMed

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

Slavov, D; Gardiner, K

2002-10-16

133

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

PubMed Central

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

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

1993-01-01

134

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.  

PubMed

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics. PMID:24574459

Adamia, Sophia; Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Tenen, Daniel G; Stone, Richard M; Griffin, James D

2014-05-01

135

Demonstration of donor specific unresponsiveness in rat islet allografts: importance of transplant site for induction by cyclosporin A and maintenance  

Microsoft Academic Search

Summary  The purpose of the present study was to evaluate the effects of transplant site (the liver via the portal vein vs the renal subcapsular space) on islet allograft survival using cyclosporin A and also to determine whether or not the unresponsiveness obtained is donor specific and how the site selected is related to the maintenance of unresponsiveness. Prolongation of islet

T. Kamei; Y. Yasunami

1989-01-01

136

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

PubMed Central

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

2013-01-01

137

RNA Splicing in a New Rhabdovirus from Culex Mosquitoes?†  

PubMed Central

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

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

2011-01-01

138

Laparoendoscopic single-site donor nephrectomy: a single-center initial experience.  

PubMed

Background This study presents our initial experience with laparoendoscopic single-site donor nephrectomy. Ten patients (8 females, 2 males; mean age 45.3±13.2 years) underwent LESS-DN. Material and Methods Transumbilical laparoscopic donor nephrectomy was performed using an SILS™ port. Standard laparoscopic instruments and a 30-degree angled camera were used during the surgery. We evaluated the following parameters: warm and cold ischemia time, duration of the operation, amount of blood loss during the operation, duration of hospitalization, creatinine level, and visual analogue scale score for pain at discharge. Results The means for duration of operation, warm ischemia time, and duration of hospitalization were 140 min, 194 s, and 1.4 days, respectively. Intraoperative and/or postoperative complications were not observed. Low pain score and cosmetic advantage were remarkable. All recipients had functional grafts. The results of our initial experience with LESS-DN appeared to be positive. Conclusions Further studies on the LESS-DN technique with larger series conducted in different centers are needed. PMID:25356806

Gurluler, Ercument; Berber, Ibrahim; Cakir, Ulkem; Gurkan, Alihan

2014-01-01

139

Functional analysis of a new splice site mutation, c.605-3C>A, in the cystinuria gene SLC7A9 leading to exon skipping.  

PubMed

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3A1 gene cause type I cystinuria, patients with non-type I cystinuria carry mutations in the SLC7A9 gene. Up to now, more than 80 mutations in SLC3A1 and 50 in SLC7A9 have been reported in the literature. While deletions, duplications, and truncating mutations can often unambiguously classified to be pathogenic, the functional relevance of base pair substitutions is often difficult to predict. To determine the functional relevance of a new splice site mutation in intron 5 of SLC7A9, c.605-3C>A, we transfected COS7 cells with expression constructs containing the wild-type and mutant allele, respectively. cDNAs derived from the resulting SLC7A9 mRNAs were sequenced. By this approach we could demonstrate that the mutant allele c.605-3A causes exon skipping and therefore represents a splice site mutation. To the best of our knowledge, this is the first splice site mutation in a cystinuria gene with a proven functional consequence. PMID:15670723

Schmidt, Christa; Lahme, Sven; Zerres, Klaus; Eggermann, Thomas

2005-02-01

140

Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome  

PubMed Central

Purpose Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, microspherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Methods Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)–PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. Results The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT–PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. Conclusions The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six. PMID:24940034

Shah, Mohd Hussain; Bhat, Vishwanath; Shetty, Jyoti S.

2014-01-01

141

SLaP mapper: A webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes  

PubMed Central

The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ?39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website. PMID:25111964

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

2014-01-01

142

Complete human gene structure of obscurin: implications for isoform generation by differential splicing.  

PubMed

The complete gene giant muscle protein obscurin, a modular protein composed largely of tandem Ig-domains, GDP/GTP exchange factor domains (GEF) for small G-proteins, and differentially spliced kinase domains, was analysed. The splice donor and acceptor sites of the 117 exons give important clues for potential splice pathways. The fusion of the conventional obscurin A, containing only the GEF domain, and obscurin B, fusing into the 3' kinase exons, was experimentally confirmed and analysed. The linker between the two kinases contains multiple predicted phosphorylation sites, as well as a predicted NFX zinc finger domain. Both kinases show only weak homology to either myosin light chain kinases or other giant muscle protein kinases, suggesting that they are functionally distinct. PMID:16625316

Fukuzawa, Atsushi; Idowu, Seraphina; Gautel, Mathias

2005-01-01

143

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

PubMed

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

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

2013-08-01

144

Factors associated with hernia and bulge formation at the donor site of the pedicled TRAM flap  

PubMed Central

The purpose of this study was to evaluate the correlation between risk factors and hernia or bulge formation at the donor site of the transverse rectus abdominis myocutaneous (TRAM) flap. A retrospective study was conducted between September 2005 and December 2008 in 206 patients who underwent breast reconstruction with pedicled TRAM flap. Eight (3.9%) of these patients had abdominal wall hernia and 26 (12.6%) had abdominal bulging. The incidence of hernia was significantly higher (P?

Abla, Luiz Eduardo Felipe; Vidal, Ronaldo; Garcia, Elvio Bueno; Gonzalez, Ricardo Joao; Gebrim, Luiz Henrique; Neto, Miguel Sabino; Ferreira, Lydia Masako

2010-01-01

145

Brief report: The use of dermal autograft for fascial repair of TRAM flap donor sites.  

PubMed

Reconstruction of the breast and other tissues by the transverse rectus abdominis myocutaneous (TRAM) flap is an accepted, reliable technique with a high success rate. Closure of the anterior rectus sheath defect that results from this flap has been the subject of debate, since hernia formation is considered a risk. We have reviewed 20 patients from a consecutive series of free TRAM and pedicled TRAM flaps using dermal autograft removed from zone IV of the flap and 117 patients using mesh, and assessed the complication of these techniques. Average follow-up time was 21.7 months (ranged 5 months to 4 years). There was one wound infection (5%) and one bulging (5%) in the dermal autograft group and 2 (1.7) bulgings, 3 (0.85) hernias and 2 (1.7) infections in the mesh group. There was no significant difference in the complication rate between the two techniques though. We conclude that the principal advantages of the dermal autograft in TRAM flap donor site closure are ease of use, economy and the benefit of using an autologous, biological material. PMID:20541484

Kheradmand, Ali Arab; Novin, Neda Ranjbar; Khazaeipour, Zahra

2011-03-01

146

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

PubMed

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. PMID:24361966

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

2014-03-01

147

Alternative splicing of the adenylyl cyclase stimulatory G-protein G alpha(s) is regulated by SF2/ASF and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and involves the use of an unusual TG 3'-splice Site.  

PubMed

The factors involved in regulating alternative splicing of the human adenylyl cyclase stimulatory G-protein G alpha(s) in different cell types remain undefined. We have designed a G alpha(s) minigene that retains the signals required for G alpha(s) alternative splicing in vivo. Employing transient transfection of human myometrial smooth muscle cells and HeLa cells, as well as in vitro splicing assays, we have provided evidence that the antagonistic splicing factors SF2/ASF and hnRNPA1 act as potent regulators of G alpha(s) isoform expression in these cells. Both SF2/ASF and hnRNPA1 control the selection of competing 5'-splice sites and respectively promote inclusion or skipping of the small cassette-type exon 3 of G alpha(s) transcripts, resulting in the generation of G alpha(s)-long and G alpha(s)-short mRNA isoforms. We have also provided evidence that SF2/ASF and hnRNPA1 play a role in 3'-splice site selection involving the use of a non-canonical TG 3'-splice site preceding exon 4. Using a score-matrix analysis to identify putative exonic enhancer sequences (ESEs), we found multiple high score ESE motifs for SF2/ASF, SC35, and SRp40 in exon 3 of G alpha(s). These results suggest that tissue-specific expression of SF2/ASF and hnRNPA1 governs the expression of alternative isoforms of G alpha(s) in these different cells types. PMID:11825891

Pollard, Alison J; Krainer, Adrian R; Robson, Stephen C; Europe-Finner, G Nicholas

2002-05-01

148

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

149

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

PubMed Central

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

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

2013-01-01

150

A sequence compilation and comparison of exons that are alternatively spliced in neurons.  

PubMed

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation. PMID:8202349

Stamm, S; Zhang, M Q; Marr, T G; Helfman, D M

1994-05-11

151

A sequence compilation and comparison of exons that are alternatively spliced in neurons.  

PubMed Central

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation. PMID:8202349

Stamm, S; Zhang, M Q; Marr, T G; Helfman, D M

1994-01-01

152

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

PubMed Central

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

Smalle, Jan A

2013-01-01

153

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

154

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

155

Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites.  

PubMed Central

We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites. Images Fig. 2. Fig. 3. Fig. 5. Fig. 6. Fig. 8. PMID:6202514

Breathnach, R

1984-01-01

156

Novel frameshift and splice site mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1) associated with hereditary sensory neuropathy type IV.  

PubMed

Congenital insensitivity to pain with anhidrosis or hereditary sensory and autonomic neuropathy type IV (HSAN IV) is the first human genetic disorder implicated in the neurotrophin signal transduction pathway. HSAN IV is characterized by absence of reaction to noxious stimuli, recurrent episodes of fever, anhidrosis, self-mutilating behavior and often mental retardation. Mutations in the neurotrophic tyrosine kinase, receptor, type 1 (NTRK1) are associated with this disorder. Here we report four homozygous mutations, two frameshift (p.Gln626fsX6 and p.Gly181fsX58), one missense (p.Arg761Trp) and one splice site (c.359+5G>T) mutation in four HSAN IV patients. The splice site mutation caused skipping of exons 2 and 3 in patient's mRNA resulting in an in-frame deletion of the second leucine-rich motif. NTRK1 mutations are only rarely reported in the European population. This report extends the spectrum of NTRK1 mutations observed in patients diagnosed with HSAN IV. PMID:16373086

Verpoorten, Nathalie; Claeys, Kristl G; Deprez, Liesbet; Jacobs, An; Van Gerwen, Veerle; Lagae, Lieven; Arts, Willem Frans; De Meirleir, Linda; Keymolen, Kathelijn; Ceuterick-de Groote, Chantal; De Jonghe, Peter; Timmerman, Vincent; Nelis, Eva

2006-01-01

157

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

PubMed

Abstract 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

2014-09-20

158

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

159

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

160

A novel HLA-B*39 allele (HLA-B*3916) due to a rare mutation causing cryptic splice site activation.  

PubMed

A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both. PMID:10773349

Tamouza, R; El Kassar, N; Schaeffer, V; Carbonnelle, E; Tatari, Z; Marzais, F; Fortier, C; Poirier, J C; Sadki, K; Bernaudin, F; Toubert, A; Krishnamoorthy, R; Charron, D

2000-05-01

161

Functional selection of splicing enhancers that stimulate trans-splicing in vitro.  

PubMed Central

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction. PMID:11421358

Boukis, L A; Bruzik, J P

2001-01-01

162

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.

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

2014-01-01

163

Donor-site morbidity after pedicled TRAM breast reconstruction: a comparison of two different types of mesh.  

PubMed

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

Sværdborg, Mille; Damsgaard, Tine Engberg

2013-11-01

164

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

PubMed Central

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

2012-01-01

165

Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.  

PubMed

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression. PMID:8085156

Cáceres, J F; Stamm, S; Helfman, D M; Krainer, A R

1994-09-16

166

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

167

Cloning and Characterization of Buffalo NANOG Gene: Alternative Transcription Start Sites, Splicing, and Polyadenylation in Embryonic Stem Cell-Like Cells  

PubMed Central

NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5?-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5?-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3?-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3?-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5?-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at ?30 and ?139?bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter. PMID:22011250

Singh, Natwar; Sharma, Ruchi; George, Aman; Singla, Suresh K.; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S.

2012-01-01

168

Rbm20 regulates titin alternative splicing as a splicing repressor  

PubMed Central

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3? splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age. PMID:23307558

Li, Shijun; Guo, Wei; Dewey, Colin N.; Greaser, Marion L.

2013-01-01

169

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions  

PubMed Central

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5? splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5? splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5? splice sites. Surprisingly, TIA binding at 5? splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3? splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5? splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing. PMID:21048981

Briese, Michael; Zarnack, Kathi; Luscombe, Nicholas M.; Rot, Gregor; Zupan, Blaz; Curk, Tomaz; Ule, Jernej

2010-01-01

170

iCLIP predicts the dual splicing effects of TIA-RNA interactions.  

PubMed

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5' splice sites. Surprisingly, TIA binding at 5' splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3' splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5' splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing. PMID:21048981

Wang, Zhen; Kayikci, Melis; Briese, Michael; Zarnack, Kathi; Luscombe, Nicholas M; Rot, Gregor; Zupan, Blaž; Curk, Tomaž; Ule, Jernej

2010-01-01

171

Composite tissue transplantation in rats: Fusion of donor muscle to the recipient site  

Microsoft Academic Search

Little information currently exists on the repair of muscular tissue at the site of an amputation stump. This study examined the healing process of muscular tissue following composite limb transplantation using transgenic rat models.

T. Ajiki; A. Kimura; Y. Sato; T. Murakami; Y. Hakamata; Y. Kariya; Y. Hoshino; E. Kobayashi

2005-01-01

172

Lymph node flap based on the right transverse cervical artery as a donor site for lymph node transfer.  

PubMed

Lymph node transfer is a novel technique in lymphedema surgery. In this study, we present our experience in harvesting lymph nodes flap based on the right transverse cervical artery. In a period of 7 months, we harvested 11 cervical lymph node flaps based on the right transverse cervical artery (TCA). The reliable anatomy of the TCA and the low complication rate of the donor site make this lymph node flap ideal for transfer in the treatment of lymphedema. Knowledge of the regional anatomy and the anatomic variations of the TCA are mandatory for safe dissection of this flap. We also present the preliminary results of our first 2 cases in which we performed cervical lymph node transfer for secondary lower extremity lymphedema. PMID:23759964

Sapountzis, Stamatis; Singhal, Dhruv; Rashid, Abid; Ciudad, Pedro; Meo, Domenico; Chen, Hung-Chi

2014-10-01

173

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

PubMed Central

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

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

1996-01-01

174

Imprinting of molecular recognition sites through electropolymerization of functionalized Au nanoparticles: development of an electrochemical TNT sensor based on pi-donor-acceptor interactions.  

PubMed

Electrochemical sensors for the analysis of TNT with enhanced sensitivities are described. The enhanced sensitivities are achieved by tailoring pi-donor-acceptor interactions between TNT and pi-donor-modified electrodes or pi-donor-cross-linked Au nanoparticles linked to the electrode. In one configuration a p-aminothiophenolate monolayer-modified electrode leads to the analysis of TNT with a sensitivity corresponding to 17 ppb (74 nM). In the second configuration, the cross-linking of Au NPs by oligothioaniline bridges to the electrode yields a functionalized electrode that detects TNT with a sensitivity that corresponds to 460 ppt (2 nM). Most impressively, the imprinting of molecular TNT recognition sites into the pi-donor oligoaniline-cross-linked Au nanoparticles yields a functionalized electrode with a sensitivity that corresponds to 46 ppt (200 pM). The electrode reveals high selectivity, reusability, and stability. PMID:18597454

Riskin, Michael; Tel-Vered, Ran; Bourenko, Tatyana; Granot, Eran; Willner, Itamar

2008-07-30

175

Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing  

PubMed Central

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5? splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic +14 mutation produce exclusively E10+ RNA. Generation of E10? RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the ?280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements. PMID:19498037

Rodriguez-Martin, Teresa; Anthony, Karen; Garcia-Blanco, Mariano A.; Mansfield, S. Gary; Anderton, Brian H.; Gallo, Jean-Marc

2009-01-01

176

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

177

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.  

PubMed

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

2014-12-01

178

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs  

PubMed Central

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

179

Novel splice-site mutations and a large intragenic deletion in PLA2G6 associated with a severe and rapidly progressive form of infantile neuroaxonal dystrophy.  

PubMed

Infantile neuroaxonal dystrophy, INAD, is a severe progressive psychomotor disorder with infantile onset and characterized by the presence of axonal spheroids throughout the central and peripheral nervous systems. A subset of INAD patients shows also brain iron accumulation which represents instead the distinctive feature of the idiopathic neurodegeneration with brain iron accumulation, NBIA. These diseases share the same causative gene, PLA2G6, encoding iPLA2-VIA, a calcium-independent phospholipase. Mutations that lead to a complete absence of protein are associated with a severe INAD profile, while compound heterozygous mutations with possibly a residual protein activity are instead associated with the less severe NBIA phenotype. Here we describe two INAD patients both with an unusually rapid disease progression and a peculiar neuroradiological presentation in one of them. Compound heterozygosity for a large intragenic deletion and a nonsense mutation was found in one of them while the other is carrying two novel splice-site mutations. Breakpoint-sequence analysis suggests a non-allelic-homologous-recombination (NAHR) event, probably underlying the rearrangement. These findings, while supporting the genotype-phenotype correlation already observed in INAD patients, provide the first sequence characterization of a genomic rearrangement in PLA2G6 gene, thus orienting the search for missing mutant alleles in PLA2G6 related diseases. PMID:20584031

Tonelli, A; Romaniello, R; Grasso, R; Cavallini, A; Righini, A; Bresolin, N; Borgatti, R; Bassi, M T

2010-11-01

180

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

181

Computational prediction of splicing regulatory elements shared by Tetrapoda organisms  

PubMed Central

Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research. PMID:19889216

Churbanov, Alexander; Vorechovsky, Igor; Hicks, Chindo

2009-01-01

182

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in Flaveria linearis.  

PubMed

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO(2)-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis. PMID:20882321

Slater, S M H; Micallef, M C; Zhang, J; Micallef, B J

2010-12-01

183

A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase alpha-subunit gene affects splicing.  

PubMed

We report the sixth mutation associated with the infantile form of Tay-Sachs disease in the Turkish population. The mutation is a single nucleotide transition (G to A) at the last nucleotide of exon 3 of hexosaminidase A (HEX A) alpha-subunit gene. The 14 exons and their flanking sequences of the HEX A gene were amplified and analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Sequencing of exon 3 showed a homozygous mutation. Cultured patient's fibroblasts produced no detectable mRNA for HEX A alpha-subunit gene by Northern blot analysis. We speculate that abnormal mRNA was rapidly degraded following transcription. Our data are consistent with the idea that the severe infantile form of Tay-Sachs disease is associated with a total lack of Hex A activity in the patient. A similar mutation (G to T) had been observed at the 5'-donor splice site of exon 3. It resulted in abnormal splicing and skipping of exon 3. The other acceptor and donor splice site mutations described in the HEX A gene ablate normal mRNA splicing. Identification of multiple mutant HEX A alleles shows molecular heterogeneity of infantile Tay-Sachs disease in our population. PMID:12689701

Ozkara, Hatice Asuman; Sandhoff, Konrad

2003-04-01

184

A general role for splicing enhancers in exon definition.  

PubMed Central

Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection. PMID:12403462

Lam, Bianca J; Hertel, Klemens J

2002-01-01

185

Modified free radial forearm fascia flap reconstruction of lower extremity and foot wounds: optimal contour and minimal donor-site morbidity.  

PubMed

Background?Free tissue transfer is commonly required for reconstruction of distal third lower extremity injuries. Injuries involving the dorsal surface of the foot require thin pliable flaps. Musculocutaneous flaps are often too bulky to accommodate shoewear. Fasciocutaneous flaps, while an improvement, need secondary contouring procedures. The modified radial forearm fascial flap (MRFFF) may offer an alternative. Methods?Twelve patients with distal third lower extremity wounds were reconstructed with MRFFF?+?split thickness skin graft. The modification in flap design leaves fascia radial to the pedicle unharvested, preserving sensibility of the dorsoradial aspect of the hand. Flaps were covered with a skin graft after inset. Donor sites were closed primarily. Results?Nine wounds were traumatic-five with exposed hardware, one burn, one diabetic ulcer, and one wound dehiscence following sarcoma resection?+?radiation. Out of 12, 11 limbs were salvaged at 1 to 2 years follow-up. All patients ambulated on the reconstructed leg and wore a shoe comfortably. Average time to weight bearing was 2 months. The donor site was limited to 25-cm scar on the volar forearm. No persistent motor/sensory deficits occurred in donor arms. Conclusion?MRFFF is an excellent flap for reconstruction of the distal lower extremity. Flap contour allows excellent shoe-fitting without secondary revisions. Replacement of the adipocutaneous flap on MRFFF donor site eliminates the need for a conspicuous donor-site skin graft. The ulnar orientation of the harvested fascia prevents sensory loss in the dorsal hand. The MRFFF provides the ideal replacement of "like with like" for selected distal lower extremity wounds. PMID:25184616

Medina, Miguel A; Salinas, Harry M; Eberlin, Kyle R; Driscoll, Daniel N; Kwon, John Y; Austen, William G; Cetrulo, Curtis L

2014-10-01

186

Receptor mutations and haplotypes in growth hormone receptor deficiency: a global survey and identification of the Ecuadorean E180splice mutation in an oriental Jewish patient.  

PubMed

Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel. PMID:7949594

Berg, M A; Peoples, R; Pérez-Jurado, L; Guevara-Aguirre, J; Rosenbloom, A L; Laron, Z; Milner, R D; Francke, U

1994-04-01

187

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

188

A novel mutation in the intron 1 splice donor site of the cholesterol ester transfer protein ( CETP) gene as a cause of hyperalphalipoproteinemia  

Microsoft Academic Search

The exchange of cholesterol ester (CE) between lipoproteins occurs through the action of cholesterol ester transfer protein (CETP). The human CETP gene is composed of 16 exons encompassing 25 kbp on chromosome 16q13. The objective of this study was to determine whether a mutation in the CETP gene accounted for severe hyperalphalipoproteinemia in an 80-year-old subject. As a secondary objective,

Tjin-Shing Jap; Yi-Chi Wu; Yi-Chu Tso; Chih-Yang Chiu

2002-01-01

189

Recognition of splice junctions on DNA sequences by BRAIN learning algorithm  

Microsoft Academic Search

Motivation: The problem addressed in this paper is theprediction of splice site locations in human DNA. The aimsof the proposed approach are explicit splicing rule description,high recognition quality, and robust and stable `oneshot\\

Salvatore Rampone

1998-01-01

190

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome  

PubMed Central

Wagner syndrome (WS) is an autosomal dominant vitreoretinopathy affecting various ocular features and is caused by mutations in the canonical splice sites of the VCAN gene, which encodes the large chondroitin sulfate proteoglycan, versican. We report the identification of novel splice acceptor and donor-site mutations (c.4004?1G>C and c.9265+2T>A) in two large WS families from France and the United Kingdom. To characterize their pathogenic mechanisms we performed qRT-PCR experiments on RNA from patient-derived tissues (venous blood and skin fibroblasts). We also analyzed RNA from the original Swiss family reported by Wagner (who has the previously reported c.9265+1G>A mutation). All three mutations resulted in a quantitative increase of transcript variants lacking exons 7 and/or 8. However, the magnitude of the increase varied between tissues and mutations. We discuss altered balance of VCAN splice variants in combination with reduction in glycosaminoglycan protein modifications as possible pathogenic mechanisms. PMID:22739342

Kloeckener-Gruissem, Barbara; Neidhardt, John; Magyar, Istvan; Plauchu, Henri; Zech, Jean-Christophe; Morle, Laurette; Palmer-Smith, Sheila M; MacDonald, Moira J; Nas, Veronique; Fry, Andrew E; Berger, Wolfgang

2013-01-01

191

Introduction to Cotranscriptional RNA Splicing  

PubMed Central

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process. PMID:24549657

Merkhofer, Evan C.; Hu, Peter; Johnson, Tracy L.

2014-01-01

192

Seroma and quilting suture at the donor site of the TRAM flap in breast reconstruction: a prospective randomized double-blind clinical trial.  

PubMed

Seroma formation at the donor site of the transverse rectus abdominis myocutaneous flap was evaluated in 48 patients who underwent breast reconstruction with either quilting sutures and suction drains (QS+DN group) or quilting sutures alone (QS group) or suction drains alone (DN group). Clinical and ultrasound examinations were performed to assess seroma formation in 5 regions of the abdominal wall on postoperative days 7 and 14. The incidence of seroma detected by ultrasound examination was significantly higher in the DN group (P = 0.008) than that in the other 2 groups. No difference in seroma volume (puncture) was found between the QS+DN and QS groups (P = 1.00). Seroma formation was observed in the iliac region in the DN group but not in the QS+DN and QS groups (P = 0.028). Quilting sutures at the transverse rectus abdominis myocutaneous flap donor site were efficient in reducing seroma formation. PMID:23407260

Rossetto, Luis Antonio; Garcia, Elvio Bueno; Abla, Luiz Eduardo Felipe; Ferreira, Lydia Masako

2014-04-01

193

A Hierarchical HMM Implementation for Vertebrate Gene Splice Site Mike Hu, Chris Ingram, Monica Sirski, Chris Pal, Sajani Swamy, Cheryl Patten  

E-print Network

in Genomic Sequences Introns are sequences of nucleotides within genes that do not code for amino acids remaining in the mRNA (called exons), which were joined after intron splicing, specify the amino acid, is involved in such processes as sex determination in fruit flies, and the synthe­ sis of alternate proteins

Pal, Chris

194

Morphomic analysis for preoperative donor site risk assessment in patients undergoing abdominal perforator flap breast reconstruction: a proof of concept study.  

PubMed

Background?Morphomics are three-dimensional measurements of aspects of the human anatomy generated by computed tomographic (CT) imaging. The purpose of this study was to generate preliminary data on the efficacy of morphomics, as a potential risk stratification tool, in predicting abdominal donor site wound-healing complications in patients undergoing abdominal perforator flap breast reconstruction. Patients and Methods?In total, 58 consecutive patients undergoing deep inferior epigastric perforator (DIEP) flap breast reconstruction were evaluated. Using preoperative CT scan data, we quantified patients' body area, visceral and subcutaneous fat, fascia area, and body depth between T12 and L4. Associations between morphomic measures and complication rates were examined using t-tests and logistic regression. Results?Of the 58 patients, 11 (19%) patients developed a wound dehiscence and 47 (81%) patients healed their abdominal incision without complications. Patients with a dehiscence had a significantly higher body mass index (BMI) (34.32 vs. 29.26 kg/m(2), p?=?0.014) than patients without a dehiscence. Multiple morphometric measures including higher visceral fat area (p?=?0.003) were significant predictors of abdominal donor site wound dehiscence. BMI (odds ratio [OR], 1.16; 95% confidence interval [CI], 1.03-1.32; p?=?0.017) and visceral fat area (OR, 1.24; 95% CI, 1.08-1.42; p?=?0.002) were independently significant predictors for wound dehiscence in the entire sample. Only visceral fat area retained its predictive ability in patients with a BMI?>?30 kg/m(2). Conclusions?Morphomic measurements correlate with the likelihood of developing postoperative donor site dehiscence after DIEP flap breast reconstruction. As a proof of concept study, this demonstrates that objective data obtained from CT scans may help in preoperatively assessing the risk for donor site wound healing complications in patients undergoing DIEP flap breast reconstruction. PMID:24911410

Levi, Benjamin; Rinkinen, Jacob; Kidwell, Kelley M; Benedict, Matthew; Stein, Isaac C; Lisiecki, Jeffrey; Enchakalody, Binu; Wang, Stewart C; Kozlow, Jeffrey H; Momoh, Adeyiza O

2014-11-01

195

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

SciTech Connect

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

Vermaas, W.F.J.; Rutherford, A.W.; Hansson, O. (Arizona State Univ., Tempe (USA))

1988-11-01

196

Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus  

PubMed Central

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that belongs to the Mononegavirales. Unlike other animal viruses of this order, BDV replicates and transcribes in the nucleus of infected cells. Previous studies have shown that BDV uses RNA splicing machinery for its mRNA expression. In the present study, we identified spliced RNAs that use an alternative 3? splice site, SA3, in BDV-infected cell lines as well as infected animal brain cells. Transient transfection analysis of cDNA clones of BDV RNA revealed that although SA3 is a favorable splice site in mammalian cells, utilization of SA3 is negatively regulated in infected cells. This negative splicing activity of the SA3 site is regulated by a putative cis-acting region, the exon splicing suppressor (ESS), within the polymerase exon of BDV. The BDV ESS contains similar motifs to other known ESSs present in viral and cellular genes. Furthermore, our results indicated that a functional polyadenylation signal just upstream of the BDV ESS is also involved in the regulation of alternative splicing of BDV. These observations represent the first documentation of complex RNA splicing in animal RNA viruses and also provide new insight into the mechanism of regulation of alternative splicing in animal viruses. PMID:11070091

Tomonaga, Keizo; Kobayashi, Takeshi; Lee, Byeong- Jae; Watanabe, Makiko; Kamitani, Wataru; Ikuta, Kazuyoshi

2000-01-01

197

Alteration of introns in a hyaluronan synthase 1 (HAS1) minigene convert Pre-mRNA [corrected] splicing to the aberrant pattern in multiple myeloma (MM): MM patients harbor similar changes.  

PubMed

Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors. PMID:23301075

Kriangkum, Jitra; Warkentin, Amanda; Warkinton, Amanda; Belch, Andrew R; Pilarski, Linda M

2013-01-01

198

Alteration of Introns in a Hyaluronan Synthase 1 (HAS1) Minigene Convert Pre-Mrna Splicing to the Aberrant Pattern in Multiple Myeloma (MM): MM Patients Harbor Similar Changes  

PubMed Central

Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3?splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3? splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors. PMID:23301075

Kriangkum, Jitra; Warkinton, Amanda; Belch, Andrew R.; Pilarski, Linda M.

2013-01-01

199

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

PubMed

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

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

2014-10-01

200

The splicing landscape is globally reprogrammed during male meiosis.  

PubMed

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

2013-12-01

201

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

202

Free hand-to-toe transfer: a method to minimise donor-site morbidity in free joint transfers.  

PubMed

Reconstruction of a congenital hand anomaly in a child using single free vascularised transfer of the proximal interphalangeal joint of a second toe with the simultaneous microvascular reconstruction of the donor toe using the stiff joint and its dorsal skin paddle from the hand is described. This is not the first reported case of a toe-finger switch, but it is the first in a free joint transfer, for which it is especially indicated. PMID:12706156

Schenker, M; Kelley, S P; Kay, S P J

2003-01-01

203

Branch point selection in alternative splicing of tropomyosin pre-mRNAs.  

PubMed

The rat tropomyosin 1 gene gives rise to two mRNAs encoding rat fibroblast TM-1 and skeletal muscle beta-tropomyosin via an alternative splicing mechanism. The gene is comprised of 11 exons. Exons 1 through 5 and exons 8 and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle whereas exons 7 and 10 are used exclusively in skeletal muscle. In the present studies we have focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). To study the mechanism and regulation of alternative splice site selection we have characterized the branch points used in processing of the tropomyosin pre-mRNAs in vitro using nuclear extracts obtained from HeLa cells. Splicing of exon 5 to exon 6 (fibroblast-type splice) involves the use of three branch points located 25, 29, and 36 nucleotides upstream of the 3' splice site of exon 6. Splicing of exon 6 (fibroblast-type splice) or exon 7 (skeletal muscle type-splice) to exon 8 involves the use of the same branch point located 24 nucleotides upstream of this shared 3' splice site. In contrast, the splicing of exon 5 to exon 7 (skeletal muscle-type splice) involves the use of three branch sites located 144, 147 and 153 nucleotides, upstream of the 3' splice site of exon 7. In addition, the pyrimidine content of the region between these unusual branch points and the 3' splice site of exon 7 was found to be greater than 80%. These studies raise the possibility that the use of branch points located a long distance from a 3' splice site may be an essential feature of some alternatively spliced exons. The possible significance of these unusual branch points as well as a role for the polypyrimidine stretch in intron 6 in splice site selection are discussed. PMID:2762151

Helfman, D M; Ricci, W M

1989-07-25

204

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

205

An intron 9 containing splice variant of PAX2  

PubMed Central

Background PAX2 is a transcription factor with an important role in embryogenic development. However, PAX2 expression was frequently identified in neoplasia responsible for the growth and survival of cancer cells. Due to alternative splicing of exon 6, exon 10 and exon 12 four isoforms of PAX2 are described so far. Methods The expression of an intron 9 containing PAX2 splice variant was analyzed in neoplastic B cell and solid tumor cell lines as well as in primary tumor samples by quantitative RT-PCR. PAX2 proteins were detected by Western Blot in a subset of cell lines. Results All 14 lymphoma cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence. This splice variant could also be detected in 35 solid tumor cell lines, in leukemia and lymphoma as well as in colon carcinoma and melanoma patient samples and in blood samples of healthy donors. Expression of this new splice variant on protein level was verified by Western Blot analysis. Conclusion We discovered a previously undescribed intron 9 and exon 10 containing splice variant of PAX2 in B-cell neoplasia and in solid tumors on mRNA and protein level. PMID:19467152

Busse, Antonia; Rietz, Anika; Schwartz, Stefan; Thiel, Eckhard; Keilholz, Ulrich

2009-01-01

206

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

207

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

PubMed

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

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

2013-03-14

208

Positive and negative intronic regulatory elements control muscle-specific alternative exon splicing of Drosophila myosin heavy chain transcripts.  

PubMed Central

Alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated to ensure the expression of specific MHC isoforms required for the distinctive contractile activities of physiologically specialized muscles. We have used transgenic expression analysis in combination with mutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11, which is encoded by five alternative exons that produce alternative "converter" domains in the MHC head. Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that control splicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function: CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elements function in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e. An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of other alternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elements establish the muscle-specific splicing of alternative exon 11s. PMID:11139507

Standiford, D M; Sun, W T; Davis, M B; Emerson, C P

2001-01-01

209

Feasibility of Use of a Barbed Suture (V-Loc 180) for Quilting the Donor Site in Latissimus Dorsi Myocutaneous Flap Breast Reconstruction  

PubMed Central

Background Latissimus dorsi (LD) myocutaneous flap is a popular method of breast reconstruction which can be associated with high incidence of seroma formation. Quilting sutures at the harvest site are used to reduce this. Barbed sutures are self anchoring sutures which avoid multiple knotting and can be useful in quilting. Methods A retrospective analysis of prospectively maintained database of patients who underwent LD flap breast reconstruction between January 2009 and January 2011 was carried out. Seroma formation at the harvest site, wound related complications, inpatient stay and duration of surgery were analysed and a comparison was made between two groups where quilting was done with barbed (V-Loc) suture and conventional polydioxanone (PDS) II sutures. Results Fifty-seven patients were included of which 33 had quilting by V-Loc sutures and in 24 patients PDS II suture was used. Median age in the PDS group was 55 years (interquartile range [IQR)], 45 to 61 years) which was comparable to the V-Loc group (53 years [IQR, 48 to 59 years]; P-value 0.948). Sixteen patients (28%) had significant seroma formation and 5 (9%) patients developed superficial wound dehiscence. Incidences of seroma or wound complications were comparable (P-value 0.378 and 1.00, respectively). Secondary outcomes such as total duration of surgery, total inpatient stay, total amount of drain at the donor site were also similar in two groups. Conclusions Use of barbed sutures for quilting the donor site in LD flap reconstruction is a feasible option and the associated seroma formation and wound complications are comparable with conventional sutures. PMID:23532830

Hussain, Tasadooq; Mahapatra, Tapan Kumar; McManus, Penelope Louise; Kneeshaw, Peter John

2013-01-01

210

A Splice Mutation and mRNA Decay of EXT2 Provoke Hereditary Multiple Exostoses  

PubMed Central

Background Hereditary multiple exostoses (HME) is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2, which are responsible for over 70% of HME cases. However, the pathological significance of the majority of these mutations is often unclear. Methods In a Chinese family with HME, EXT1 and EXT2 genes were screened by direct sequencing. The consequence of a detected mutant was predicted by in silico analysis and confirmed by mRNA analysis. The EXT1 and EXT2 mRNA and protein levels and the HS patterns in the HME patients were compared with those in healthy controls. Results A heterozygous transition (c.743+1G>A) in the EXT2 gene, which co-segregated with the HME phenotype in this family, was identified. The G residue at position +1 in intron 4 of EXT2 was predicted to be a 5? donor splice site. The mRNA analysis revealed an alternative transcript with a cryptic splice site 5 bp downstream of the wild-type site, which harbored a premature stop codon. However, the predicted truncated protein was not detected by western blot analysis. Decay of the mutant mRNA was shown by clone sequencing and quantification analysis. The corresponding downregulation of the EXT2 mRNA will contribute to the abnormal EXT1/EXT2 ratio and HS pattern that were detected in the patients with HME. Conclusion The heterozygous mutation c.743+1G>A in the EXT2 gene causes HME as a result of abnormal splicing, mRNA decay, and the resulting haploinsufficiency of EXT2. PMID:24728384

Tian, Chen; Yan, Rengna; Wen, Shuzhen; Li, Xueling; Li, Tianfeng; Cai, Zhenming; Li, Xinxiu; Du, Hong; Chen, Huimei

2014-01-01

211

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

212

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

PubMed Central

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

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

2001-01-01

213

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

214

Mdm2 Splice Variants.  

National Technical Information Service (NTIS)

The invention provides polypeptide and polynucleotide splice variants of the mouse Mdm2 gene, including Mdm2-b, which is homologous to the human Hdm2-b variant, as well as host cells, vectors and transgenic mice comprising the variants, and methods for th...

S. N. Jones, H. Steinman

2004-01-01

215

The transcription factor c-Myb affects pre-mRNA splicing.  

PubMed

c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF(65) and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing. PMID:18498763

Orvain, Christophe; Matre, Vilborg; Gabrielsen, Odd S

2008-07-25

216

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

217

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

218

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.

2013-07-08

219

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

PubMed Central

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

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

2008-01-01

220

Copper-Sulfur Complexes Supported by N-Donor Ligands: Towards Models of the Cu(Z) Site in Nitrous Oxide Reductase.  

PubMed

The distinctive structure of the [(his)(7)Cu(4)(?-S)](n+) cluster in the "Cu(Z)" active site of nitrous oxide reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper-sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper-sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper-sulfur bonding interactions in the complexes. PMID:19262681

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

2008-03-01

221

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

PubMed Central

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

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

1998-01-01

222

Retroperitoneal LESS donor nephrectomy.  

PubMed

Donor nephrectomy with laparo-endoscopic single site (LESS) surgery has been reported via the transperitoneal approach. We describe a novel technique of retroperitoneal donor nephrectomy using a single surgical incision in the groin, below the abdominal skin crease or "bikini line". The LESS groin incision offers superior cosmesis, while the retroperitoneal approach has distinct advantages, such as the ability to identify the renal vessels early. The new procedure has been performed in two obese patients (body mass index 32 and 33 kg/m2, respectively). The operative times were 4 and 5 hours, warm ischemic times 135 and 315 seconds, blood loss 100 and 250 mL, and hospitalization 3 and 2 days, respectively. Retroperitoneal LESS donor nephrectomy through a single, inconspicuous groin incision is feasible and safe. Further evaluation of the technique in a larger patient cohort is indicated. PMID:21044377

van der Merwe, A; Bachmann, A; Heyns, C F

2010-01-01

223

Identification of motifs that function in the splicing of non-canonical introns  

PubMed Central

Background While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment. Results Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts. Conclusion The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif. PMID:18549497

Murray, Jill I; Voelker, Rodger B; Henscheid, Kristy L; Warf, M Bryan; Berglund, J Andrew

2008-01-01

224

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.  

PubMed Central

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA. PMID:11421362

Hastings, M L; Wilson, C M; Munroe, S H

2001-01-01

225

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

226

Splicing Wires Permanently With Explosives  

NASA Technical Reports Server (NTRS)

Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

Bement, Laurence J.; Kushnick, Anne C.

1990-01-01

227

Donor Tag Game  

MedlinePLUS

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

228

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

PubMed Central

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

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

1999-01-01

229

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

230

The Intronic GABRG2 Mutation, IVS6+2T->G, Associated with CAE Altered Subunit mRNA Intron Splicing, Activated Nonsense-Mediated Decay and Produced a Stable Truncated ?2 Subunit  

PubMed Central

The intronic GABRG2 mutation, IVS6+2T?G, was identified in an Australian family with childhood absence epilepsy (CAE) and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wildtype and mutant ?2S subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wildtype and mutant ?2S subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wildtype and mutant GABRG2 mRNA splicing patterns were determined in both BAC transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention and produced a frame shift in exon 7 that created a premature translation-termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense mediated mRNA decay (NMD) or translated to a stable, truncated subunit (the ?2-PTC subunit) containing the first 6 GABRG2 exons and a novel frame-shifted 29 aa C terminal tail. The ?2-PTC subunit was homologous to the mollusk acetylcholine binding protein (AChBP) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with ?1 and ?2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T?G, reduced surface ???2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant negative effect on ???2 receptor assembly. PMID:22539854

Tian, Mengnan; Macdonald, Robert L.

2012-01-01

231

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

PubMed

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

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

2012-01-01

232

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

233

A splice site mutation in the methyltransferase gene FTSJ1 in Xp11.23 is associated with non-syndromic mental retardation in a large Belgian family (MRX9).  

PubMed

Mental retardation is the most frequent cause of serious handicap in children and young adults. The underlying causes of this heterogeneous condition are both acquired and genetically based. A recently performed refinement of the linkage interval in a large Belgian family with mild to severe non-syndromic X linked mental retardation, classified as MRX9, revealed a candidate region of 11.3 Mb between markers DXS228 and DXS1204 on the short arm of the X chromosome. In order to identify the underlying disease gene in the MRX9 family, we established a gene catalogue for the candidate region and performed comprehensive mutation analysis by direct sequencing. A human homologue of the bacterial 23S rRNA methyltransferase Fstj, the FTSJ1 gene, is located within this region and displayed a sequence alteration in the conserved acceptor splice site of intron 3 (IVS3-2A>G) in all tested patients and carrier females of this family. In contrast, it was absent in all unaffected male family members tested. The mutation results in skipping of exon 4 and introduces a premature stop codon in exon 5, probably leading to a severely truncated protein. Our finding indicates that a protein, possibly associated with ribosomal stability, can be linked to X linked mental retardation (XLMR). PMID:15342698

Ramser, J; Winnepenninckx, B; Lenski, C; Errijgers, V; Platzer, M; Schwartz, C E; Meindl, A; Kooy, R F

2004-09-01

234

Trans-splicing with the group I intron ribozyme from Azoarcus.  

PubMed

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme. PMID:24344321

Dolan, Gregory F; Müller, Ulrich F

2014-02-01

235

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

236

Alternative splicing of TAF6: downstream transcriptome impacts and upstream RNA splice control elements.  

PubMed

The TAF6? pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6? is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6? has been shown to be a pivotal event in triggering death via the TAF6? pathway, yet nothing is currently known about the mechanisms that promote TAF6? splicing. Furthermore the transcriptome impact of the gain of function of TAF6? versus the loss of function of the major TAF6? splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6? drives a transcriptome profile distinct from that resulting from depletion of TAF6?. To define the cis-acting RNA elements responsible for TAF6? alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6? and also reveal a role for RNA secondary structure in the selection of TAF6?. PMID:25025302

Kamtchueng, Catherine; Stébenne, Marie-Éve; Delannoy, Aurélie; Wilhelm, Emmanuelle; Léger, Hélène; Benecke, Arndt G; Bell, Brendan

2014-01-01

237

Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo.  

PubMed

A single rat gene encodes both fibroblast TM-1 and skeletal muscle beta-tropomyosin by an alternative RNA-processing mechanism. The gene contains 11 exons: Exons 1-5 and exons 8 and 9 are constitutive exons common to all mRNAs expressed from this gene; exons 6 and 11 are used in fibroblasts as well as smooth muscle; exons 7 and 10 are used exclusively in skeletal muscle. We have studied the internal alternative RNA splice choice (exons 6 and 7) of the rat tropomyosin 1 gene in vitro, using nuclear extracts obtained from HeLa cells. Use of alternative splice sites in vitro is dependent on the ionic conditions of the assay, and correct splicing occurs only under well-defined salt conditions. Splicing of exon 5 to exon 6 (fibroblast-type splice) and exon 5 to exon 7 (skeletal muscle-type splice) was dependent on precursors in which exon 6 or 7 was first joined to exon 8. The same patterns of alternatively spliced RNAs were formed when similar templates were introduced in HeLa cells by transfection. Thus, there appears to be an ordered pathway of splicing in which the internal alternatively spliced exons must first be joined to the downstream constitutive exon before they can be spliced to the upstream constitutive exon. The data are consistent with a model in which the critical event in alternative splicing occurs during the joining of exon 6 to exon 8 (fibroblast-type splice) or exon 7 to exon 8 (skeletal muscle-type splice). PMID:3215513

Helfman, D M; Ricci, W M; Finn, L A

1988-12-01

238

Molecular characterization of two novel mutations causing factor XI deficiency: A splicing defect and a missense mutation responsible for a CRM+ defect.  

PubMed

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency. PMID:18327400

Guella, Ilaria; Soldà, Giulia; Spena, Silvia; Asselta, Rosanna; Ghiotto, Rossella; Tenchini, Maria Luisa; Castaman, Giancarlo; Duga, Stefano

2008-03-01

239

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

PubMed

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. PMID:22205025

Ramirez-Gomez, Francisco; Greene, Whitney; Rego, Katherine; Hansen, John D; Costa, Greg; Kataria, Priti; Bromage, Erin S

2012-02-01

240

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

241

Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA.  

PubMed Central

The genome of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains a functional gene denominated x-lor that may be important in HTLV-I transformation of human T cells. To study the role of x-lor and other HTLV-I genes in cellular transformation, we obtained a transformed nonproducer human T-cell line containing a single defective HTLV-I provirus (HTLV-I 55/PL). This 7-kilobase provirus had undergone a deletion involving the entire envelope gene and the nonconserved region. The point of the deletion corresponded to the junction of a donor splice site, located between the polymerase gene and the envelope gene (nucleotide 5183), and the acceptor site for the mRNA of the x-lor gene (nucleotide 7302). The juxtaposition of nucleotides 5182 and 7302 brings the initiating methionine codon of the envelope gene immediately 5' to the x-lor region, leaving the DNA sequence in frame for expression of a protein product. This finding suggests that a double splicing mechanism is used to express the x-lor gene, and that the defective provirus 55/PL was generated through the reverse transcription of a partially spliced mRNA. Analysis of the x-lor mRNA of other HTLV-I-transformed cell lines revealed that a double splicing process is commonly used. Furthermore, since 55/PL can be faithfully transmitted and is able to immortalize recipient T cells, we can conclude that the envelope gene is not necessary for in vitro transformation by HTLV-I. Images PMID:3001724

Aldovini, A; De Rossi, A; Feinberg, M B; Wong-Staal, F; Franchini, G

1986-01-01

242

Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains.  

PubMed

In order to study the potential role of the steroid molting hormone (20-hydroxyecdysone) in regulating molt-induced claw muscle atrophy, full-length cDNAs encoding retinoid-X receptor (Gl-RXR) and E75 early ecdysone inducible gene (Gl-E75) were obtained from land crab (Gecarcinus lateralis) skeletal muscle mRNA using RT-PCR and 3' and 5' RACE. Gl-E75A (3528bp), which encoded a protein of 828 amino acids, had highest sequence identity to Me-E75A from a shrimp (Metapenaeus ensis). It was expressed in skeletal muscle and gonads. The deduced amino acid sequence of Gl-RXR was highly similar to that of the fiddler crab RXR (Up-RXR) and insect ultraspiracle (USP). Nine variant sequences occurred in Gl-RXR mRNAs at three alternative splicing sites, one in the "T box" in the linker D domain and two in the ligand-binding domain (LBD). The three T-box variants, termed T(+8), T(+7), and T(+12), contained insertions of 8, 7, or 12 amino acids, respectively. Four variants were generated at the first site in the LBD. Two of the LBD site 1 variants differed in the presence (+33) or absence (-33) of a 33-amino acid sequence; the other two were LBD truncations with or without the 33 amino acid sequence (+33DeltaE/F and -33DeltaE/F, respectively). Two variants differing in the presence (+35) or absence (-35) of a 35-amino acid sequence were generated at the second site in the LBD. The Gl-RXRa isoform (1516 bp) with the longest open reading frame (+12/+33/+35) encoded a protein of 436 amino acids. Thoracic muscle expressed only isoforms with the T(+12) sequence. In contrast, claw muscle expressed isoforms with T(+7) or T(+12) and fewer isoforms with T(+8). Ovary and testis expressed a greater number of RXR isoforms than skeletal muscle. All tissues expressed full-length and truncated RXR isoforms. These data suggest that differences in response of claw and thoracic muscles to elevated ecdysteroid are due in part to differences in the expression of RXR isoforms. PMID:16150535

Kim, Hyun-Woo; Lee, Sung Gu; Mykles, Donald L

2005-10-20

243

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

244

A splice junction mutation in muscle carnitine palmitoyltransferase II deficiency.  

PubMed

We report the first splice junction mutation to be described in the carnitine palmitoyltransferase (CPT) 2 gene in a patient with the muscle form of CPT II deficiency. The patient, a 25-year-old man, suffered from attacks of myalgia and muscle weakness in early adult life. There was biochemical evidence of CPT II deficiency. Molecular genetic analysis revealed the common S113L mutation on one allele whilst a novel mutation at the splice donor junction in intron 3 was identified on the other allele. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products clearly demonstrated that this mutation causes the skipping of exon 3, thus establishing its pathogenic role. PMID:12809643

Deschauer, Marcus; Chrzanowska-Lightowlers, Zofia M A; Biekmann, Eckhard; Pourfarzam, Morteza; Taylor, Robert W; Turnbull, Douglass M; Zierz, Stephan

2003-06-01

245

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

PubMed Central

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

2014-01-01

246

Manipulation of alternative splicing by a newly developed inhibitor of Clks.  

PubMed

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing. PMID:15010457

Muraki, Michiko; Ohkawara, Bisei; Hosoya, Takamitsu; Onogi, Hiroshi; Koizumi, Jun; Koizumi, Tomonobu; Sumi, Kengo; Yomoda, Jun-ichiro; Murray, Michael V; Kimura, Hiroshi; Furuichi, Kiyoshi; Shibuya, Hiroshi; Krainer, Adrian R; Suzuki, Masaaki; Hagiwara, Masatoshi

2004-06-01

247

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

248

Frequent Gain and Loss of Intronic Splicing Regulatory Elements during the Evolution of Vertebrates  

PubMed Central

Splicing regulatory elements (SREs) are sequences bound by proteins that influence splicing of nearby splice sites. Constitutively spliced introns have evolved to utilize many different splicing factors. The evolutionary processes that influenced which splicing factors are used for splicing of individual introns are generally unclear. We demonstrate that in the lineage that gave rise to mammals, many introns lost U-rich sequences and gained G-rich sequences, both of which resemble known SREs. The apparent conversion of U-rich to G-rich SREs suggests that the associated splicing factors are functionally equivalent. In support of this we demonstrated that U-rich and G-rich SREs are both capable of promoting splicing of an SRE-dependent splicing reporter. Furthermore, we demonstrate, using the heterologous MS2 tethering system (bacterial MS2 coat fusion-protein and its RNA stem-loop binding site), that both the U-rich SRE-binding protein (TIA1) and the G-rich SRE-binding protein (HNRNPF) can promote splicing of the same intron. We also observed that gain of G-rich SREs is significantly associated with G/C-rich genomic isochores, suggesting that gain or loss of SREs was driven by the same processes that ultimately resulted in the formation of mammalian genomic isochores. We propose the following model for the gain and loss of mammalian SREs. Ancestral U-rich SREs located in genomic regions that were experiencing high rates of A/T to G/C conversion would have suffered frequent deleterious mutations. However, this same process resulted in increased formation of functionally equivalent G-rich SREs, and acquisition of new G-rich SREs decreased purifying selection on the U-rich SREs, which were then free to decay. PMID:22619362

Voelker, Rodger B.; Erkelenz, Steffen; Reynoso, Vinicio; Schaal, Heiner; Berglund, J. Andrew

2012-01-01

249

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.  

PubMed

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster. PMID:2307372

Helfman, D M; Roscigno, R F; Mulligan, G J; Finn, L A; Weber, K S

1990-01-01

250

Fatigue and corrosion in aircraft pressure cabin lap splices  

Microsoft Academic Search

Aircraft structures are susceptible to fatigue and corrosion damage, notably at joints. There may be interactions between fatigue and corrosion, especially as aircraft age. Longitudinal lap splices from several types of transport aircraft pressure cabins were disassembled and investigated for Multiple Site Damage (MSD) fatigue cracking and corrosion. The results are compared with NASA data for a full-scale fatigue test.

R. J. H Wanhill; M. F. J Koolloos

2001-01-01

251

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

PubMed Central

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

2014-01-01

252

Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage? †  

PubMed Central

We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3? splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3?-end processing of this exon can be distinguished. First, the 3? splice site establishes connections to enhance 3?-end processing, while the nascent 3?-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3?-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3? end, and then finishing with splicing of the last intron. PMID:17967872

Rigo, Frank; Martinson, Harold G.

2008-01-01

253

Functional coupling of last-intron splicing and 3'-end processing to transcription in vitro: the poly(A) signal couples to splicing before committing to cleavage.  

PubMed

We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3' splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3'-end processing of this exon can be distinguished. First, the 3' splice site establishes connections to enhance 3'-end processing, while the nascent 3'-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3'-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3' end, and then finishing with splicing of the last intron. PMID:17967872

Rigo, Frank; Martinson, Harold G

2008-01-01

254

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

PubMed

X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the QA site semiquinone (SQA) with nitrogens from the local protein environment in natural abundance (14)N and in (15)N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 N? and Ala-M260 peptide nitrogen (Np) were estimated through simultaneous simulation of the Q-band (15)N Davies ENDOR, X- and Q-band (14,15)N HYSCORE, and X-band (14)N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a((14)N) = 2.3 MHz, T = 0.3 MHz for His-M219 N? and a((14)N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 N? is established as the stronger of the two H-bond donors, Ala-M260 Np is found to have the larger value of a((14)N). The nuclear quadrupole coupling constants were estimated as e(2)Qq/4h = 0.38 MHz, ? = 0.97 and e(2)Qq/4h = 0.74 MHz, ? = 0.59 for His-M219 N? and Ala-M260 Np, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes-Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter ? to be a sensitive probe of the histidine N?-semiquinone hydrogen bond strength. This is supported by a strong correlation observed between ? and the isotropic coupling constant a((14)N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a((14)N) and ? will provide an important structural characterization tool in future studies of semiquinone-binding proteins. PMID:25026433

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

2014-08-01

255

Splign: algorithms for computing spliced alignments with identification of paralogs  

PubMed Central

Background The computation of accurate alignments of cDNA sequences against a genome is at the foundation of modern genome annotation pipelines. Several factors such as presence of paralogs, small exons, non-consensus splice signals, sequencing errors and polymorphic sites pose recognized difficulties to existing spliced alignment algorithms. Results We describe a set of algorithms behind a tool called Splign for computing cDNA-to-Genome alignments. The algorithms include a high-performance preliminary alignment, a compartment identification based on a formally defined model of adjacent duplicated regions, and a refined sequence alignment. In a series of tests, Splign has produced more accurate results than other tools commonly used to compute spliced alignments, in a reasonable amount of time. Conclusion Splign's ability to deal with various issues complicating the spliced alignment problem makes it a helpful tool in eukaryotic genome annotation processes and alternative splicing studies. Its performance is enough to align the largest currently available pools of cDNA data such as the human EST set on a moderate-sized computing cluster in a matter of hours. The duplications identification (compartmentization) algorithm can be used independently in other areas such as the study of pseudogenes. Reviewers This article was reviewed by: Steven Salzberg, Arcady Mushegian and Andrey Mironov (nominated by Mikhail Gelfand). PMID:18495041

Kapustin, Yuri; Souvorov, Alexander; Tatusova, Tatiana; Lipman, David

2008-01-01

256

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries  

PubMed Central

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ? subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G?T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing. PMID:16061559

Ohno, K; Tsujino, A; Shen, X; Milone, M; Engel, A

2005-01-01

257

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.

258

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, Maria E.; Eckhold, Juliane; Puisac, Beatriz; Dalski, Andreas; Gil-Rodriguez, Maria C.; Braunholz, Diana; Baquero, Carolina; Hernandez-Marcos, Maria; de Karam, Juan C.; Ciero, Milagros; Santos-Simarro, Fernando; Lapunzina, Pablo; Wierzba, Jolanta; Casale, Cesar H.; Ramos, Feliciano J.; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J.; Pie, Juan

2014-01-01

259

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.  

PubMed

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

260

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

261

Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors  

PubMed Central

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1?, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3? splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ?20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. PMID:23152763

Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

2012-01-01

262

Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments.  

PubMed

Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting. PMID:17698277

Englert, Markus; Latz, Andreas; Becker, Dirk; Gimple, Olaf; Beier, Hildburg; Akama, Kazuhito

2007-11-01

263

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

264

TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription  

PubMed Central

Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-xS 5? splice site. PMID:22158966

Montes, Marta; Cloutier, Alexandre; Sánchez-Hernández, Noemí; Michelle, Laetitia; Lemieux, Bruno; Blanchette, Marco; Hernández-Munain, Cristina; Chabot, Benoit

2012-01-01

265

Promoter usage and alternative splicing.  

PubMed

Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation. PMID:15901495

Kornblihtt, Alberto R

2005-06-01

266

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

Grodecka, Lucie; Lockerova, Pavla; Ravcukova, Barbora; Buratti, Emanuele; Baralle, Francisco E.; Dusek, Ladislav; Freiberger, Tomas

2014-01-01

267

An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing  

PubMed Central

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

2010-01-01

268

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

269

Some Characterizations in Splicing Systems  

NASA Astrophysics Data System (ADS)

The splitting and recombinant of deoxyribonucleic acid or DNA by specified enzymes using concepts in Formal Language Theory was first mathematically modeled by Head in 1987. This splicing system, S can be presented as a set of initial string I over an alphabet A that acts upon 5' or 3' overhangs of restriction enzymes and can be simply viewed as S = (A, I, B, C). In this paper, a great interest in presenting some relations on certain types of splicing system namely null-context, uniform, simple, semi-simple, semi-null and Sk based on differentiating their rules are given as proposition, corollaries and counterexamples.

Sarmin, Nor Haniza; Yusof, Yuhani; Wan Heng, Fong

2010-11-01

270

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

PubMed Central

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

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

2012-01-01

271

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron  

PubMed Central

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation. PMID:19920126

Elina, Helen; Brown, Gregory G.

2010-01-01

272

Alternative Splicing: Therapeutic Target and Tool  

Microsoft Academic Search

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for ther- apy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense

Mariano A. Garcia-Blanco

273

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

274

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

275

Donor Return Following Temporary Deferral  

PubMed Central

Background The consequences of temporary pre-donation donor deferrals are unsatisfactorily understood. Previous studies have found that deferral negatively impacts future return for donation in both first time and repeat donors. However, the applicability of these findings across centers has not been established. Methods Using a cohort design, presenting donors with a temporary deferral in the years 2006 – 2008 in 1 of 6 categories (low hematocrit, blood pressure or pulse, feeling unwell, malaria travel, tattoos/piercing and related exposures, or couldn’t wait/second thoughts) were passively followed for up to a 3-year period for the time to first return after the expiration of the deferral at 6 US blood centers. Time-to-event methods were used to assess return following the receipt of each deferral. We also analyzed which donor characteristics are associated with return following temporary deferral using multivariable logistic regression. Results Of 3.9 million donor presentations, 505,623 resulted in deferral in 1 of the 6 categories. Low hematocrit was the most common deferral, had the shortest median time to return, and largest cumulative number of donors returning. Deferrals of shorter duration had better return. Longer term deferrals (up to 1-year in length) had the lowest cumulative return which did not exceed 50% during the study period for malaria travel or tattoo/piercing and related exposures. In multivariable logistic regression modeling, return following deferral was associated with previously identified factors such as repeat donor status, older age, and higher educational attainment regardless of the type of deferral. In addition, return was associated with having been born in the USA, Asian race/ethnicity, and donation at fixed sites regardless of the type of deferral. Conclusion The category of temporary deferral influences the likelihood of future return, but the demographic and donation factors associated with return are consistent regardless of the deferral. PMID:21155833

Custer, Brian; Schlumpf, Karen S; Wright, David; Simon, Toby; Wilkinson, Susan; Ness, Paul

2012-01-01

276

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

277

Donor Cell Myeloid Sarcoma  

PubMed Central

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

Walshauser, Mark A.; Sojitra, Payal

2014-01-01

278

Fine-Scale Variation and Genetic Determinants of Alternative Splicing across Individuals  

PubMed Central

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases. PMID:20011102

Coulombe-Huntington, Jasmin; Lam, Kevin C. L.; Dias, Christel; Majewski, Jacek

2009-01-01

279

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

280

RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.  

PubMed

Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility. PMID:10413481

Saldanha, R; Chen, B; Wank, H; Matsuura, M; Edwards, J; Lambowitz, A M

1999-07-13

281

Nuclear pre-mRNA Compartmentalization: Trafficking of Released Transcripts to Splicing Factor Reservoirs  

PubMed Central

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein–Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors. PMID:10679009

Melcak, Ivo; Cermanova, Stepanka; Jirsova, Katerina; Koberna, Karel; Malinsky, Jan; Raska, Ivan

2000-01-01

282

Transcriptional expression of cis-acting and trans-acting splicing mutations cause autosomal dominant retinitis pigmentosa.  

PubMed

Two types of mutations may lead to deficient pre-mRNA splicing: cis-acting mutations that inactivate a constitutive or alternative splice site within the pre-mRNA, and trans-acting mutations that affect the function of a basal factor of the splicing machinery. Autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in at least 12 genes, with mutations in rhodopsin being the most prevalent. Two cis-acting mutations, g.3811A>G and g.5167G>T at the splice site in the rhodopsin gene (RHO; GenBank U49742.1) are linked to adRP in a Spanish population; while a cis-acting mutation, g.4335G>T, has been linked to recessive RP (arRP). Transcriptional expression analysis showed that the cis-acting splicing mutations linked to adRP promoted alternative splice sites, while the arRP linked mutation results in exclusion of exon 4. Trans-acting splicing mutations associated with adRP have also been found, and mutations in the pre-mRNA splicing factors PRPF3, PRPF8, PRPF31, and RP9 are associated with adRP in several populations. This report describes a new mutation in PRPF3 in a Spanish adRP family. We also investigated the transcriptional patterns in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells from patients carrying a mutation in PRPF8. Despite the role of PRPF8 in the minor U12 splicing processes, microarray analysis revealed that mutations in PRPF8 not only did not result in significant differences in splicing efficiency of rhodopsin, but no apparent changes in expression of U12-type intron genes and splicing processes was observed. Microarray analysis revealed a panel of differentially expressed genes mapped to the RP loci, and future work will determine their role in RP. PMID:18412284

Gamundi, María José; Hernan, Imma; Muntanyola, Marta; Maseras, Miquel; López-Romero, Pedro; Alvarez, Rebeca; Dopazo, Ana; Borrego, Salud; Carballo, Miguel

2008-06-01

283

Atypical Splicing of the Latency-Associated Transcripts of Herpes Simplex Type 1  

Microsoft Academic Search

We previously have shown that two latency-associated transcripts (LATs) of herpes simplex type 1 (HSV-1) are probably lariats, produced during splicing. By RNaseH digestion analysis, we now show that the major branchpoint of the 2.0-kb LAT was within 46 nt 5? of the splice acceptor site. A more detailed mapping by primer extension revealed the branchpoint as an adenosine 29

Ting-Ting Wu; Ying-Hsiu Su; Timothy M. Block; John M. Taylor

1998-01-01

284

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation  

PubMed Central

The molecular basis of cell signal-regulated alternative splicing at the 3? splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3? splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3? splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3? splice site usage. PMID:22684629

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G.; Xie, Jiuyong

2012-01-01

285

An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing  

Microsoft Academic Search

BACKGROUND: Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each

Keven R Johnson; Jessie Nicodemus-Johnson; Robert S Danziger

2010-01-01

286

Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons  

PubMed Central

Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3? and 5? splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

2012-01-01

287

Functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway.  

PubMed Central

SR proteins play critical roles in the major pre-mRNA splicing pathway. A second pathway processes U12-dependent AT-AC introns. We demonstrate, by biochemical complementation, the requirement for SR proteins in splicing of AT-AC introns. Whereas SR proteins were sufficient to activate splicing of a P120 AT-AC intron, splicing of a sodium channel AT-AC intron required an additional nuclear fraction. Individual recombinant SR proteins promoted splicing of both substrates, but displayed marked preferences. SR proteins supported basal AT-AC splicing, and also splicing stimulation via a downstream enhancer or conventional 5' splice site. Analysis of chimeric transcripts revealed that information dispersed throughout exons and introns dictates SR protein specificity and the requirement for the additional nuclear fraction. Thus, SR proteins function in both major and minor splicing pathways, and in coordinating the activities of both spliceosomes via exon definition. These results suggest that despite the substantial differences in intron consensus sequences and in four of the five snRNPs in each spliceosome, at least some of the interactions involving SR proteins are conserved between the two pathways. PMID:11333026

Hastings, M L; Krainer, A R

2001-01-01

288

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

PubMed

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+1G>T) in affected family members and carriers, 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, Jing Yu; Dai, Xiaohua; Sheng, Jiqun; Cui, Xin; Wang, Xu; Jiang, Xueqing; Tu, Xin; Tang, Zhaohui; Bai, Yan; Liu, Mugen; Wang, Qing K

2008-03-01

289

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy  

PubMed Central

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing. PMID:20186123

Pedrotti, Simona; Bielli, Pamela; Paronetto, Maria Paola; Ciccosanti, Fabiola; Fimia, Gian Maria; Stamm, Stefan; Manley, James L; Sette, Claudio

2010-01-01

290

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

291

Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum ? †  

PubMed Central

Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5? and 3? splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5? and 3? splice sites. However, the 5? consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3? splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack. PMID:21926333

Zhang, Xiaohong; Tolzmann, Caitlin A.; Melcher, Martin; Haas, Brian J.; Gardner, Malcolm J.; Smith, Joseph D.; Feagin, Jean E.

2011-01-01

292

Branch point identification and sequence requirements for intron splicing in Plasmodium falciparum.  

PubMed

Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5' and 3' splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5' and 3' splice sites. However, the 5' consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3' splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack. PMID:21926333

Zhang, Xiaohong; Tolzmann, Caitlin A; Melcher, Martin; Haas, Brian J; Gardner, Malcolm J; Smith, Joseph D; Feagin, Jean E

2011-11-01

293

Next-Generation Sequencing Reveals Deep Intronic Cryptic ABCC8 and HADH Splicing Founder Mutations Causing Hyperinsulinism by Pseudoexon Activation  

PubMed Central

Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ?10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals’ fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease. PMID:23273570

Flanagan, Sarah E.; Xie, Weijia; Caswell, Richard; Damhuis, Annet; Vianey-Saban, Christine; Akcay, Teoman; Darendeliler, Feyza; Bas, Firdevs; Guven, Ayla; Siklar, Zeynep; Ocal, Gonul; Berberoglu, Merih; Murphy, Nuala; O'Sullivan, Maureen; Green, Andrew; Clayton, Peter E.; Banerjee, Indraneel; Clayton, Peter T.; Hussain, Khalid; Weedon, Michael N.; Ellard, Sian

2013-01-01

294

Lung donor selection criteria  

PubMed Central

The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant. PMID:25132970

Chaney, John; Suzuki, Yoshikazu; Cantu, Edward

2014-01-01

295

Lung donor selection criteria.  

PubMed

The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant. PMID:25132970

Chaney, John; Suzuki, Yoshikazu; Cantu, Edward; van Berkel, Victor

2014-08-01

296

Automatic detection of exonic splicing enhancers (ESEs) using SVMs  

PubMed Central

Background Exonic splicing enhancers (ESEs) activate nearby splice sites and promote the inclusion (vs. exclusion) of exons in which they reside, while being a binding site for SR proteins. To study the impact of ESEs on alternative splicing it would be useful to have a possibility to detect them in exons. Identifying SR protein-binding sites in human DNA sequences by machine learning techniques is a formidable task, since the exon sequences are also constrained by their functional role in coding for proteins. Results The choice of training examples needed for machine learning approaches is difficult since there are only few exact locations of human ESEs described in the literature which could be considered as positive examples. Additionally, it is unclear which sequences are suitable as negative examples. Therefore, we developed a motif-oriented data-extraction method that extracts exon sequences around experimentally or theoretically determined ESE patterns. Positive examples are restricted by heuristics based on known properties of ESEs, e.g. location in the vicinity of a splice site, whereas negative examples are taken in the same way from the middle of long exons. We show that a suitably chosen SVM using optimized sequence kernels (e.g., combined oligo kernel) can extract meaningful properties from these training examples. Once the classifier is trained, every potential ESE sequence can be passed to the SVM for verification. Using SVMs with the combined oligo kernel yields a high accuracy of about 90 percent and well interpretable parameters. Conclusion The motif-oriented data-extraction method seems to produce consistent training and test data leading to good classification rates and thus allows verification of potential ESE motifs. The best results were obtained using an SVM with the combined oligo kernel, while oligo kernels with oligomers of a certain length could be used to extract relevant features. PMID:18783607

Mersch, Britta; Gepperth, Alexander; Suhai, Sandor; Hotz-Wagenblatt, Agnes

2008-01-01

297

Underwater splice for submarine coaxial cable  

SciTech Connect

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

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

1984-10-30

298

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses1  

PubMed Central

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDP-glucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156–188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed. PMID:16169969

Thors?e, Karina Sinding; Bak, S?ren; Olsen, Carl Erik; Imberty, Anne; Breton, Christelle; M?ller, Birger Lindberg

2005-01-01

299

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

300

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.

2013-07-08

301

A Unique, Consistent Identifier for Alternatively Spliced Transcript Variants  

PubMed Central

Background As research into alternative splicing reveals the fundamental importance of this phenomenon in the genome expression of higher organisms, there is an increasing need for a standardized, consistent and unique identifier for alternatively spliced isoforms. Such an identifier would be useful to eliminate ambiguities in references to gene isoforms, and would allow for the reliable comparison of isoforms from different sources (e.g., known genes vs. computational predictions). Commonly used identifiers for gene transcripts prove to be unsuitable for this purpose. Methodology We propose an algorithm to compute an isoform signature based on the arrangement of exons and introns in a primary transcript. The isoform signature uniquely identifies a transcript structure, and can therefore be used as a key in databases of alternatively spliced isoforms, or to compare alternative splicing predictions produced by different methods. In this paper we present the algorithm to generate isoform signatures, we provide some examples of its application, and we describe a web-based resource to generate isoform signatures and use them in database searches. Conclusions Isoform signatures are simple, so that they can be easily generated and included in publications and databases, but flexible enough to unambiguously represent all possible isoform structures, including information about coding sequence position and variable transcription start and end sites. We believe that the adoption of isoform signatures can help establish a consistent, unambiguous nomenclature for alternative splicing isoforms. The system described in this paper is freely available at http://genome.ufl.edu/genesig/, and supplementary materials can be found at http://genome.ufl.edu/genesig-files/. PMID:19865484

Riva, Alberto; Pesole, Graziano

2009-01-01

302

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation  

PubMed Central

Precise 5? splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5? splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation. PMID:21468032

Rosel, Tanja Dorothe; Hung, Lee-Hsueh; Medenbach, Jan; Donde, Katrin; Starke, Stefan; Benes, Vladimir; Ratsch, Gunnar; Bindereif, Albrecht

2011-01-01

303

hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing.  

PubMed

Homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1) causes spinal muscular atrophy. SMN1 has been duplicated in humans to create SMN2, which produces a low level of functional SMN protein. However, most SMN2 transcripts lack exon 7, resulting in a non-functional protein. A single nucleotide difference near the 5' end of exon 7 largely accounts for SMN2 exon 7 skipping, an effect that has been attributed to loss of an exonic splicing enhancer (ESE) dependent on the SR protein splicing factor ASF/SF2 or to the creation of an exonic splicing silencer (ESS) element that functions by binding of the splicing repressor hnRNP A1. Our earlier experiments favored the latter mechanism and here we provide further evidence supporting the ESS model. We demonstrate that the striking effect of hnRNP A1 depletion on SMN2 exon 7 splicing is specific, as hnRNP A1 depletion has little or no effect on other inefficient splicing events tested, and ASF/SF2 depletion does not affect SMN1/2 splicing. By two different methods, we find a strong and specific interaction of hnRNPA1 with SMN2 exon 7 and only weak and equivalent interactions between ASF/SF2 and other SR proteins with the 5' ends of SMN1 and SMN2 exon 7. Finally, we describe two disease-related exon-skipping mutations that create hnRNP A1 binding sites, but show that splicing can be restored only modestly or not at all by hnRNP A1 depletion. Together our results provide strong support for the idea that SMN2 exon 7 splicing is repressed by an hnRNPA1-dependent ESS, but also indicate that creation of such elements is context-dependent. PMID:17884807

Kashima, Tsuyoshi; Rao, Nishta; David, Charles J; Manley, James L

2007-12-15

304

In Brief: (mis)splicing in disease.  

PubMed

Splicing of pre-mRNAs is a crucial step in the gene expression pathway. Disruption of splicing has been linked to the pathogenesis of several human diseases and is particularly widespread in cancer. Recently, a number of mutations affecting genes of the core spliceosome machinery have been identified in haematological malignancies, yet the effect of such mutations on RNA splicing is unclear. A better understanding of how mis-splicing contributes to malignancies may provide diagnostic or prognostic information and new drug targets for therapeutic approaches. PMID:24615176

Pedrotti, Simona; Cooper, Thomas A

2014-05-01

305

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

306

46 CFR 111.60-19 - Cable splices.  

Code of Federal Regulations, 2010 CFR

...2010-10-01 2010-10-01 false Cable splices. 111.60-19 ...HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL...Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in a...

2010-10-01

307

Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction.  

PubMed

The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression. PMID:9443963

Antoniou, M; Geraghty, F; Hurst, J; Grosveld, F

1998-02-01

308

Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction.  

PubMed Central

The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression. PMID:9443963

Antoniou, M; Geraghty, F; Hurst, J; Grosveld, F

1998-01-01

309

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing  

PubMed Central

Summary Angiogenesis is regulated by the balance of pro-angiogenic VEGF165 and anti-angiogenic VEGF165b splice isoforms. Mutations in WT1, the Wilms’ tumour suppressor gene, suppress VEGF165b and cause abnormal gonadogenesis, renal failure and Wilms’ tumours. In WT1 mutant cells, reduced VEGF165b was due to lack of WT1 mediated transcriptional repression of the splicing factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding-site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF, and rendered WT1 mutant cells pro-angiogenic. Altered VEGF splicing was reversed by wildtype WT1, knockdown of SRSF1 or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumour growth. PMID:22172722

Amin, Elianna M; Oltean, Sebastian; Hua, Jing; Gammons, Melissa VR; Hamdollah-Zadeh, Maryam; Welsh, Gavin I; Cheung, Man-Kim; Ni, Lan; Kase, Satoru; Rennel, Emma S; Symonds, Kirsty E; Nowak, Dawid G; Pokora-Royer, Brigitte; Saleem, Moin A; Hagiwara, Masatoshi; Schumacher, Valerie A; Harper, Steven J; Hinton, David R; Bates, David O; Ladomery, Michael R

2013-01-01

310

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

311

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

312

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

313

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

PubMed

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

2014-08-01

314

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

315

ATPase\\/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome  

Microsoft Academic Search

We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5 splice site (5ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding

Chunru Lin; Liuqing Yang; Jenny J. Yang; Youliang Huang; Zhi-Ren Liu

2005-01-01

316

Professional donors protest.  

PubMed

In New Delhi, India, professional blood donors, a group of impoverished men who sell blood for a living, reject the accusation that they have spread HIV infection through India's blood supply. Lax sterilization practices by blood banks are blamed. Secrecy surrounds those professional donors, HIV positive, due to social ostracism, and a HIV-positive report among 6 children donating blood is reported to have led to suicide attempts. Estimates of the donor population range from 20,000 to 30,000. 15-16 units at 350 ml/unit are sold a month for sometimes as little as Rs25/unit or US$.85. It is a painful profession due to the thick needles used to make blood draws and the inability to find veins particularly in winter. The Indian Medical Research Council (ICMR) in December 1991 stated that all types of blood donors were found to be HIV positive. The highest seropositivity was found among those who regularly sold blood for blood product manufacture and lowest among voluntary donors. Estimates of HIV-positive incidence in Madras was .38% for voluntary donors and 4.1% for paid donors. An explanation for the paid donor incidence according to the ICMR is that donors are reinfected with the blood portion of donated products after the separation of the plasma. If the plasma-separating machine has not been properly sterilized after each use, 1 unit of HIV-positive blood can infect several donors. For a short period beginning in 1989, blood manufacture was prohibited due to this concern, but the ban has been lifted and a technical committee has been set up to check safety. The ICMR position is compatible with the explanation offered by the leader of the Professional Blood Donors Welfare Association (PBDWA) that donors' physical and economic condition prevents the men from sexual promiscuity with prostitutes or expensive drug use. Reuse of needles and lack of proper sterilization techniques is attributed by the PBDWA as the reason for seropositivity among blood donors. No blood donors tested positive until 1987 even though testing was extensive since 1985. Official case reports of positive HIV infection as of March 1992 are 7183 people and 130 AIDS cases. Blood donors feel victimized. PMID:12318131

Girimaji, P

1992-07-01

317

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

318

Two novel phosphatidylinositol-4-phosphate 5-kinase type I? splice variants expressed in human cells display distinctive cellular targeting  

PubMed Central

The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger PtdIns(4,5)P2 is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type I? isoform of PIPK (PtdIns4P 5-kinase), which generate PtdIns(4,5)P2, have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKI? in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in multiple tissue types and display PIPK activity in vitro. Interestingly, both of these novel splice variants display distinct subcellular targeting. With the addition of these two new splice isoforms, there are minimally five PIPKI? splice variants that have been identified in mammals. Therefore, we propose the use of the HUGO (Human Genome Organization) nomenclature in the naming of the splice isoforms. PIPKI?_i4 (700 amino acids) is present in the nucleus, a targeting pattern that has not been previously observed in any PIPKI? splice variant. PIPKI?_i5 (707 amino acids) is targeted to intracellular vesicle-like structures, where it co-localizes with markers of several types of endosomal compartments. As occurs with other PIPKI? splice variants, the distinctive C-terminal sequences of PIPKI?_i4 and PIPKI?_i5 may facilitate association with unique protein targeting factors, thereby localizing the kinases to their appropriate cellular subdomains for the site-specific generation of PtdIns(4,5)P2. PMID:19548880

Schill, Nicholas J.; Anderson, Richard A.

2009-01-01

319

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

320

Distinctive Characteristics of Educational Donors  

ERIC Educational Resources Information Center

Examining the charitable behavior of 56,663 US households, this paper evaluates the distinctive characteristics of educational donors as compared with donors to noneducational charitable organizations and with nondonors. In general, educational donors had significantly greater income, wealth, and education than other donors. Educational donors

James, Russell N., III.

2008-01-01

321

Multiple links between transcription and splicing.  

PubMed

Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes. PMID:15383674

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

2004-10-01

322

Intron mis-splicing: no alternative?  

PubMed Central

A recent report reveals widespread mis-splicing of RNA transcripts in eukaryotes, with mis-spliced RNA destroyed by nonsense-mediated mRNA decay. This striking inefficiency deepens the mystery of the proliferation and persistence of introns. PMID:18304372

Roy, Scott William; Irimia, Manuel

2008-01-01

323

Use of RNase H and primer extension to analyze RNA splicing.  

PubMed

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo. PMID:2840638

Erster, S H; Finn, L A; Frendewey, D A; Helfman, D M

1988-07-11

324

The Characterizations of Different Splicing Systems  

NASA Astrophysics Data System (ADS)

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

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

325

New connections between splicing and human disease  

PubMed Central

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles called spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in certain cancers and pre-cancerous conditions suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Missplicing of critical genes may underlie the pathologies of both these diseases. Identifying these genes and understanding the mechanisms involved in their missplicing may lead to advancements in diagnosis and treatment. PMID:22397991

Padgett, Richard A.

2012-01-01

326

Phosphoregulation of Ire1 RNase splicing activity  

NASA Astrophysics Data System (ADS)

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

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

2014-04-01

327

Tumor microenvironment–associated modifications of alternative splicing  

PubMed Central

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ?20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors. PMID:24335142

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

2014-01-01

328

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

329

Hydrogen enhancement of silicon thermal donor formation  

NASA Astrophysics Data System (ADS)

Oxygen-related thermal donor formation in Czochralski silicon is characterized by the capacitance-voltage and deep level transient spectroscopy techniques as a function of 450 °C anneal time following hydrogenation. Increases in the formation rate and number of thermal donor (TD) defects found after hydrogenation are reported. This study finds an increase in TD+/++ concentration in the near-surface region at short anneal times, but at longer times an elevated concentration was not observed. No acceleration through the sequence of thermal donor defects was detected. This fails to support the model of hydrogen lowering the barrier to oxygen diffusion and accelerating the TDn?TDn+1 transitions. This study does, however, support a model in which the hydrogen increases the available thermal donor core sites.

Lamp, C. D.; James, D. J., II

1993-04-01

330

Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.  

PubMed Central

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner. PMID:8816452

Zhang, W J; Wu, J Y

1996-01-01

331

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.  

PubMed

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

332

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing  

PubMed Central

Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

Liu, Jing; Hu, Jiaxin; Corey, David R.

2012-01-01

333

A Widespread and Unusual RNA Trans-Splicing Type in Dinoflagellate Mitochondria  

PubMed Central

Cytochrome oxidase subunit 3 (Cox3) is a mitochondrion-encoded core membrane protein of complex IV of the mitochondrial respiratory chain, and consists of seven trans-membrane helices. Here we show that in diverse later-branching dinoflagellates, cox3 is consistently split into two exons in the mitochondrial genome between helices six and seven. Gene exons are transcribed as two discrete oligoadenylated precursor RNAs, and these are subsequently trans-spliced to form a complete coding mRNA. This trans-splicing is highly unusual in that some of the oligoadenylated tail is incorporated at the splice site, such that a short string of adenosines links the two coding exons. This feature is consistently represented in diverse dinoflagellates, however the number of adenosines added varies according to the size of the coding gap between the two exons. Thus we observed between zero (Amphidinium carterae) and 10 (Symbiodinium sp.) adenosines added in different taxa, but the final coding sequence length is identical with the reading frame maintained. Northern analyses show that precursor cox3 transcripts are approximately equally abundant as mature cox3 mRNAs, suggesting a slow or regulated maturation process. These data indicate that the splicing mechanism in dinoflagellate mitochondria is tolerant of variations in the length of the precursor coding sequence, and implicates the use of a splicing template, or guide molecule, during splicing that controls mature mRNA length. PMID:23437234

Jackson, Christopher J.; Waller, Ross F.

2013-01-01

334

Modulation of alternative splicing by expression of small nuclear ribonucleoprotein polypeptide N.  

PubMed

Alternative splicing of pre-mRNA, catalyzed by small nuclear ribonucleoproteins (snRNPs), plays an important role in proteome complexity and the modulation of cellular functions. snRNP polypeptide N (SmN), is tissue-specifically expressed, where it replaces snRNP polypeptide B (SmB)/B' in the Sm core assembly of snRNPs. Recent studies have demonstrated that perturbation of snRNPs leads to alternative splicing, but whether SmN modulates functions of the splicing machinery remains unclear. In this study, we found that ectopic expression of SmN increased utilization of the proximal 5' splice site on an adenovirus early gene 1A reporter. To evaluate the molecular mechanisms underlying SmN-dependent alternative splicing, we generated a HeLa cell line with an inducible expression system for SmN. Upon SmN induction, SmB/B' expression decreased dramatically, despite only small changes in the level and splicing pattern of SNRPB mRNA. In addition, SmN was incorporated into the U2 snRNP but not into the U1 snRNP after induction. Sedimentation analysis revealed a decrease in the level of mature U2 snRNP. This result suggests that SmN incorporation into the Sm core may impede processing, decreasing the level of functional U2 snRNP. We also found that the inclusion frequencies of alternatively spliced exons in the bridging integrator 1 and exocyst complex component 7 (EXOC7) genes were modulated by SmN expression. An enhanced GFP-EXOC7 reporter was used to confirm that SmN increases the inclusion frequency of EXOC7 exon 7. Taken together, our findings indicate that SmN expression reduces the level of mature U2 snRNP, leading to alternative splicing. PMID:25238490

Lee, Moon-Sing; Lin, Yu-Shan; Deng, Yi-Fang; Hsu, Wan-Ting; Shen, Chiung-Chun; Cheng, Yi-Hsin; Huang, Yao-Ting; Li, Chin

2014-12-01

335

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.  

PubMed

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1?, Nrxn1?, Nrxn2?, Nrxn3?, and Nrxn3? mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1? and Nrxn3? (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-?, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that ?-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing. PMID:24639501

Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

2014-04-01

336

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing  

PubMed Central

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1?, Nrxn1?, Nrxn2?, Nrxn3?, and Nrxn3? mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1? and Nrxn3? (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-?, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that ?-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing. PMID:24639501

Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R.; Sudhof, Thomas C.

2014-01-01

337

Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation†  

PubMed Central

Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation. PMID:15684395

Trembley, Janeen H.; Tatsumi, Sawako; Sakashita, Eiji; Loyer, Pascal; Slaughter, Clive A.; Suzuki, Hitoshi; Endo, Hitoshi; Kidd, Vincent J.; Mayeda, Akila

2005-01-01

338

The evolving roles of alternative splicing Liana F Lareau1  

E-print Network

The evolving roles of alternative splicing Liana F Lareau1 , Richard E Green1 , Rajiv S Bhatnagar2,3 and Steven E Brenner1,2Ã? Alternative splicing is now commonly thought to affect more than half of all human forms. Comparisons of alternative splicing between human and mouse genes show that predominant splice

339

Residual Strength Analyses of Riveted Lap-Splice Joints  

NASA Technical Reports Server (NTRS)

The objective of this paper was to analyze the crack-linkup behavior in riveted-stiffened lap-splice joint panels with small multiple-site damage (MSD) cracks at several adjacent rivet holes. Analyses are based on the STAGS (STructural Analysis of General Shells) code with the critical crack-tip-opening angle (CTOA) fracture criterion. To account for high constraint around a crack front, the "plane strain core" option in STAGS was used. The importance of modeling rivet flexibility with fastener elements that accurately model load transfer across the joint is discussed. Fastener holes are not modeled but rivet connectivity is accounted for by attaching rivets to the sheet on one side of the cracks that simulated both the rivet diameter and MSD cracks. Residual strength analyses made on 2024-T3 alloy (1.6-mm thick) riveted-lap-splice joints with a lead crack and various size MSD cracks were compared with test data from Boeing Airplane Company. Analyses were conducted for both restrained and unrestrained buckling conditions. Comparison of results from these analyses and results from lap-splice-joint test panels, which were partially restrained against buckling indicate that the test results were bounded by the failure loads predicted by the analyses with restrained and unrestrained conditions.

Seshadri, B. R.; Newman, J. C., Jr.

2000-01-01

340

Behind the Scene Role of Conserved Threonine in Intein Splicing  

NASA Astrophysics Data System (ADS)

Protein splicing is an autocatalytic process where an ``intein'' self-cleaves from a precursor protein and catalyzes ligation of the flanking fragments. Inteins occur in all domains of life and have myriad uses in biotechnology. While reaction steps of intein splicing are known, mechanistic details remain incomplete. Here, we investigate the possible role of a highly conserved active-site Threonine residue in bringing about the initial step of splicing: peptide bond rearrangement at a conserved Glycine-Cysteine motif. We report that although not part of the active transition state in this reaction, Threonine plays an important role in reducing the energy barrier through charge screening of active residues in the transition state. Interestingly, Threonine-Glycine hydrogen bonding makes sulfur of the attacking Cysteine less nucleophilic, thereby minimizing Coulomb repulsion in the transition state. These non-intuitive results are obtained through a combination of crystal structure, quantum mechanical simulations, and mutagenesis data. Our results further predict that the sluggish reaction rates observed with intein mutants harboring Threonine-Alanine substitutions can be accelerated in the presence of non-aqueous solvents.

Dearden, Albert; Callahan, Brian; Belfort, Marlene; Nayak, Saroj

2012-02-01

341

On the effect of nuclear bridge modes on donor-acceptor electronic coupling in donor-bridge-acceptor molecules  

NASA Astrophysics Data System (ADS)

We report a theoretical study of intra-molecular electronic coupling in a symmetric DBA (donor-bridge-acceptor) complex, in which a donor electronic site is coupled to an acceptor site by way of intervening orbitals of a molecular bridge unit. In the off-resonant (deep tunneling) regime of electronic transport, the lowest unoccupied molecular orbitals (MO's) of the DBA system are split into distinguishable donor/acceptor and bridge orbitals. The effect of geometrical changes at the bridge on the donor/acceptor electronic energy manifold is studied for local stretching and bending modes. It is demonstrated that the energy splitting in the manifold of donor/acceptor unoccupied MOs changes in response to such changes, as assumed in simple McConnell-type models. Limitations of the simple models are revealed where the electronic charging of the bridge orbitals correlates with increasing donor/acceptor orbital energy splitting only for stretching but not for bending bridge modes.

Davis, Daly; Toroker, Maytal Caspary; Speiser, Shammai; Peskin, Uri

2009-03-01

342

Regular languages, regular grammars and automata in splicing systems  

NASA Astrophysics Data System (ADS)

Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

2013-04-01

343

Rous Sarcoma Virus Negative Regulator of Splicing Selectively Suppresses src mRNA Splicing and Promotes Polyadenylation  

Microsoft Academic Search

Retroviruses require a balance of spliced and unspliced RNA for efficient replication. Here, we examined the effect of mutations in a splicing suppressor sequence called the negative regulator of splicing (NRS), located within the gag gene of Rous sarcoma virus. While the NRS mutant viruses showed only small changes in the levels of spliced env mRNAs, they had significant increases

Christopher T. O'Sullivan; Tatjana S. Polony; Robert E. Paca; Karen L. Beemon

2002-01-01

344

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. The 105 participating surgeons at 80 sites were masked to information about the donor cornea including donor age. Surgery and post-operative care were performed according to the surgeons’ usual routines. Subjects were followed for five years. Main Outcome Measures Graft failure, defined as a regraft or a cloudy cornea that was sufficiently opaque as to compromise vision for a minimum of three consecutive months. Results The 5-year cumulative probability of graft survival was 86% in both the <66.0 donor age group and the ?66.0 donor age group (difference = 0%, upper limit of one-sided 95% confidence interval = 4%). In a statistical model with donor age as a continuous variable, there was not a significant relationship between donor age and outcome (P=0.11). Three graft failures were due to primary donor failure, 8 to uncorrectable refractive error, 48 to graft rejection, 46 to endothelial decompensation (23 of which had a prior, resolved episode of probable or definite graft rejection), and 30 to other causes. The distribution of the causes of graft failure did not differ between donor age groups. Conclusions Five-year graft survival for cornea transplants at moderate risk for failure is similar using corneas from donors ? 66.0 years and donors < 66.0 years. Surgeons and patients now have evidence that corneas comparable in quality to those used in this study from donors through age 75 years are suitable for transplantation. PMID:18387407

2009-01-01

345

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.  

PubMed

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection. PMID:8474457

Mayeda, A; Helfman, D M; Krainer, A R

1993-05-01

346

Alternative splicing in disease and therapy  

Microsoft Academic Search

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting

Andrew P Baraniak; Erika L Lasda; Mariano A Garcia-Blanco

2004-01-01

347

Regulation of Neurexin 1[beta] Tertiary Structure and Ligand Binding through Alternative Splicing  

SciTech Connect

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{beta} carrying an alternative splice insert at site SS{number_sign}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{beta}+SS{number_sign}4 reveals dramatic rearrangements to the 'hypervariable surface', the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop {beta}10-{beta}11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca{sup 2+}-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1{beta} isoforms acquire neuroligin splice isoform selectivity.

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

2008-08-04

348

Characterization of a Disease-associated Mutation Affecting a Putative Splicing Regulatory Element in Intron 6b of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene*  

PubMed Central

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5?- and 3?-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75. PMID:19759008

Faa, Valeria; Incani, Federica; Meloni, Alessandra; Corda, Denise; Masala, Maddalena; Baffico, A. Maria; Seia, Manuela; Cao, Antonio; Rosatelli, M. Cristina

2009-01-01

349

Purifying Selection on Splice-Related Motifs, Not Expression Level nor RNA Folding, Explains Nearly All Constraint on Human lincRNAs.  

PubMed

There are two strong and equally important predictors of rates of human protein evolution: The amount the gene is expressed and the proportion of exonic sequence devoted to control splicing, mediated largely by selection on exonic splice enhancer (ESE) motifs. Is the same true for noncoding RNAs, known to be under very weak purifying selection? Prior evidence suggests that selection at splice sites in long intergenic noncoding RNAs (lincRNAs) is important. We now report multiple lines of evidence indicating that the great majority of purifying selection operating on lincRNAs in humans is splice related. Splice-related parameters explain much of the between-gene variation in evolutionary rate in humans. Expression rate is not a relevant predictor, although expression breadth is weakly so. In contrast to protein-coding RNAs, we observe no relationship between evolutionary rate and lincRNA stability. As in protein-coding genes, ESEs are especially abundant near splice junctions and evolve slower than non-ESE sequence equidistant from boundaries. Nearly all constraint in lincRNAs is at exon ends (N.B. the same is not witnessed in Drosophila). Although we cannot definitely answer the question as to why splice-related selection is so important, we find no evidence that splicing might enable the nonsense-mediated decay pathway to capture transcripts incorrectly processed by ribosomes. We find evidence consistent with the notion that splicing modifies the underlying chromatin through recruitment of splice-coupled chromatin modifiers, such as CHD1, which in turn might modulate neighbor gene activity. We conclude that most selection on human lincRNAs is splice mediated and suggest that the possibility of splice-chromatin coupling is worthy of further scrutiny. PMID:25158797

Schüler, Andreas; Ghanbarian, Avazeh T; Hurst, Laurence D

2014-12-01

350

Purifying Selection on Splice-Related Motifs, Not Expression Level nor RNA Folding, Explains Nearly All Constraint on Human lincRNAs  

PubMed Central

There are two strong and equally important predictors of rates of human protein evolution: The amount the gene is expressed and the proportion of exonic sequence devoted to control splicing, mediated largely by selection on exonic splice enhancer (ESE) motifs. Is the same true for noncoding RNAs, known to be under very weak purifying selection? Prior evidence suggests that selection at splice sites in long intergenic noncoding RNAs (lincRNAs) is important. We now report multiple lines of evidence indicating that the great majority of purifying selection operating on lincRNAs in humans is splice related. Splice-related parameters explain much of the between-gene variation in evolutionary rate in humans. Expression rate is not a relevant predictor, although expression breadth is weakly so. In contrast to protein-coding RNAs, we observe no relationship between evolutionary rate and lincRNA stability. As in protein-coding genes, ESEs are especially abundant near splice junctions and evolve slower than non-ESE sequence equidistant from boundaries. Nearly all constraint in lincRNAs is at exon ends (N.B. the same is not witnessed in Drosophila). Although we cannot definitely answer the question as to why splice-related selection is so important, we find no evidence that splicing might enable the nonsense-mediated decay pathway to capture transcripts incorrectly processed by ribosomes. We find evidence consistent with the notion that splicing modifies the underlying chromatin through recruitment of splice-coupled chromatin modifiers, such as CHD1, which in turn might modulate neighbor gene activity. We conclude that most selection on human lincRNAs is splice mediated and suggest that the possibility of splice–chromatin coupling is worthy of further scrutiny. PMID:25158797

Schüler, Andreas; Ghanbarian, Avazeh T.; Hurst, Laurence D.

2014-01-01

351

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating  

PubMed Central

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ?50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development. PMID:22719839

Senatore, Adriano; Spafford, J. David

2012-01-01

352

Targeting RNA-Splicing for SMA Treatment  

PubMed Central

The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease. PMID:22382684

Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

2012-01-01

353

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein.  

PubMed Central

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays. PMID:11911361

Liu, Haiying; Zhang, Wenqing; Reed, Robyn B; Liu, Weiqun; Grabowski, Paula J

2002-01-01

354

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells  

PubMed Central

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing. PMID:24872507

Ammon, Tim; Mishra, Shravan Kumar; Kowalska, Kaja; Popowicz, Grzegorz M.; Holak, Tad A.; Jentsch, Stefan

2014-01-01

355

Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs.  

PubMed Central

Two forms of human thyroid peroxidase cDNAs were isolated from a lambda gt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTPO-1 and hTPO-2, we show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. These results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2. Images PMID:3475693

Kimura, S; Kotani, T; McBride, O W; Umeki, K; Hirai, K; Nakayama, T; Ohtaki, S

1987-01-01

356

Highly n -doped silicon: Deactivating defects of donors  

NASA Astrophysics Data System (ADS)

We report insight into the deactivation mechanisms of group V donors in heavily doped silicon. Based on our ab initio calculations, we suggest a three step model for the donor deactivation. In highly n -type Si grown at low temperatures, in the absence of excess native point defects, the intrinsic limit to ne seems to rise in part by means of donor deactivating distortions of the silicon lattice in the proximity of two or more donor atoms that share close sites. Also, donor dimers play an important part in the deactivation at high doping concentrations. While the dimers constitute a stable or metastable inactive donor configuration, the lattice distortions lower the donor levels gradually below the impurity band in degenerate silicon. On the other hand, we find that, in general, none of the earlier proposed deactivating donor pair defects is stable at any position of the Fermi level. The lattice distortions may be viewed as a precursor to Frenkel pair generation and donor-vacancy clustering process (step 2) that account for deactivation at elevated temperature and longer annealing times. Ultimately, and most prominently in the case of the large Sb atoms, precipitation of the donor atoms may set in as the last step of the deactivation process chain.

Mueller, D. Christoph; Fichtner, Wolfgang

2004-12-01

357

Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues  

PubMed Central

Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads).  Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60%) were novel, involving new exons (~3000), new introns (~16000), or both. When tracing these events across the sixteen tissues, only a small number (4-7%) appeared to be differentially expressed (‘switched’) between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared.  Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository ( http://zenodo.org/record/7068; doi: 10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile. PMID:24555089

Florea, Liliana

2013-01-01

358

SplicePlot: a utility for visualizing splicing quantitative trait loci  

PubMed Central

Summary: RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Availability and implementation: Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available. Contact: wu.eric.g@gmail.com or smontgom@stanford.edu PMID:24363378

Wu, Eric; Nance, Tracy; Montgomery, Stephen B.

2014-01-01

359

Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing.  

PubMed

Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns. PMID:20123975

Younis, Ihab; Berg, Michael; Kaida, Daisuke; Dittmar, Kimberly; Wang, Congli; Dreyfuss, Gideon

2010-04-01

360

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes  

E-print Network

computa- tional predictions. As cancer-related genes such as Mdm2 (ref. 13) or Cdkn2a14 are regulated melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line

Cai, Long